Molecular requirements for PLK1 activation by T-loop phosphorylation

Molecular requirements for PLK1 activation by T-loop phosphorylation | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Molecular requirements for PLK1 activation by T-loop phosphorylation Arianna Esposito Verza , View ORCID Profile Duccio Conti , Paulo D. Rodrigues Pedroso , View ORCID Profile Lina Oberste-Lehn , Carolin Koerner , Sabine Wohlgemuth , Artem Mansurkhodzhaev , View ORCID Profile Ingrid R. Vetter , View ORCID Profile Marion E. Pesenti , View ORCID Profile Andrea Musacchio doi: https://doi.org/10.1101/2025.07.25.666769 Arianna Esposito Verza 1 Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology , Otto-Hahn-Straße 11, 44227 Dortmund, Germany 2 Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen , Essen, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: arianna.espositoverza{at}mpi-dortmund.mpg.de andrea.musacchio{at}mpi-dortmund.mpg.de Duccio Conti 1 Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology , Otto-Hahn-Straße 11, 44227 Dortmund, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Duccio Conti Paulo D. Rodrigues Pedroso 1 Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology , Otto-Hahn-Straße 11, 44227 Dortmund, Germany 3 i3S - Instituto de Investigação e Inovação em Saúde , Rua Alfredo Allen, 208, 4200-135 Porto (PT) Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lina Oberste-Lehn 1 Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology , Otto-Hahn-Straße 11, 44227 Dortmund, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lina Oberste-Lehn Carolin Koerner 1 Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology , Otto-Hahn-Straße 11, 44227 Dortmund, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sabine Wohlgemuth 1 Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology , Otto-Hahn-Straße 11, 44227 Dortmund, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Artem Mansurkhodzhaev 1 Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology , Otto-Hahn-Straße 11, 44227 Dortmund, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ingrid R. Vetter 1 Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology , Otto-Hahn-Straße 11, 44227 Dortmund, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ingrid R. Vetter Marion E. Pesenti 1 Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology , Otto-Hahn-Straße 11, 44227 Dortmund, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Marion E. Pesenti Andrea Musacchio 1 Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology , Otto-Hahn-Straße 11, 44227 Dortmund, Germany 2 Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen , Essen, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Andrea Musacchio For correspondence: arianna.espositoverza{at}mpi-dortmund.mpg.de andrea.musacchio{at}mpi-dortmund.mpg.de Abstract Full Text Info/History Metrics Preview PDF ABSTRACT Activation of PLK1, a master mitotic kinase, requires phosphorylation of its activation segment on Thr210, within a basic consensus sequence for Aurora kinases. Aurora B-dependent phosphorylation of Thr210 has been reported, but other evidence identified a strict requirement for the Aurora A partner Bora for Thr210 phosphorylation. Here, we investigate the elusive mechanistic basis for this requirement. We show that Aurora A:Bora phosphorylates Thr210 of PLK1 in vitro, while Aurora A, other Aurora A:activator complexes, and Aurora B:INCENP fail to target T210 even at high kinase/substrate ratios. A transient interaction of Bora and PLK1, identified by structural modelling and probed mutationally, is uniquely required for Thr210 phosphorylation. Dependency on Bora for Thr210 phosphorylation is eliminated after mutating Lys208, in the Aurora consensus, into arginine. This conservative mutation turns PLK1 into an excellent substrate of nearly all tested active Aurora kinases, especially Aurora B. Collectively, these results shine a new light on the specificity of the PLK1 activation mechanism. INTRODUCTION The spatial and temporal orchestration of a cell’s entry into mitosis reflects the ordered activation of several master mitotic serine/threonine (Ser/Thr) kinases, including cyclin-dependent kinase 1 (CDK1), Aurora A and B, and Polo-like kinase 1 (PLK1) ( 1 , 2 ). Besides reciprocally controlling their own activation and the activity of several downstream targets, these kinases are also embedded in a regulated network of protein phosphatases, including different forms of the protein phosphatases 1 and 2A (PP1 and PP2A, respectively)( 2 , 3 ). To reach full activity, many protein kinases require post-translational phosphorylation of their activation loop (or T-loop) either by a distinct trans-activating kinase or by auto-phosphorylation ( 4 – 6 ). The phosphate group, typically attached to a threonine or serine residue, interacts with positively charged amino acids to induce conformational changes in the kinase that stabilize its active conformation. This consists of an inward (or “DFG-in”, where DFG stands for aspartate-phenylalanine-glycine) conformation of the activation loop that is ideally suited for substrate binding and phosphotransfer ( 4 – 6 ). This activation mechanism has a prominent role in the temporal and spatial control of kinase activity. A full comprehension of its regulation is therefore a crucial aspect of dissecting how the activity of protein kinases is controlled in their specific biological context ( 4 – 6 ). PLK1 activity is required for the success of several distinct events occurring predominantly between the G2 phase and the subsequent M and G1 phases, and that include, among several others, nuclear envelope breakdown, centrosome maturation and spindle assembly, chromosome bi-orientation, cytokinesis, as well as the deposition of the specialized centromere histone CENP-A in early G1 ( 7 , 8 ). In G2, PLK1 becomes progressively activated through phosphorylation of Thr210 (in human PLK1) on the activation loop by trans-activation ( 4 , 9 – 15 ). PLK1 then promotes mitotic entry by activating Cell Division Cycle 25 (CDC25), which dephosphorylates and activates the master regulator of mitosis, Cyclin-dependent kinase 1 (CDK1) ( 16 – 19 ). PLK1 phosphorylates target motifs matching the consensus sequence [D/N/E]-X-[ S/T ]-[I/L/M/V/F/W/Y] ( 20 – 22 ). This substrate preference likely explains why PLK1 is unable to auto-phosphorylate on Thr210, a residue embedded in a sequence motif that is typically targeted by basophilic kinases of the AGC group, which includes Protein Kinase A, and closely related members such as the Aurora kinase family ( 23 ). The activation segments of Aurora A and B kinases share high sequence similarity to that of PLK1 ( Figure 1A ), and these basophilic kinases can auto-activate through phosphorylation on T288 and T232, respectively ( 24 – 26 ). Both Aurora A and Aurora B have been proposed to phosphorylate PLK1 on Thr210 to activate it ( 9 ). Aurora B has been implicated in PLK1 activation in the model organism Drosophila melanogaster ( 27 , 28 ). Evidence of an Aurora B involvement in PLK1 activation in other organisms, however, remains limited. Conversely, there is ample evidence of a ubiquitous role of Aurora A in PLK1 activation in late G2 phase as well as in maintaining PLK1 activation during mitosis ( 16 , 18 , 19 , 29 – 32 ). Download figure Open in new tab Figure 1. PLK1 is a poor substrate for Aurora kinases A ) Sequence Logo for human PLK1, human Aurora A and human Aurora B T-loop sequences. Sequences were obtained from TreeFam database and Logos were generated with WebLogo (Berkeley University). B ) AlphaFold 2 (AF2) multimer model of Aurora A in complex with Bora is shown in comparison to the experimental models of Aurora A bound to TPX2 (PDB code: 1OL5), CEP192 (PDB code: 8PR7), and to Aurora B in complex with INCENP (PDB code: 2BFY). Aurora A and Aurora B models are displayed as green cartoons, protein cofactors are displayed as coral red cartoons. C ) Top view of the N-lobe of Aurora A bound to Bora (AF2 model) and TPX2 (PDB code: 1OL5). Coloring scheme as in (B). D ) Western blot showing activation loop phosphorylation of all Aurora kinase complexes. The anti-Aurora A pThr288 antibody cross-reacts with Aurora B pThr232 (indicated by an asterisk). E ) Schematic representation of proteins used for the biochemical assay, including Aurora A and B, the listed protein cofactors and the substrates PLK1, NDC80 bonsai , H3 nucleosome, and CENP-A nucleosome. F-I ) Phosphorylation of PLK1 Thr210 (F), Ndc80 Ser55 (G), H3 Ser10 (H), and CENP-A Ser7 (I) was monitored by Western blotting with phospho-specific antibodies and quantified with Aurora:Bora A activity as reference (equal to 1.0). The arithmetic mean of two independent experiments is reported. A Coomassie Brilliant Blue stained SDS-PAGE gel of the same samples was combined to verify equal protein loading between conditions. Aurora A promotes PLK1 activation in complex with the protein cofactor Bora ( 30 – 34 ). Bona fide activators of Aurora A, such as TPX2 and CEP192, contain unfolded segments that bind the kinase’s N-terminal lobe to stabilize the active, inward conformation of the activation loop ( 35 – 38 ). A similar mechanism leads to activation of Aurora B by the IN-box segment of INCENP ( 39 – 42 ). Bora’s intrinsic disorder and recognizable sequence similarities with TPX2 led to propose that it may activate Aurora A through a structural mechanism reminiscent of that of other Aurora A activators ( 32 ). Besides interacting with Aurora A’s kinase domain, Bora also interacts with PLK1 ( 43 – 45 ). Phosphorylation of Ser252 of human Bora (hBora) by CDK1 promotes this interaction with the polo-box domain of PLK1 (PBD) ( 15 , 16 , 19 , 30 , 32 , 46 – 49 ). The PBD is believed to stabilize an auto-inhibited conformation of PLK1. Its engagement through a phosphopeptide has been proposed to relieve auto-inhibition and also to promote phosphorylation of the PLK1 activation loop ( 30 , 50 ). Binding of the PLK1 PBD to pSer252 of Bora, however, is not required for Thr210 phosphorylation, which proceeds normally on the isolated kinase domain of PLK1 in the absence of the PBD ( 32 ). Moreover, Bora 1-224 , a minimal Bora fragment lacking the PLK1 docking site but still capable of interacting with Aurora A, remains proficient in PLK1 phosphorylation ( 32 , 48 ). PLK1 binding to Ser252 is instead involved in partial degradation of Bora at the onset of mitosis through b β-TrCP( 49 , 51 ). More recent studies have implicated phosphorylation of Ser252 in PLK1 dimerization and its controlled activation ( 52 ). Another way in which CDK activity regulates Bora towards PLK1 is through phosphorylation of Ser112 of Bora. After phosphorylation, this residue can act in trans as mimic of Aurora A Thr288 phosphorylation, activating the dephosphorylated kinase and even bypassing the deleterious effects on Aurora A activity resulting from mutating Thr288 into valine ( 32 ). Mutation of Ser112 to a non-phosphorylatable residue also prevents timely mitotic entry ( 32 ), indicating that its phosphorylation enables exquisite control of the Aurora A:Bora complex by CDK activity around mitotic entry. If the only contribution of Bora to PLK1 T210 phosphorylation were through the activation of Aurora A, other Aurora:cofactor complexes, such as Aurora B:INCENP or Aurora A:CEP192, may also be expected to activate PLK1, unless prevented by strict lack of co-localization. Indeed, besides the already mentioned evidence of PLK1 activation by the Aurora B:INCENP complex at centromeres and the central spindle during cytokinesis, evidence of PLK1 phosphorylation by Aurora A:CEP192 or Aurora A:Protein furry homolog (FRY) at centrosomes has also been reported ( 27 , 28 , 53 – 59 ). Puzzlingly, however, compensation by other kinases of the decrease in pThr210 levels observed upon Bora depletion has not been observed ( 29 , 31 ), supporting the specificity of the Bora-dependent mechanism. To shed further light on this issue, it is therefore important to investigate the precise mechanism through which Bora activates Aurora A and targets it to PLK1, and to explore whether comparable mechanisms are employed by other cofactors of Aurora kinases. Here, we dissected the biochemical underpinnings of the Bora:Aurora A catalytic complex and of its activity towards PLK1 in vitro, and validated our findings in human cells when applicable. Our results indicate that Aurora A:Bora relies on a transient interaction to the PLK1 kinase domain for Thr210 phosphorylation. This interaction overcomes an intrinsic inefficiency of Aurora kinases in phosphorylating the non-canonical Thr210 motif that is effectively bypassed by the mutation of a single crucial gatekeeper residue. RESULTS A structural prediction of the Aurora A:Bora complex Previous structural work identified distinct but related binding modes for Aurora kinase activators (herewith referred to as Aurora cofactors). These 40-to 100-residue fragments are devoid of a stable own conformation, but adopt one upon binding to the small lobe of the cognate Aurora kinase, embracing it (as in the case for CEP192 and INCENP), or simply lining it (as is the case for TPX2) (see gallery in Figure 1B ). We used AlphaFold (AF) ( 60 ) to obtain a structural model of the complex of Aurora A with a previously identified ( 32 ) minimal activation segment of Bora (residues 18-120). AF predicted with high confidence that Bora 18-120 wraps around the small lobe of Aurora A, extending even beyond a full circumference ( Figure 1C ). In the complex of Aurora A with TPX2, two fragments of TPX2 (previously identified as M1 and M2, and corresponding approximately to segments TPX 7-21 and TPX 30-43 ( 32 )) form a hairpin near the αC helix of Aurora A ( Figure 1C ). The contact with Bora predicted by AF is significantly more extensive, but segments encompassing Bora 22-48 and Bora 89-107 seem to correspond to the TPX2 M1 and M2 segments, as predicted ( 32 ), with significant differences (e.g., the M1 regions run in opposite directions in the Bora and TPX2 models, Figure 1C ). In both cases, there is a predominance of contacts with the kinase through aromatic residues. In M1, the side chains of Phe25, Leu39, and Phe45 of Bora occupy positions equivalent to those of Tyr8, Tyr10, and Phe19 of TPX2, respectively. In M2, Phe103 and Phe104 are equivalent to Trp34 and Phe35 of TPX2, respectively. Kinases and cofactors contribute to substrate preference in vitro We assembled several Aurora kinase:cofactor complexes and assessed their ability to phosphorylate PLK1 and other substrates in vitro in comparison to Aurora A:Bora. Specifically, we expressed and purified recombinant human Aurora A, and combined it with fragments of Bora (residues 1-224), TPX2 (full length), and CEP192 (residues 400-600) encompassing the predicted or experimentally validated binding sites for Aurora A ( Figure S1A ). We also co-expressed Aurora B kinases with INCENP (residues 351-C) (a detailed procedure is described in Material and Methods). Successful binding of the cofactors to Aurora A was verified by analytical size exclusion chromatography (SEC) ( Figure S1B ). With or without cofactor, T288 in the activation segment of Aurora A was found to be phosphorylated ( Figure 1D , top ). Next, we used these Aurora:cofactor complexes in kinase assays in vitro with various substrates ( Figure 1E ). Substrates included, besides Thr210 in the PLK1 activation loop, Ser55 in the N-terminal tail of the kinetochore protein NDC80, Ser10 in N-terminal tail of histone H3, and Ser7 in the N-terminal tail of the centromere-specific histone H3 variant CENP-A ( 61 – 64 ) ( Figure 1E-H ). Phosphorylation was monitored by using phospho-specific antibodies and a procedure adapted from previous studies ( 32 ). The Aurora A:Bora complex phosphorylated PLK1’s Thr210 very efficiently in the conditions of our assay. Conversely, Aurora A alone, or its complexes with TPX2 or CEP192, were unable to phosphorylate Thr210. Phosphorylation of PLK1 by Aurora B in complex with its activating cofactor INCENP ( 30 , 31 , 39 , 40 , 42 ) was also below the detection limit ( Figure 1F ). Note that the anti-pThr210 antibody cross-reacts with the auto-phosphorylated activation segment of Aurora B (as indicated by an asterisk in Figure 1F ) but not with the one of Aurora A, which was nonetheless phosphorylated in all cases ( Figure 1D ). Thus, only Aurora A:Bora can phosphorylate PLK1 efficiently. Unlike Thr210 of PLK1, Ser55 of Ndc80 was efficiently phosphorylated by all the Aurora:cofactor complexes in the same time frame, including Aurora A:Bora, while Aurora A:CEP192 was the least efficient kinase on this target ( Figure 1G ). Histone H3 was also phosphorylated by all kinases except Aurora A:CEP192 ( Figure 1H ). CENP-A, on the other hand, was preferentially phosphorylated by Aurora B:INCENP and was poorly phosphorylated by Aurora A and its variants ( Figure 1I ). Taken as a whole, these experiments indicate that the Aurora A: CEP192 is the least active of the tested Aurora complexes, at least towards the limited number of substrates in our set, in line with a recent report ( 36 ). These results also indicate that the activity of the Aurora A:Bora complex, at least in vitro, is not limited to PLK1, as the complex can target additional substrates. Conversely, by identifying Aurora A:Bora as a highly selective activator of PLK1, our experiments suggest that further work in vitro may unveil the mechanistic basis of this phenomenon. Bora promotes transient binding to the PLK1 kinase domain To investigate how Bora directs Aurora A to the PLK1 activation loop, we hypothesized that Bora may directly bind PLK1, as suggested by previous observations that both the PBD and kinase domain of PLK1 bind Bora in cells( 30 ). While the dispensability of the PBD for PLK1 activation has been demonstrated( 32 , 48 ), the requirement of Bora binding to the PLK1 kinase domain remains unclear. Attempts to reconstitute a stable complex using Bora, Aurora A, and PLK1 in pull-down and nuclear magnetic resonance binding assays did not yield a stable complex ( 32 ), a finding we independently confirmed (AEV and AM, unpublished results). Nevertheless, we reasoned that in the rapid phosphotransfer reaction, even a transient interaction may contribute to target selection. We therefore queried AlphaFold (AF) Multimer ( 65 ) to identify possible interactions of the Aurora A:Bora complex with PLK1. In a highly reproducible prediction of the ternary complex, AF identified a binding interface built up by a segment encompassing residues 55-69 of Bora and a region encompassing the N-terminal extension, the αB-αC helices, and the β4 strand of PLK1, all in the small lobe ( Figure 2A-B , and Figure S2A-B ). Residues 55-69 of Bora are positioned between the two TPX2-like M1 and M2 motifs of Bora, and their sequence is conserved across species ( Figure 1C , Figure 2B and Figure S2C ). We refer to this region as the PLK1-binding motif of Bora (abbreviated as P1 motif). The conformation of this segment is largely identical in the predicted binary (Aurora A:Bora) and ternary (Aurora A:Bora:PLK1) complexes, and is stabilized by its extensive interactions with the Aurora A small lobe. In the prediction of the ternary complex, the Aurora A catalytic cleft is positioned in front of the PLK1 activation-loop, poised for phosphotransfer. The ternary complex as depicted in Figure 2A reveals an additional interaction whereby the PLK1 PBD docks on Ser252 of Bora. However, the interaction of the Bora P1 motif with PLK1 was predicted even with a fragment of Bora lacking the PBD docking site, as well as in the absence of Aurora A. Thus, collectively, the predictions suggest that a favorable reciprocal configuration of kinase domains in an Aurora A:Bora:PLK1 ternary complex, orchestrated by the P1 motif of Bora, may direct Aurora A to the PLK1 activation loop. Download figure Open in new tab Figure 2. PLK1-Bora interface drives T-loop phosphorylation catalysis A ) Cartoon representation of the Aurora A:Bora 18-280 : PLK1 AlphaFold 2 model. PLK1 Kinase Domain (KD) = dark blue; PLK1 Polo-Box Domain (PBD) = light blue, Aurora A kinase domain (KD) = green; Bora = coral red. The close-up on the right shows the residues mainly involved in the interaction between the PLK1 kinase domain and Bora. B ) Sequence conservation of the Bora “P1” element and of PLK1 region of interaction with Bora, across species. Residues selected for point mutagenesis are marked with asterisks. White coloring represents minimum level of sequence conservation; dark orange (Bora) and dark blue (PLK1) the highest. The complete version of the alignment of the sequences from all species included in our analysis is shown in Figure S2C-D . C ) T-loop phosphorylation of PLK1 variants in vitro was checked by Western blotting after 15, 30, 45, 60 minutes at 30°C, in presence of the Aurora A:Bora complex (n=2). D ) T-loop phosphorylation of PLK1 by Aurora A in presence of WT or the indicated Bora variants was checked by Western blotting after 60 minutes at 30°C (n=2). E ) Quantification of data in panels D and F. The arithmetic mean of two independent experiments is reported. F ) The same Bora variants as in (D) were phosphorylated with CDK1-phosphorylated Bora for 60 minutes at 30°C and then added to the reaction mixture. Phosphorylation of PLK1 was monitored by Western blotting (n=2). To validate this model, we generated single amino acid substitutions to alanine of highly conserved exposed residues of PLK1 predicted by AF to interact with the P1 motif, and including Arg48, Arg95, Arg106, and Glu121 ( Figure 2A , right panel , and Figure S2D ). None of the mutations appeared to destabilize PLK1, as demonstrated by essentially identical catalytic activity on a C-terminal truncation of SA2, a bona fide substrate of PLK1 ( 66 ) ( Figure S2E ). We then tested the effects of the mutations on PLK1 Thr210 phosphorylation. The replacement of Arg48 and Arg106 with alanine displayed a very moderate reduction in the levels of Thr210 phosphorylation in a time course assay, which was only visible at the earliest timepoint of 15’. On the contrary, mutations of Arg95 and Glu121 led to a highly potent inhibition of Thr210 phosphorylation ( Figure 2C ). Next, we introduced alanine mutations in the Bora P1 motif, and specifically at residues Phe56, Trp58, Ser59, and Ile66, predicted to be exposed and in contact with PLK1 in our AF model. All the amino acid substitutions tested produced a deleterious effect on PLK1 Thr210 phosphorylation in single timepoint assays, with mutations of Phe56 and Trp58 appearing most penetrant, with almost no residual Thr210 phosphorylation ( Figure 2D , quantified in Figure 2E ). CDK phosphorylation on Bora’s Ser112 reinforces binding to Aurora A, enhancing its activity towards PLK1 Thr210 ( 32 ). Furthermore, Bora phosphorylation activates Aurora A towards PLK1 Thr210 in the absence of Aurora A activation loop phosphorylation at Thr288, even when this residue is mutated to prevent phosphorylation ( 32 ). We therefore asked if the deleterious effects on PLK1 phosphorylation from the mutations in the Bora P1 motif could be bypassed by pre-phosphorylation of Bora with CDK, a condition expected to mimic cellular events near the G2-M transition. To avoid confounding effects, we mutated Aurora A Thr288 to valine to entirely eliminate phosphorylation of the Aurora A activation loop (shown schematically in Figure S3C ), as previously reported ( 32 ). In the absence of CDK activity, Bora failed to activate Aurora A T288V to promote PLK1 Thr210 phosphorylation, whereas addition of CDK1 resulted in robust PLK1 phosphorylation ( Figure 2F , quantified in Figure 2E ). Addition of CDK1 resulted in an apparent rescue of the negative effects of alanine mutations of Ser59 and Ile66, but the mutations of Phe56 and Trp58 remained very penetrant even under these conditions. The same mutations did not affect the ability of the Aurora A:Bora complex to phosphorylate NDC80 on Ser55, while H3 Ser10 phosphorylation appeared very slightly affected in the same reaction conditions ( Figure S3A ). Even when combined, the mutations did not affect the interaction of Bora with Aurora A, as shown by analytical SEC binding assay ( Figure S3B ). Collectively, these results validate the hypothesis, supported by a high-confidence AF model, that Bora and PLK1 physically interact to allow Aurora A to phosphorylate the PLK1 activation loop. Preventing binding to Bora affects PLK1 activation in cells Bora reaches its expression peak during G2 and is then degraded ( 49 , 51 ), but a small residual pool of Bora is retained in mitosis ( 29 , 67 ). Correspondingly, mitotic cells depleted of Bora or acutely treated with an Aurora A inhibitor were shown to possess decreased levels of phospho-PLK1 ( 29 ). To evaluate the cellular consequences from expressing Bora mutants defective in PLK1 activation, we generated stable doxycycline-inducible cell lines to express mNeonGreen (mNG)-tagged versions of the most penetrant PLK1 and Bora mutated variants, including PLK1 R95A , PLK1 E121A , Bora F56A , and Bora W58A . After inducing the expression of the relevant transgenes, we performed pull-downs from mitotically-arrested cells to enrich PLK1 and monitor pT210 phosphorylation by Western blotting (pre-purification of PLK1 via pull-down was necessary as pT210 detection on the whole cell lysate was sub-optimal and did not result in reproducible quantifications). Both PLK1 R95A and PLK1 E121A caused a reduced level of T210 phosphorylation, with E121A having a stronger effect than R95A ( Figure 3A and Figure S3D ). To assess the function of Bora mutants, endogenous Bora was depleted and replaced by re-expression of a transgenic construct. Bora depletion resulted in a marked reduction of PLK1 phosphorylation in mitotic cells. While seemingly in contrast with an early report( 30 ), this observation is instead consistent with more recent work ( 29 ). Expression of Bora WT rescued the reduced phosphorylation of T210 of PLK1, while expression of Bora F56A or Bora W58A did not promote any phosphorylation of T210 ( Figure 3B and Figure S3E ). Download figure Open in new tab Figure 3. PLK1-Bora interface mutants decrease PLK1 phosphorylation in human cells A ) mNeonGreen-PLK1 WT , PLK1 R95A , or PLK1 E121A were purified via pull-down from mitotic HeLa cells inducibly expressing the PLK1 variants. The bar plot represents the arithmetic mean of three replicates and black dots are the normalized values (over PLK1 signal) of signal intensity for each replicate. OE= Over-expressed. B ) Pull-down from mitotic HeLa cell lysates expressing mCherry-PLK1 WT , depleted of Bora, and transfected to express mNeonGreen-Bora WT , Bora F56A , or Bora W58A . The bar plot represents the arithmetic mean of four replicates and black dots are the normalized values (over PLK1 signal) of signal intensity for each replicate. OE= Over-expressed; siBora= siRNA-mediated depletion of endogenous Bora. C ) Scheme of the experiment presented in panel (D). D-E ) HeLa cell lines expressing mNeonGreen-PLK1 (wild-type or mutants), mNeonGreen-Bora (wild-type or mutants) were synchronized via double thymidine block and released to follow their entry into mitosis. Dot plots show the distributions of mitotic entry time of individual cells since double-thymidine release (DTR). Each dot represents an individual cell; the horizontal line represents the median and the vertical line the 95% confidence interval (CI) of the median, which can be used for statistical comparison of different conditions (see Materials and Methods). OE= Over-expressed; siBora= siRNA-mediated depletion of endogenous Bora. The effects of Bora depletion have been studied mainly in the context of early mitotic functions, where a delay in mitotic entry as well as spindle alterations, were observed. At least the spindle alterations, however, were less dramatic than those observed upon acute small-molecule inhibition of PLK1 activity ( 16 , 17 , 29 – 31 , 49 , 51 , 68 ). Based on these previous results, we decided to assess the effects of inhibiting the Bora:PLK1 interaction on the timing of mitotic entry. Towards this goal, we depleted endogenous PLK1, or co-depleted endogenous PLK1 and Bora, and re-expressed mNG-PLK1 E121A or mNG-PLK1 WT . To monitor mitotic entry, we synchronized cells at the G1/S transition with a double-thymidine block, released them from the arrest, and followed their progression into G2 and M phase for 23 hours using time-lapse fluorescence microscopy ( Figure 3C ). Over-expression of mNG-PLK1 WT rescued, at least partly, the delay caused by depletion of endogenous PLK1 ( Figure 3D ). By co-depleting Bora, over-expressed mNG-PLK1 WT was not properly activated, as testified from the further, albeit modest, increase in the mitotic entry delay. Depletion of endogenous PLK1 combined with over-expression of mNG-PLK1 E121A produced a mitotic entry delay similar or even more severe that the one observed after Bora and PLK1 co-depletion ( Figure 3D and Figure S3F ). Expression of the Bora mutants led to a similar trend: cells expressing Bora WT were more proficient than Bora F56A or Bora W58A in rescuing the mitotic delay caused by Bora depletion ( Figure 3E and Figure S3G ). A gatekeeper residue for Bora selectivity in the PLK1 T-loop Substrate selectivity of Aurora kinase family members has been extensively characterized. The minimal consensus sequence consists of positively charged residues upstream of the phospho-acceptor site and a hydrophobic residue immediately downstream of it (R/K/N-R/K-X- S/T -Φ, where X stands for any amino acid and Φ for hydrophobic amino acids except a proline; the phosphorylated residue is shown in bold) ( 20 , 69 – 71 ). This consensus sequence, also present in the activation loops of Aurora A and B, is the basis of their auto-activation mechanism ( Figure 1A ). Despite its similarity to the activation loop of Aurora kinases, the PLK1 activation loop carries lysine rather than arginine at the -2 residue (position 208 in the human enzyme; Figure 1A ). This substitution, albeit conservative, is a largely evolutionary conserved feature of the PLK1 activation loop ( Figure S4A ). Presence of a -2 lysine does not universally prevent efficient Aurora phosphorylation of substrates, but arginine is preferred over lysine at this position ( 20 , 69 – 74 ). We therefore hypothesized that this substitution may limit the ability of Aurora kinases to phosphorylate the PLK1 activation loop, and that the limitation might be overcome by the direct interaction of the Aurora A:Bora complex with PLK1. To test this hypothesis, we replaced Lys208 with arginine (PLK1 K208R ) and asked if this amino acid substitution facilitated phosphorylation of PLK1 Thr210 by additional Aurora kinase complexes. In an in vitro kinase assay using SA2 as a substrate, PLK1 K208R appeared as active as PLK1 WT , indicating that the mutation does not cause overt structural or functional perturbations ( Figure S4B ). Again, we compared the ability of Aurora kinases to phosphorylate PLK1 in presence or absence of the K208R mutation. In agreement with our hypothesis, the PLK1 K208R mutant allowed every Aurora species we tested to phosphorylate PLK1 Thr210, with the Aurora A:TPX2 and Aurora B:INCENP complexes being apparently as active on Thr210 as Aurora A:Bora, in sharp contrast with their complete inability to phosphorylate wild-type PLK1 within the reaction time ( Figure 4A ; a schematic description of this experiment is presented in Figure S4C ). Mutations of two additional residues neighboring Lys208 – i.e. Glu206 to leucine (the corresponding residue in the Aurora B activation-loop) and Arg207 to lysine, did not recapitulate this effect. PLK1 E206L was phosphorylated essentially indistinguishably from PLK1 WT , showing the same selectivity for the Aurora A:Bora complex ( Figure S4D , lanes 1-6 ). PLK1 R207K , on the other hand, was also phosphorylated selectively, but to lower levels, in line with the general importance of the -3 arginine residue ( Figure S4D , lanes 7-12 ). So, neither mutation made PLK1 accessible to other Aurora kinases, a property that, among the mutants we tested, was only conferred by the Lys208 to arginine mutation. Download figure Open in new tab Figure 4. The primary sequence of PLK1 T-loop renders phosphorylation contingent on Bora A ) PLK1 WT or PLK1 K208R were assayed in parallel for activation with the same Aurora complexes. A western blot is shown to assess relative phosphorylation of the substrates, quantified with Aurora A:Bora activity on PLK1 K208R as reference (equal to 1.0). The arithmetic mean of two independent experiments is shown and each black dot represents the signal intensity for a single experiment. A Coomassie Brilliant Blue stained gel of the same samples was combined to verify equal protein loading between conditions. B ) An MBP fused PLK1 kinase domain WT or K208R (1–326) was assayed under the same conditions as for PLK1 in (A). C ) An MBP fused peptide containing PLK1 T-loop sequence WT or K208R was assayed under the same conditions as for PLK1 in (A). D ) PLK1 WT was assayed for activation in presence or absence of MAP205 and BUB1 docking sites. Samples were collected during the phosphorylation reaction at 5 and 60 minutes. Another set of samples was incubated for 15 hours at 10°C, to saturate the reaction. To further probe the specificity of this effect, we increased the concentration of PLK1 substrate ∼12-fold (to a concentration of 2.5 µM) and tested Thr210 phosphorylation with increasing concentration of Aurora A:Bora or Aurora B:INCENP, up to an equimolar concentration with the substrate. Wild type PLK1 continued to remain entirely impervious to phosphorylation by the Aurora B:INCENP complex, even under these extremely facilitating conditions, while it was readily phosphorylated by Aurora A:Bora, as anticipated. In contrast, PLK1 K208R was phosphorylated at all concentrations of Aurora B:INCENP and with apparently indistinguishable rates in comparison to Aurora A:Bora, likely reflecting saturation over the entire range of conditions ( Figure S4E ). Thus, Lys208 of PLK1 is a crucial gatekeeper deterring Aurora kinase:cofactor variants other than the Aurora A:Bora complex from phosphorylating PLK1 Thr210. The PLK1 activation loop is an intrinsically poor Aurora substrate The “closed”, autoinhibited conformation of PLK1 is stabilized by the interaction of the PBD and kinase domains. It has been proposed that in this closed conformation, the activation segment of PLK1 may be inaccessible to phosphorylation, and that the crucial step for the Aurora A:Bora complex to be granted access to PLK1 Thr210 is the release of the closed conformation upon engagement of the PBD on phosphorylated Bora ( 30 ). Our new observations now suggest that the aminoacid sequence of the activation segment is also a crucial determinant of Aurora kinase selectivity. To investigate whether the sequence of the activation segment plays a role independently of its accessibility, we truncated PLK1 at the beginning of the interdomain linker (aa 326), a few residues downstream the C-side end of the kinase domain. Confirming our results that the selectivity for Aurora A:Bora is attributable to features largely localized to the kinase domain, the phosphorylation patterns of PLK1 1-326 WT and of PLK1 1-326 K208R remained unchanged, with Aurora B gaining access to the mutant but not to the wild-type ( Figure 4B ). These results suggest that the PLK1 activation loop is intrinsically a better substrate for the Aurora A:Bora complexes. However, it is also possible that the mutation of Lys208 to arginine affects the overall exposure of the PLK1 activation loop, improving its accessibility to Aurora kinases other than Aurora A:Bora. We therefore asked if selective phosphorylation of Thr210 by the various Aurora species was also retained when the activation loop was fully exposed. For this, we fused a 15-residue peptide encompassing Thr210 and the seven residues downstream and upstream to His-MBP, and subjected it to in vitro phosphorylation. The T-loop peptide with wild type sequence was poorly phosphorylated by any Aurora species, including the Aurora A:Bora complex ( Figure 4C ). On the contrary, an equivalent construct carrying the K208R mutation was efficiently phosphorylated by all Aurora complexes, recapitulating results obtained with the full-length PLK1 K208R mutant ( Figure 4C ). These observations show that the sequence of the PLK1 activation loop is an intrinsically poor Aurora substrate, and that its incorporation in the context of the kinase domain, and possibly also of other PLK1 features, builds the selectivity for Aurora A:Bora. The PLK1 activation loop in closed-PLK1 is accessible to Aurora A:Bora In principle, a requirement for a transition from a closed to an open conformation as a condition for PLK1 Thr210 phosphorylation is not mutually exclusive with the observed selectivity of Thr210 phosphorylation on Aurora A:Bora. For instance, the Aurora A:Bora complex may perform both functions, first “opening” PLK1 and then accessing the activation loop. A counterintuitive aspect of this model, however, is that it would imply that Aurora B:INCENP, which turned into an excellent PLK1 Thr210 kinase after mutating Lys208 to arginine, can also “open” PLK1 as a precondition for its phosphorylation. To further investigate this issue, we focused on the coupling between conformational changes of PLK1 and its activation. For this, we measured Aurora A:Bora accessibility to the activation loop of wild type PLK1 under two conditions known to modify the reciprocal conformation of the PBD and kinase domains. First, we combined PLK1 with a peptide from Drosophila melanogaster MAP205 that binds the PBD to lock PLK1 in an inactive, closed conformation, a condition predicted to restrict access to the PLK1 activation loop( 28 , 50 ). Second, we combined PLK1 with a BUB1 peptide pre-phosphorylated with CDK1 to generate a high-affinity binding site for the PBD. The phospho-dependent interaction of BUB1 is expected to release the auto-inhibitory interaction between PLK1 PBD and kinase domain ( 28 , 50 , 75 ). As a control, we used PLK1 in the absence of PBD ligands, assuming that it might thermally oscillate between open and closed states. Both MAP205 and pBUB1 are able to bind to PLK1 in analytical SEC binding assay ( Figure S5A ) These experiments revealed that presence of the MAP205 or BUB1 peptides did not have significant effects on PLK1 Thr210 phosphorylation, with levels being indistinguishable also in comparison to the sample without peptide ( Figure 4D ). Furthermore, the presence of PBD-binding peptides did not increase the accessibility of Thr210 to the non-cognate kinases Aurora A:TPX2 and Aurora B:INCENP ( Figure 4D ). Collectively, our results demonstrate that the activation segment of PLK1 is, at least in first approximation, equally accessible when PLK1 is locked in a closed conformation or open by an activator, in line with a structural model of full-length PLK1 ( 75 ). PLK1 K208R mutant phosphorylation in the absence of Bora Because the PLK1 K208R mutant we have identified is efficiently phosphorylated by various Aurora kinase species in vitro , we asked if its activation had been made independent of Bora also in the cellular environment. For this, we over-expressed mNG-PLK1 WT or K208R, and monitored the levels of T210 phosphorylation in Nocodazole-arrested cells, depleted of Bora. Phosphorylation of PLK1 K208R was comparable to the one of PLK1 WT, and depletion of Bora strongly affected phosphorylation of both protein variants ( Figure 5A and Figure S5B ). Reduced Thr210 phosphorylation of PLK1 WT upon Bora depletion was not rescued by okadaic acid, a PP1/PP2A inhibitor, suggesting that in the adopted experimental conditions no alternative kinase phosphorylates Thr210 in the absence of Bora ( Figure 5B and Figure S5C ). In contrast, PLK1 K208R exhibited higher overall Thr210 phosphorylation than PLK1 WT upon addition of okadaic acid, and combined Bora depletion treatment resulted in reduced but still substantial phosphorylation levels ( Figure 5B and Figure S5C ). Download figure Open in new tab Figure 5. PLK1 K208R mutant remains dependent on Bora for T-loop phosphorylation A ) Pull-down of mNeonGreen-PLK1 WT or mNeonGreen-PLK1 K208R from mitotic HeLa cell lysates with or without RNAi-mediated Bora depletion, as schematized. The bar plot represents the arithmetic mean of three replicates and black dots are the normalized values (over PLK1 signal) of signal intensity for each replicate. OE= Over-expressed. B ) Pull-down performed as in (A), except for the addition of Okadaic acid (100 nM OA, 1 hour before cell harvesting), with or without Bora depletion, as schematized. The bar plot represents the arithmetic mean of three replicates and black dots are the normalized values (over PLK1 signal) of signal intensity for each replicate. OE= Over-expressed; siBora= siRNA-mediated depletion of endogenous Bora; PPPi=Phospho-protein phosphatase inhibitor. C ) HeLa cell lines expressing mNeonGreen-PLK1 WT or mNeonGreen-PLK1 K208R were synchronized with a double thymidine arrest, released in the cell cycle, and followed as they entered mitosis. Dot plots show the distribution of mitotic entry times of individual cells after double-thymidine release (DTR). Each dot represents an individual cell, the horizontal line represents the median and the vertical one the 95% CI of the median, which can be used for statistical comparison of different conditions (see Materials and Methods). OE= Over-expressed; siBora/siPLK1= siRNA-mediated depletion of endogenous Bora or endogenous PLK1; PLK1i=Polo-like kinase 1 inhibitor (referred to BI2536 compound). D ) In vitro assay with Aurora A:Bora and PLK1 WT , PLK1 R95A , PLK1 E121A, , PLK1 R95A-K208R , and PLK1 E121A-K208R. . After 60 minutes at 30°C, activation loop phosphorylation of PLK1 was monitored. E ) Pull-down from mitotic cell lysates to assess the extent of T-loop phosphorylation of mNeonGreen-PLK1 WT , mNeonGreen-PLK1 E121A , and mNeonGreen-PLK1 E121A-K208R . The experimental regime was like in Figure 3A . The bar plot represents the arithmetic mean of three replicates and black dots are the normalized values of signal intensity for each replicate. OE= Over-expressed. HeLa cell lines stably expressing mNG-PLK1 WT or mNG-PLK1 K208R were synchronized at the G1/S boundary and then released into the cell cycle as in the experiments described above (e.g Figure 3D ). While the overexpression of mNG-PLK1 WT partly compensated PLK1 depletion, it did not compensate Bora depletion, as evidence by a robust mitotic entry delay. Expression of mNG-PLK1 K208R in absence of Bora led to a mitotic entry delay comparable to that observed with the expression of mNG-PLK1 WT ( Figure 5C and Figure S5E-F ). Mitotic entry in control cells expressing mNG-PLK1 K208R was significantly faster, although we could not unequivocally ascribe this effect to an enhanced Thr210 phosphorylation ( Figure 5C ). Thus, the Aurora:Bora complex remains important for timely mitotic entry even in cells expressing PLK1 K208R . The continued requirement for Bora may reflect additional regulatory controls on PLK1 activation, for instance through co-localization. Introduction of the K208R mutation into the Bora-binding deficient PLK1 R95A or PLK1 E121A mutants caused a substantial increase of pT210 levels phosphorylation in vitro, confirming the dispensability of the Bora P1 motif for PLK1 K208R T210 phosphorylation ( Figure 5D ). In line with these results, the PLK1 E121A-K208R double mutant variant was substantially phosphorylated also upon expression in HeLa cells, contrary to the PLK1 E121A single mutant ( Figure 5E and Figure S5D ). While the identity of the kinase responsible for this phosphorylation remains unknown, this finding demonstrates that the K208R mutation increases the efficiency of PLK1 phosphorylation in cells. Resilience of PLK1 pThr210 to protein phosphatases Bora levels decline upon mitotic entry but Bora remains essential for PLK1 Thr210 phosphorylation also in mitosis ( 29 ). Our above observations confirm that there is no mitotic kinase able to phosphorylate PLK1 WT on Thr210 after Bora depletion. Conversely, accumulation of pThr210 on PLK1 K208R is still dependent on Bora, but this requirement can be partly overcome if phosphatase activity is inhibited. These observations raise interest in the accessibility of the PLK1 activation segment to phosphatase activity, a question that remains poorly studied. Specifically, we were curious to know whether the maintenance of high levels of pThr210 in spite of declining levels of Bora may reflect a poor accessibility of pThr210 by phosphatase activity. Furthermore, we also wanted to investigate the possibility that the presence of the gatekeeper Lys208 may not only make the PLK1 activation segment impervious to “ordinary” Aurora kinases, but also protect it from dephosphorylation by protein phosphatases. Faster dephosphorylation of the arginine-containing motif in PLK1 K208R may explain why it does not accumulate in cells unless phosphatase activity is temporarily blocked. To test this hypothesis, we pre-phosphorylated PLK1 with the Aurora A:Bora complex and then subjected it to dephosphorylation reactions in vitro with three different protein phosphatases (PP) playing critical roles before, during, and after mitosis: PP1 (ψ isoform), PP2A:B55 (ο isoform), and PP2A:B56 (ψ isoform) ( Figure 6A ). As a control, we performed the same dephosphorylation assay with the broadly specific bacteriophage lambda-phosphatase ( 76 ). pThr210 of PLK1 WT was remarkably resilient to dephosphorylation by PP2A-B55, and retained a substantial level of phosphorylation even when exposed at an approximately stoichiometric ratio to the action of the phosphatase for 60’. As a comparison, we monitored the dephosphorylation of pThr288 on the Aurora A activation segment, using PP2A-B55 in equimolar amount, revealing that pThr288 of Aurora A underwent rapid and complete dephosphorylation by PP2A:B55ο under all tested conditions ( Figure 6B ). Similar results were obtained with PP2A:B56ψ, which was only slightly less effective at dephosphorylating pThr288 of Aurora A ( Figure 6C ). On the other hand, PP1ψ appeared to be the least active of the three phosphatases on the two kinases tested in this assay ( Figure S6A ), while Lambda-phosphatase did not show significant differences in the ability to dephosphorylate Aurora A and PLK1 ( Figure S6B ). Download figure Open in new tab Figure 6. Thr210 of PLK1 is a poor substrate of mitotic protein phosphatases A ) Experimental scheme adopted to analyse susceptibility of PLK1 to phosphatases. PLK1 WT or K208R was incubated overnight with Aurora A:Bora, then dephosphorylated with increasing concentrations of PP2A-B55, PP2A-B56, PP1 and lambda-phosphatase. B ) Dephosphorylation assay in presence of PP2A-B55 from 0.05 to 1 μM. Phosphorylation of PLK1 WT or PLK1 K208R , and of Aurora A Thr288 was followed by Western blotting with phospho-antibodies. A Western blot of Aurora A and PLK1 and a Coomassie blue stained gel were included to verify equal protein loading between conditions. C ) Dephosphorylation assay in presence of PP2A-B56 from 0.05 to 1 μM. Phosphorylation of PLK1 WT or PLK1 K208R , and of Aurora A Thr288 was followed by Western blotting with the indicated phospho-specific antibodies. A Western blot of Aurora A and PLK1, and a Coomassie blue stained gel were included to demonstrate equal protein loading between conditions. These results provide an initial demonstration that the activation loop of PLK1 is comparatively highly resilient to dephosphorylation by mitotic phosphatases, which may explain why its phosphorylation can be maintained even in spite of Bora’s massive degradation upon mitotic entry. In comparison, the activation segment of PLK1 K208R , was slightly but reproducibly less resilient to dephosphorylation by PP2A ( Figure 6B-C and Figure S6 ), supporting the conclusion that Lys208 protects pThr210 from phosphatase activity more effectively than when this residue is replaced with arginine. DISCUSSION Activation of PLK1 requires Bora as an Aurora A cofactor, but the molecular basis for this selective activation mechanism have remained obscure. Here, we developed an assay that, by using purified proteins, recapitulates the exquisitely selective requirement of Bora for PLK1 activation loop phosphorylation. The establishment of this assay allowed us to identify the major determinants of Aurora kinases selectivity towards PLK1. Using PLK1 kinase dead variant (K82R), we excluded that PLK1 autoactivates in presence of the Aurora A:Bora complex, in line with a previous study ( 32 ). Demonstrating that the PLK1 T-loop consensus sequence is a suboptimal Aurora substrate, a single conservative substitution from Lysine to Arginine in position -2 from Thr210 was sufficient to promote phosphorylation by nearly all Aurora complexes tested in vitro . Despite a well-documented preference of Aurora kinases for arginine over lysine at position -2, the presence of the latter residue does not universally prevent efficient Aurora phosphorylation of substrates ( 20 , 69 – 74 ). For instance, NDC80 Ser55, used as a control substrate ( Figure 1C ), also has a lysine at position -2 but it is rapidly phosphorylated in the same time frame by all tested Aurora kinases. Thus, we cannot affirm that Lys208 is the sole element controlling the gatekeeping mechanism we have discovered. However, PLK1 variants bearing other amino acid substitutions at Thr210 neighboring sites (i.e. PLK1 E206L and PLK1 R207K ) displayed a very different behaviour compared to PLK1 K208R . We also considered a potential role of steric hindrance in making the PLK1 activation loop impervious to Aurora kinases in the absence of Bora. A hypothetical accessibility barrier to the PLK1 activation loop may arise from an intramolecular interaction of the PLK1 kinase domain and the PBD. It was proposed that in the resulting “closed” state of PLK1, the region spanning residues 312-360 of the interdomain linker may mask the activation loop, making it inaccessible to Aurora A and inhibiting Thr210 phosphorylation.( 50 ). We were unable to perform experiments on PLK1 1-312 , the construct used by Xu and colleagues (reference ( 50 )), as it appeared poorly stable. Nevertheless, their conclusion seems inconsistent with our findings, as we show that Thr210 is equally accessible when the intramolecular interaction between PLK1’s kinase domain and the PBD is actively stabilized (by using an inhibitory MAP205 peptide) or destabilized (by adding a CDK-phosphorylated BUB1 peptide). Furthermore, a short peptide exclusively encompassing the activation loop of PLK1 and exposing a lysine residue at position 208 was a poorer substrate than the same peptide exposing an arginine, indicating that the main obstacle that Bora needs to overcome is intrinsic to the specific sequence of the PLK1’s activation loop, rather than to its conformation. Instead, our work suggests that overcoming the intrinsic barrier to phosphorylation requires the Aurora A:Bora complex to interact directly, albeit transiently, with PLK1. While we were not able to trap the complex biochemically, due to its instability, we validated a high-confidence AF prediction by mutationally targeting the complementary interacting interfaces predicted by structural modelling. The αC helix is an extremely well-conserved feature in the small lobe of protein kinases ( 4 ). It contributes to the folding of the small lobe and positions residues that stabilize ATP in the active site, promoting catalysis ( 77 ). Unlike the standard regulation mechanisms involving the spatial orientation of the αC helix by post-translational modifications or cofactor binding ( 4 , 6 ), the PLK1 αC helix and neighboring residues dynamically bridge the interaction with a short N-terminal α-helix of Bora (the P1 element). Mutations in these regions of Bora and PLK1 impaired Thr210 phosphorylation. Particularly, the predicted hydrogen bond between PLK1 Glu121 and Bora Trp58 may be essential for efficient catalysis. We conclude that Bora bypasses the unfavorable phosphorylation reaction of the PLK1 activation-loop by forming a physical connection between Aurora A and the PLK1 kinase domain. We speculate therefore that the main role of Bora is to prolong the residency of the PLK1 activation loop in the proximity of the Aurora A active site, ultimately overcoming a kinetic barrier to phosphorylation introduced by the peculiar sequence of the PLK1’s activation loop, which deviates from the ideal Aurora consensus. The effects on cell cycle progression from depleting Bora in different model systems range from a modest to a complete blockade to mitotic entry ( 19 , 30 – 32 , 47 ). In cultured human cells, the main consequence is a delayed mitotic entry, but the effect is strongly exacerbated if cells are forced to recover from a temporary cell cycle arrest caused by DNA damage ( 31 ). We mimicked this effect with mutants preventing the Bora:PLK1 interaction. In a study that developed independently in parallel with ours, Pillan and colleagues also studied the mechanistic basis of Bora activation of PLK1 phosphorylation, coming to conclusions that are perfectly in line with ours ( 78 ). Using model systems that emphasize the role of Bora for mitotic entry in comparison to human cells, their study also provides a compelling biological demonstration that the interaction of Bora and PLK1 is essential for PLK1 activation and function in Xenopus egg extracts. Contrary to the mutations at the Bora:PLK1 interface, substitution of Lys208 to arginine showed a more nuanced situation than the one observed in vitro . Thr210 phosphorylation of the PLK1 K208R variant remained dependent on Bora. Only upon phosphatase inhibition, we observed Bora-independent PLK1 Thr210 phosphorylation of the mutant motif, but not of its wild-type counterpart. The reason why the activation loop motif of PLK1 remain impervious to Aurora kinases even after introduction of the conservative arginine mutation at the -2 position will need further investigation. It may reflect the low cellular concentrations of PLK1 and its activating kinase in vivo, which may justify a decreased but continuous requirement for Bora. A non-mutually exclusive additional explanation is that the lysine-to-arginine conservative substitution at position 208 makes pThr210 a better phosphatase substrate. In line with this idea, we observed a small but highly reproducible acceleration of pThr210 dephosphorylation of the activation loop of PLK1 K208R relative to that in PLK1 WT . Whether the effect we observed in vitro explains our results in vivo will require further studies. Nonetheless, the observation that Thr210 of PLK1 is largely impervious to PP2A and PP1 phosphatases is tantalizing, as it may explain the puzzling observation that the decrease of Bora levels in mitosis does not have major consequences for the PLK1 activation state, provided that low residual levels of Bora are present ( 29 ). While it was initially speculated that the persistence of pThr210 under these conditions or after Aurora A inhibition may reflect temporal inactivation of the appropriate phosphatase during mitosis ( 29 ) or protection by additional cofactor proteins such as Apolo1 (reference ( 79 )), our results suggest that also sequence or structural elements around pThr210 site hamper its dephosphorylation by active phosphatases. There are some corollary observations in our data that deserve a brief comment. First, our experiments with the Aurora A:Bora complex demonstrate that it can phosphorylate other substrates in addition to PLK1. Whether this property of the Aurora A:Bora complex is physiologically significant will require further analysis. Second, our observations indicate that CEP192 acts as a partial inhibitor of Aurora A towards the substrates tested ( Figure 1C ). This result is partly in line with recent work showing CEP192 does not activate ( 37 ) or even inhibits Aurora A ( 36 ). The result is also consistent with the AlphaFold prediction of the Aurora A:CEP192 complex, where the cofactor is shown to intrude deeply into the catalytic pocket of the kinase, predicting inhibition. It has been proposed that oligomerization of Aurora A on CEP192 in presence of an oxidative environment promotes activation loop auto-phosphorylation and activation of Aurora A ( 80 – 82 ). The specified condition cannot be straightforwardly tested using in vitro phosphorylation assays, and therefore we cannot exclude that CEP192-bound Aurora A at the centrosome is able to activate PLK1. Nevertheless, we can say that at least intrinsically, the complex phosphorylates PLK1 inefficiently on the activation-loop. Our results are graphically summarized in Figure 7 . We identify PLK1 as a suboptimal Aurora kinase substrate whose phosphorylation requires Bora. Such requirement can be bypassed by replacing lysine 208 of the activation loop with arginine. On the other hand, mutating residues at the Bora:PLK1 interface that we have identified abrogates Thr210 phosphorylation altogether, an effect that is rescued by introducing the K208R mutation. Thus, the two crucial elements of this mechanism are a) the suboptimal target sequence of the motif containing Thr210, which creates a major barrier to phosphorylation, and b) the overcoming of this limitation through a direct interaction of Bora with PLK1’s kinase domain. Although additional studies will be needed to fully comprehend the network generated by the binding of Bora to crucial mitotic kinases such as CDK1, Aurora A, and PLK1, our data offer the long-sought-for molecular explanation for why Bora is required as an Aurora A cofactor to phosphorylate Thr210 of PLK1. Download figure Open in new tab Figure 7. Mutations of PLK1 used in the study and their effect on T-loop phosphorylation Scheme summarizing the dependency of activation of PLK1 from Aurora A:Bora upon mutation of either the Bora:PLK1 interface or the activation-loop region, with a distinction on how those two manipulations altered activation-loop phosphorylation in cells or with recombinant proteins. Materials and Methods Mutagenesis and sub-cloning into expression plasmids All DNA constructs were generated by PCR amplification of the DNA fragment encoding the protein (insert) and the expression vector of choice or PCR amplification of the insert only and vector cleavage with restriction enzymes. Both strategies were followed by a ligation step via Gibson Assembly (enzymes were purchased from New England Biolabs and the master mix was produced in house). Mutations were introduced via site-directed mutagenesis with a single forward primer. The protein encoding region of all the final constructs was verified by Sanger sequencing (Microsynth Seqlab). Oligonucleotides used for site-directed mutagenesis and cloning were purchased from Sigma. A list of plasmids used in this study can be found in Table S1 . View this table: View inline View popup Table S1 List of the plasmids used in the study. Expression of recombinant proteins Full-length human His-Aurora A WT , His-Aurora A T288V , Halo-Aurora B FL co-expressed with MBP-INCENP 351-918 , GST-B56γ3, GST-PPP2R1A, as well as all human MBP-Bora-His fragments and mutants, His-MBP-PLK1 WT and its truncations or mutants, MBP-TPX2 FL -His, MBP-CEP192 400-600 -His, MBP-Map205 264-322 -His, MBP-Bub1 569-616 -His were expressed in Escherichia coli BL21 CodonPlus (DE3)-RIL (Agilent Technologies). Transformed bacteria were incubated in Luria Broth (Bora, B56γ3 and TPX2) or Terrific Broth (Aurora A, Aurora B:INCENP, PLK1, PPP2R1A, MAP205, BUB1 and CEP192) media at 37°C under agitation and the cultures were shifted at 18°C at OD 600 = 0.6-0.8. Protein expression was induced by the addition of IPTG (final concentration 100 µM) and the cultures were incubated 18 hours at 18°C under agitation. Cyclin B-CDK1-CKS1 was obtained from insect cells TnaO38 expressing GST-CDK1 with a 1:25 of viral stock dilution in the expression culture (for a standard protocol of virus generation, see reference ( 83 )). His-CKS1 and MBP-scCAK1 (also with a 1:25 dilution) were cultured for three days at 27°C ( 84 ). His-PPP2CA was purified from insect cells (Tnao38), kept in culture after transfection of the virus at a 1:20 ratio, for 4 days at 19°C ( 85 ). Ndc80 bonsai expression in E. coli BL21(DE3) was induced with 400 μM IPTG at OD 600 = 0.45–0.6 for 12–16 hr at 20–25°C ( 86 ).PP2Ac:B55δ were co-expressed in Expi293F cells (Thermo Fisher Scientific), grown in suspension in Expi293 Expression Medium (Thermo Fisher Scientific), using the BigBac system to generate the virus for transfection. Briefly, the DNA fragments encoding full length B55 delta (B55d) and PP2A catalytic (PP2Ac) subunits were cloned into pcDNA4/TO vector (containing CMV promotor for the expression in mammalian cells) downstream to and in frame with a region encoding an N-terminal enhanced Green Fluorescent Protein (eGFP) followed by the HRV 3C/Prescission cleavage site, or an N-terminal StreptagII followed by TEV protease cleavage site, respectively. The coding sequences were then cloned from pcDNA4/TO into pBIG1a vector, using cassette primers, to generate the bacmid. For the expression in 1L of Expi293F cells, they were infected with V2 virus culture precipitated overnight. To precipitate V2 virus, 4x Polyethylene glycol (PEG) solution was used -32% PEG6000 (Merck) -, 400 mM of NaCl, 40 mM of HEPES pH 7.4). The virus was mixed with 4xPEG solution in 3:1 ratio and incubated at 4°C in the dark overnight. The day before the infection Expi293F cells were split to 2-2.5 x10 6 /mL, so that on the day of infection the density was 3.5-4.5 x10 6 /mL. The following day, the mixture of V2 virus and PEG was centrifuged at 4000 RPM, at 4°C, for 30 min. The supernatant was removed, and the pellet was resuspended in 5 mL of Expi293 Expression Medium (Thermo Fisher Scientific) prewarmed to 37C. Once the cell density reached the desired range (3.5-4.5 x10 6 /mL), resuspended virus was added at a 1:200 ratio. 6-8 hours post infection, sodium butyrate (Sigma Aldrich) was added to Expi293F culture to 5 mM final concentration. The expression was performed at 37°C, 8% CO 2 under agitation. 48-52 hours post infection cells were pelleted by centrifugation, snap frozen in liquid nitrogen and stored at -80C until further use. Purification of recombinant proteins All steps of the purification procedures below were performed on ice or at 4°C. Aurora A WT and T288V was purified as a 6xHis-thrombin site-T7 tag fusion. Bacteria were pelleted and resuspended in lysis buffer (50 mM Hepes pH 7.5, 300 mM NaCl, 5% vol/vol glycerol, 1 mM TCEP, 2 mM PMSF), lysed by ultrasonication on ice and cleared by centrifugation at 80,000 g at 4°C for 30-45 min. The resulting soluble lysate was subjected to Nickel or Cobalt-based chromatography by loading it on 5-mL of packed NiNTA or Talon column (Cytiva), pre-equilibrated in affinity buffer (50 mM Hepes pH 7.5, 300 mM NaCl, 5% vol/vol glycerol, 1 mM TCEP). After extensive washes, protein was eluted with 300 mM Imidazole in affinity buffer. The eluted sample was concentrated using an ultrafiltration centrifugal protein concentrator (MerckMillipore). The protein was loaded on a HiLoad 16/600 Superdex 200 pg sizing column (Cytiva), equilibrated in SEC buffer (50 mM Hepes pH 7.5, 300 mM NaCl, 5% vol/vol glycerol, 1 mM TCEP). The protein containing fractions were pooled and concentrated as described above. The purified protein was aliquoted, flash frozen in liquid nitrogen, and stored at -80°C. Human Bora 1-224 , human TPX2 FL , human CEP192 400-600 , MAP205 264-322 from Drosophila melanogaster, and human BUB1 569-616 , were expressed and purified as tagged proteins, bearing an N-terminal MBP tag and a C-terminal 8xHis fusion. Bacteria from 1-liter culture were harvested by centrifugation and resuspended in lysis buffer (20 mM Tris pH 8, 500 mM NaCl, 1 mM TCEP), supplemented with HP plus protease inhibitor mix (Serva) and DNase I (Roche). The same protocol described before has been applied for protein purification and storage, except that buffer containing 20 mM Tris pH 8, 500 mM NaCl, 1 mM TCEP was used throughout the entire procedure. Human PLK1 wt and its variants were expressed as TEV cleavable 6xHis-MBP fusions, which leaves ten non-native residues (GPGASASALA) at the N-terminus upon TEV digestion. Bacteria from 2-liter culture were pelleted and resuspended in lysis buffer (50 mM Hepes pH 7.5, 300 mM NaCl, 5% vol/vol glycerol, 1 mM TCEP, 2 mM PMSF). Every subsequent step was performed in lysis buffer devoid of PMSF. The purification protocol described above has been modified to allow the TEV-mediated cleavage of His-MBP after elution. His-TEV protease (produced in house) has been added and incubated overnight with the eluted protein. The protein was finally resolved on a HiLoad 16/600 Superdex 200 pg sizing column (Cytiva) and additionally separated from His-TEV by mounting a 5 mL NiNTA pre-packed column downstream of the size-exclusion chromatography column. B56γ3 was expressed as a H3RVC-cleavable GST-fusion, which leaves five non-native residues (GPLGS) at the N-terminus upon Prescission digestion. Bacteria from 1 Liter culture were harvested by centrifugation and resuspended in lysis buffer (50 mM Hepes pH 7.5, 300 mM NaCl, 5% vol/vol glycerol, 1 mM TCEP) complemented with HP plus protease inhibitor mix (Serva) and DNase I (Roche). The cell resuspension was lysed by ultrasonication on ice and cleared by centrifugation at 80,000 g at 4°C for 30-45 min. The resulting clarified lysate was subjected to GSH-based affinity chromatography by loading it on 5 mL of pre-equilibrated Glutathione agarose beads (Serva) slurry. After extensive washes, GST-Prescission protease (produced in house) was added to lysis buffer and the beads were kept shaking gently to allow the removal of the GST-tag, overnight. The sample containing cleaved B56 protein was collected and concentrated by ultrafiltration as described for other protein purifications. The protein was resolved on a pre-equilibrated HiLoad 16/600 Superdex 200 pg sizing column (Cytiva). The protein-containing fractions were pooled and concentrated as described above. The purified protein was aliquoted, flash frozen in liquid nitrogen, and stored at -80°C. PPP2R1A was cloned as a TEV-cleavable GST-fusion, which leaves one non-native residue (G) at the N-terminus after TEV digestion. The same protocol as for GST-B56 was applied, except that cleavage was performed with His-TEV protease, that was separated by size-exclusion chromatography from the purified protein. 6xHis-PPP2CA-expressing cells from a 2-liter culture were resuspended in lysis buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 2 mM TCEP, 5% vol/vol glycerol) supplemented with HP plus protease inhibitor mix (Serva) and DNase I (Roche). After ultrasonication and centrifugation at 85000 g for 45 minutes, the supernatant was filtered through a 0.8 µm filter. The sample was loaded on 2×5-mL Talon column and washed extensively with lysis buffer, to then be eluted in 2-mL fractions with elution buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 2 mM TCEP, 5% vol/vol glycerol, 250 mM imidazole). Fractions containing the protein were selected by using Bradford reagent (Thermo Fisher Scientific). The selected fractions were pooled and diluted 1:4 in ion exchange buffer (50 mM Hepes pH 7.5, 50 mM NaCl, 5% vol/vol glycerol, 1 mM TCEP), to be loaded on a 6-mL ResourceQ column (Cytiva). After a washing step with ion exchange buffer, protein was eluted with a gradient up to 400 mM NaCl in ion exchange buffer A. Protein content was inspected via Coomassie Blue-stained SDS-PAGE gel and fractions were pooled and concentrated by ultrafiltration. The resulting sample was loaded and resolved on a Superdex 10/300 75 pg sizing column (Cytiva), pre-equilibrated in SEC buffer (same composition as lysis buffer). Fractions from size-exclusion chromatography were pooled, concentrated as described above and aliquoted. Aliquots were snap-frozen and stored at -80°C. PP2A-B56 complex formation was carried out prior to the experiment, by mixing a 1:1:1 ratio of PPP2R1A, PPP2CA and B56. PP2A-B55 complex was purified as previously described, with few adjustments reported below. The PP2Aa subunit (residues 9–589) was expressed as a His-MBP-tagged fusion. Cells from 500 mL of culture were resuspended in lysis buffer (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole, 0.5 mM TCEP, 0.1% Triton X-100) supplemented with protease inhibitors (Serva), and DNase (Roche) and lysed by ultrasonication. The lysate was clarified by centrifugation at 80000 g for 45 min at 4 °C, and the supernatant was loaded onto 2×5 mL HisTrap FF columns (Cytiva) pre-equilibrated with affinity buffer (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole, 0.5 mM TCEP). Following extensive washing, the protein was eluted in a single step with 500 mM imidazole in the same buffer. Eluted fractions were pooled and dialyzed overnight at 10 °C with in-house purified His-TEV protease in SnakeSkin tubing (Thermo Fisher) to cleave the His-MBP tag. The dialyzed mixture was incubated with equilibrated Ni-NTA agarose beads (Roche) to remove the tag, and the flow-through containing cleaved PP2Aa was collected. The sample was concentrated by ultrafiltration, diluted to 100 mM NaCl with low-salt buffer (20 mM Tris pH 8.0, 0 mM NaCl, 0.5 mM TCEP), and brought to 100 mL total volume with buffer A (20 mM Tris pH 8.0, 100 mM NaCl, 0.5 mM TCEP). The solution was filtered (0.2 µm) and loaded onto a 6 mL HiTrap Q column (Cytiva). Bound protein was eluted over a linear 100–1000 mM NaCl gradient. Relevant fractions were pooled, concentrated by ultrafiltration, and subjected to size exclusion chromatography (HiLoad 16/600 Superdex 200 pg, Cytiva) in SEC buffer (20 mM Tris pH 8.0, 150 mM NaCl, 0.5 mM TCEP). Fractions containing PP2Aa were pooled, concentrated, aliquoted, flash frozen in liquid nitrogen, and stored at –80 °C. The His-PPP2CA:B55δ holoenzyme was purified from 1 L of Expi293F cell pellets co-expressing StrepII-PP2Ac and eGFP-B55δ. All steps were performed on ice or at 4 °C. Cells were resuspended in lysis buffer (20 mM Tris pH 8.0, 500 mM NaCl, 0.5 mM TCEP, 0.1% Triton X-100) supplemented with protease inhibitors (Serva), and DNase (Roche) and lysed by sonication. To promote holoenzyme assembly, 6 mg of purified PP2Aa was added to the lysate before clarification by centrifugation at 80000 g for 45 min at 4 °C. The supernatant was filtered (0.8 µm) and then incubated with GSH agarose resin (Serva) pre-bound to GST-GFP nanobodies (see nanobody purification section). After washing, beads were resuspended directly on the column in cleavage buffer (20 mM Tris pH 8.0, 250 mM NaCl, 1 mM MnCl₂, 0.5 mM TCEP), and in-house purified GST-PreScission protease was added for overnight cleavage at 10 °C. The flowthrough containing the eluted complex was collected, concentrated by ultrafiltration and diluted to 100 mM NaCl by addition of low salt buffer (20 mM Tris pH 8.0, 0 mM NaCl, 1 mM MnCl₂, 0.5 mM TCEP). The total volume was adjusted to 100 mL with buffer A (20 mM Tris pH 8.0, 100 mM NaCl, 1 mM MnCl₂, 0.5 mM TCEP), filtered through a 0.2 µm membrane (Cytiva), and loaded onto a 6 mL HiTrap Q column (Cytiva). The column was washed with buffer A and eluted over a linear gradient from 100 mM to 1000 mM NaCl in buffer A. Fractions containing the PP2A:B55δ holoenzyme were pooled, concentrated in storage buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM MnCl₂, 0.5 mM TCEP) by ultrafiltration, aliquoted, snap frozen in liquid nitrogen, and stored at –80 °C. For purification of GST-GFP nanobody, 4 liters of bacterial cell pellets expressing GST-GFP nanobody were resuspended in lysis buffer (50 mM Hepes pH 7.5, 300 mM NaCl, 5% glycerol, 1 mM TCEP) with 1 mM phenylmethane sulfonyl fluoride (PMSF) (Serva). The suspension was lysed by ultrasonication and centrifuged (80000 g , 45 min). The supernatant was loaded onto 5 mL FF GST-trap column (Cytiva), equilibrated in lysis buffer. The column was washed and the bound protein was eluted using elution buffer (50 mM Hepes pH 7.5, 300 mM NaCl, 5% glycerol, 1 mM TCEP, 20 mM Glutathione (GSH) (Roth)). The eluted protein was concentrated by ultrafiltration and further purified, using Size Exclusion Chromatography (SEC), HiLoad 16/600 Superdex 200 pg (Cytiva), in SEC buffer (50 mM Hepes pH 7.5, 300 mM NaCl, 5% glycerol, 1 mM TCEP). The fractions containing the protein were pooled, concentrated by ultrafiltration, snap frozen in liquid nitrogen and stored at -80C. The day of PP2A purification, the GST-eGFP nanobodies were mixed with GSH agarose resin (Serva) equilibrated with the PP2A:B55d resuspension buffer (20 mM Tris pH 8.0, 500 mM NaCl, 0.5 mM TCEP) for 3 hours at 10°C to immobilize the nanobodies to the agarose beads, using a magnetic stirrer at low speed. The beads were collected into 10 mL drop columns, and washed with the resuspension buffer, to be then directly used for mixing with the supernatant of Expi293F cells expressing PP2Ac and B55d subunits. Halo-Aurora B-8xHis / MBP INCENP 351-C (co-expressed via transformation of BL21CodonPlus(DE3)-RIL with a pETDuet-1 plasmid) from 4 liters of expression culture was resuspended in Lysis buffer (50 mM Hepes pH 7.5, 500 mM NaCl, 5% vol/vol glycerol, 1 mM TCEP, 5mM MgCl 2 , 1mM PMSF). After lysis via sonication, Dnase (Roche) was added and incubated 30 minutes at 4°C. Lysate was clarified by centrifugation at 75000xg for 30 minutes. Cleared lysate was supplemented of Polyethylenimine (PEI, from Sigma) at 0.25% vol/vol and sample was spun down at 75000xg for 30 minutes. The supernatant was collected and loaded on Ni-NTA slurry beads placed in a drop-column at 4°C. After washing, a 1 mM ATP wash in lysis buffer E was added to remove chaperones. The elution was performed with elution buffer B (50 mM Hepes pH 7.5, 300mM NaCl, 5% vol/vol glycerol, 1mM TCEP and 300 mM imidazole). The eluate was incubated with His-TEV at a ratio of 1:20 overnight. The protein after cleavage was concentrated, spun down and finally resolved on a HiLoad 16/600 Superdex 200 pg sizing column (Cytiva), pre-equilibrated in SEC buffer (50mM Hepes pH 7.5, 300mM NaCl, 5% vol/vol glycerol, 1mM TCEP). Fractions containing the soluble complex of Halo-AuroraB-8xHis and INCENP 351-C were pooled, spun down aliquoted and snap-frozen, to be stored at -80°C for long-term storage. Cyclin B-CDK1-Cks1 complex was purified as previously described ( 84 ). In brief, frozen pellets were thawed and resuspended in lysis buffer (50 mM HEPES pH 7.4, 250mM NaCl, 2 mM TCEP, 5% vol/vol glycerol) supplemented with HP plus protease inhibitor mix (Serva) and DNase I (Roche). Cell lysates were prepared by sonication and cleared by centrifugation at 80,000g at 4°C for 30–45 min. The soluble lysate was passed through a 0,8 µm filter and loaded onto a column with 20 ml Glutathione Sepharose 4 Fast Flow resin (Cytiva). After washing with 25 column volumes of wash buffer (same composition as lysis buffer), CDK1 was cleaved with Prescission protease and His-TEV protease (both produced in house) for 16 hr. The eluate was concentrated through centrifugation and separated on a Superdex 200 16/600 column equilibrated in buffer A. To remove GST, uncleaved GST-CDK1, GST-3C-PreScission, and uncleaved Cks1 or His-TEV, a 5 ml GSH column (Cytiva) and a 5-mL NiNTA (Cytiva) were mounted after the size-exclusion column. His-TEV-Cyclin-B containing lysates were prepared as described above with 15 mM imidazole in lysis and wash buffers. After loading onto a 5 ml or 10 ml Talon (Clontech) or Ni-NTA (GE Healthcare) column, and washing with approximately 50 column volumes, Cyclin-B was eluted in buffer with 250 mM imidazole and concentrated to 2 ml through centrifugation with a 30 k Amicon filter (Millipore). To remove the polyhistidine tag, Cyclin-B was exposed to TEV protease for 16 hr. Cyclin-B was further purified on a Superdex 200 16/600 column equilibrated in buffer A. To remove His-TEV protease and uncleaved His-Cyclin-B, a 5 ml Talon column (GE Healthcare) was mounted after the size-exclusion column. Purified CDK1:Cks1 and Cyclin-B were mixed in a 1:1 ratio for 1–2 hr on ice and complex formation was assessed by size-exclusion chromatography on a Superdex 200 16/600 column in buffer A. Fractions containing CCC were concentrated through centrifugation, flash-frozen in liquid nitrogen, and stored at −80°C. Ndc80 bonsai purification was performed as described ( 86 ). Bacterial pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.6, 300 mM NaCl, 1 mM DTT, 1 mM EDTA, Dnase (Roche), protease-inhibitor mix HP Plus (Serva). Sonicated lysates were cleared by centrifugation at 80000g for 45–60 min. The cleared lysate was bound to Glutathion-Agarose beads (3 ml resin for 5L expression culture, Serva) equilibrated in washing buffer (lysis buffer without protease inhibitors). The beads were washed extensively and protein was cleaved of the beads by overnight cleavage with 3C PreScission protease (generated in-house). The eluate was concentrated using via ultrafiltration and applied to a Superdex 200 10/300 column (Cytiva) equilibrated in 50 mM Hepes, pH 8.0, 250 mM NaCl, 2 mM TCEP, 5% v/v glycerol. Relevant fractions were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80°C. CENP-A and H3 containing mononucleosomes assembled on 145-bp DNA were reconstituted precisely as described ( 87 ). For the assembly of dinucleosomes, CENP-A and H3 containing NCPs were first reconstituted using two different CEN1-like DNA fragments. One micromolar of NCPs reconstituted on CEN1-like DNA fragment 1 and CEN1-like DNA fragment 2 were ligated to each other for 16 hours at 4°C using 8xHis-tagged T4 DNA ligase. The ligated NCPs were incubated with cOmplete nickel beads (Roche) for 7 hours at 4°C to remove the T4 DNA ligase. The ligated dinucleosomes were concentrated, and glycerol was added at a final concentration of 2% (v/v). The mixture was loaded on a cylindrical gel containing 5% reduced bis-acrylamide. Native PAGE was carried out on a Mini Prep Cell apparatus (Bio-Rad) at 1 W of constant power. Dinucleosomes were eluted at a constant flow rate of 0.1 ml/min overnight at 4°C into a nucleosome buffer (50 mM NaCl, 10 mM triethanolamine, and 1 mM EDTA) and collected in 250 μl of fractions on 96-well plates. The OD260 (optical density at 260 nm) and OD280 of the individual fractions were measured using a CLARIOstar Plus plate reader (BMG Labtech). Fractions containing dinucleosomes were pooled, concentrated, and stored at 4°C. Phosphorylation assays The PLK1 phosphorylation assay consisted of the addition of 200 nM of PLK1 K82R (dead) or other variants as indicated in the figure legends, to 200 nM Aurora A, in presence of 400 nM of Bora, TPX2, CEP192 or to 200 nM Aurora B:INCENP. The kinase buffer (50 mM HEPES 7.5, 150 mM NaCl, 5 mM MgCl 2 , 1 mM TCEP, 2.5 mM ATP) was used to reach a final reaction volume of 60 µL, incubated for 1 h at 30°C (or as otherwise indicated in figure legends and schemes). The reaction was stopped by adding 5X Laemmli sample buffer. The assay with CDK1 was performed in two steps. Step 1 consisted of the phosphorylation of 4 μM Bora 1-224 with 100 nM Cyclin B1-CDK1-CKS1 in 30 µL of kinase buffer incubated for 1 h at 30°C. The step 2 consisted of the dilution of Bora to 400 nM in the reaction mixture. The final concentration of the components and the experimental setup was the same as described above. The phosphorylation of His-MBP-SA2 1217-1231 was performed by the addition of 10 nM PLK1 kinase WT or mutants, to 200 nM substrate, in 60 µL of kinase buffer (50 mM HEPES 7.5, 150 mM NaCl, 5 mM MgCl 2 , 1 mM TCEP, 2.5 mM ATP) incubated for the timepoints indicated in the figures, at 30°C. The reaction was stopped by addition of 5X Laemmli sample buffer. Purified phospho-BUB1 (MBP-Bub1 569-616 -His) was generated by thawing 10 mg of pure protein (diluted at a 500 μΜ concentration) and incubating it with 2.5 μΜ CDK1/CyclinB1 in kinase buffer for 15 hours at 10°C. Phosphorylated BUB1 was loaded on a pre-equilibrated Superdex 10/300 200 pg sizing column (Cytiva), concentrated, aliquoted and snap-frozen for subsequent use in binding assays. Dehosphorylation assays PLK1 wt or PLK1 K208R was pre-phosphorylated for 15 hours at 10°C using the kinase buffer described above and the following protein concentration: 400 nM PLK1, 400 nM Aurora A, 800 nM Bora 1-224 . Phospho-PLK1 was diluted 1:2 in a reaction mixture containing 50, 100, 250, 500, 1000 nM phosphatase (PP2A-B56γ, PP2A-B55δ, PP1γ, λ-PPP) in phosphatase buffer (50 mM HEPES 7.5, 150 mM NaCl, 1 mM TCEP; 1 mM MnCl2 was added only in the case of PP1 and λ-PPP because needed to promote dephosphorylation). A final reaction volume of 60 µL was incubated for 1 h at 30°C. The reaction was stopped by addition of 5X Laemmli sample buffer. Analytical Size Exclusion Chromatography (SEC) binding assays Analytical Size Exclusion Chromatography was performed on a Superdex 5/150 Increase 200 pg sizing column (Cytiva), mounted on an ÄKTA™ micro system (Cytiva). 50 µL samples at 5 µM single protein concentration have been incubated for 1 hour on ice, then centrifuged at 16,900 g for 20 minutes. After sample injection, the protein absorbance was monitored at 280 nm and the proteins were eluted under isocratic condition, at a flow of 0.1 mL/min, in 100 µl fractions at 4°C. Fractions were analysed by SDS-PAGE and Coomassie Blue staining. Fractions from 1 mL elution volume to 2.3 mL have been loaded on SDS-PAGE gels, subsequently stained with Coomassie brilliant blue (produced in house). Pull-down/immunoprecipitation assays Hela FlpIn T-Rex cells stably expressing mNG-PLK1 WT/R95A/E121A or mCherry-PLK1 WT were seeded and after 24 hours, if stated in figure legends, RNAi depletion of Bora was performed by using 20 ( Fig. 5A and B ) or 50 ( Fig. 3B ) nM oligo and RNAiMAX reagent (Thermo Fisher Scientific). If stated in figure legends, after 19 hours, a transient transfection of mNG-Bora WT/W58A/F56A was performed by adding 1ng/µL of pCDNA5 vector containing the Bora gene and Lipofectamine 2000 (Thermo Fisher Scientific). After 8 hours from transfection (27 hours from depletion), cells media was supplemented of Nocodazole 3.3 µM ( Fig. 5A and B ) or 0.33 µM ( Fig. 3B ). After 24 hours, cells were harvested by centrifugation, washed once with 1 mL with Phosphate Buffered Saline (PBS) and snap-frozen to be stored at -80°C until pull-down experiment. Cells were thawed the day of the experiment, resuspended in IP buffer (50 mM Hepes 7.5, 150 mM NaCl, 5% glycerol, 1 mM TCEP, 0.1 % NP-40, 1 phosSTOP tablet/10 mL IP buffer, DNAase from Roche, Protease Inhibitors from Serva) and 1.5-3 mg of total protein (clarified cell lysate) was mixed with pre-equilibrated 20 µL slurry anti-mNeonGreen magnetic beads or Red Fluorescent Protein magnetic beads (for mCherry-tagged PLK1) and incubated 3 hours at 4°C. Input sample was collected right before beads were washed twice with 500 µL of IP buffer and elution was performed by addition 5X Laemmli sample buffer. Samples were boiled for 5 minutes at 95°C. Structural modeling Alpha-Fold predictions of Aurora A:Bora, Aurora A:CEP192, Aurora A:TPX2 and Aurora B:INCENP in Figure 1B-C were performed using AF2 version 2.2.0, while the model in Figure 2A was predicted using AlphaFold2 version 2.3.1. All figures for structural models were created using PyMOL v2.5.2 (Schrödinger, LLC). Each protein subunit was shown in all figures using the following colours: PLK1 Kinase Domain= dark blue; Polo-box domain= light blue; Aurora A and B= green; Bora, = coral red. Western blotting The samples from in vitro phosphorylation assays and cell lysates were loaded on SDS-PAGE gels (produced in house) and transferred onto a nitrocellulose membrane (BioRad) via the Trans-Blot system (BioRad) with pre-defined transfer protocols. The blotted membrane was blocked with either 5% milk or 5% BSA, and probed with the antibodies listed in Table S3 , diluted in TBS-T (Tris Buffered Saline with 0.1% Tween-20) as indicated. After 18-hour incubation at 4°C, the membrane was washed with 10 mL TBS-T three times and mixed with secondary antibodies (diluted at 1:10000 ratio in TBS-T, or 1:5000 for Bora antibody) for 1 hour at 20°C. As secondary antibodies, anti-mouse or anti-rabbit (NXA931 and NA934; Amersham, 1:5000) conjugated to horseradish peroxidase were used. After incubation with ECL Western blotting reagent (Cytiva), images were acquired with the ChemiDoc MP System (Bio-Rad) and analysed using Image Lab 6.0.1 software. View this table: View inline View popup Download powerpoint Table S2. List of the stable cell lines and siRNA oligos used in this study. View this table: View inline View popup Table S3. List of the primary antibodies used in this study. Cell culture HeLa Flp-In T-REx cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% tetracycline-free FBS, 50 µg/ml Penicillin/Streptomycin, and 2 mM L-glutamine (all reagents/media were from PAN Biotech). Flp-In T-REx HeLa expression cell lines for PLK1 were generated and maintained as previously described ( 88 ). DNA fragments encoding mNeonGreen-PLK1 were sub-cloned into a pCDNA5/FRT/TO-EGFP-IRES plasmid and co-transfected with pOG44 (Invitrogen), encoding the Flp recombinase, into cells using X-tremeGENE (Roche) according to the manufacturer’s instructions. Subsequently, cells were selected for 2 weeks in DMEM supplemented with hygromycin B (250 µg/ml; Thermo Fisher Scientific) and blasticidin (4 µg/ml; Thermo Fisher Scientific). Single-cell colonies were isolated, expanded and transgeme expression was induced by addition of 300 ng/ml doxycycline (Sigma-Aldrich) and checked by immunofluorescence microscopy and immunoblotting. Transgene expression during experiments was induced by addition of 50 ng/ml doxycycline (Sigma Aldrich) for 24 hr. To achieve single or double depletion of PLK1 and Bora, cells were transfected with RNAiMax 1:25 ratio in Optimem (Invitrogen), Bora siRNA(5′-TAACTAGTCCTTCGCCTATTT-3) (reference ( 49 )) and PLK1 siRNA (5’-AUACCUUGUUAGUGGGCAATT-3’) at 20 nM and 50 nM respectively for 48-72 h (see experimental scheme of the individual experiments in the figures) following manufacturer’s protocol. To perform transient transfection of mNeongreen-Bora WT, W58A and F56A, HeLa FRT cells were supplemented with 1 ng/µL pCDNA5 mNeongreen-Bora plasmid and Lipofectamine 2000 1:50 ratio in Optimem. After a 5-minute incubation of the Lipofectamine 2000 in Optimem, DNA and the reagent were mixed 1:1 and incubated for 20 minutes at 20°C. A list of the cell lines used and the siRNA oligos can be found in Table S2 . Live-cell imaging For time-lapse experiments, 60,000 cells were seeded into each well of a µ-Slide 8 well microscopy chamber (Ibidi). The proteins of interest were knocked down during seeding by reverse siRNA transfection. 24 hours after seeding, cells were exposed to 2 mM Thymidine (Sigma-Aldrich) and synchronized for 16 hours. The following morning, cells were released from the first Thymidine block into media containing 50 ng/ml doxycycline (Sigma-Aldrich). After 8-10 hours, cells were exposed to 2 mM Thymidine and 50 ng/ml doxycycline for 20h. The following morning, cells were released in L15 supplemented with 5% FCS, including 300 nM of BI2536 (Focus Biomolecules), if required by the experiment. 5 hours after release, cells were imaged at 37°C every 20 minutes for 18 hours (for a total tracking of 23 hours) on a DeltaVision Elite (GE Healthcare) deconvolution microscope, equipped with an IX71 inverted microscope (Olympus, Japan), a PLAPON x60/1.42NA oil objective (Olympus) and a pco.edge sCMOS camera (PCO-ECH Inc., USA). For each field of view, 3 z-sections spaced at 6 µm each using were taken. Images were converted into maximal intensity projections in SoftWoRx (Cytiva) for analysis. For each experimental repeat, at least 50 cells were tracked. Quantification was performed manually using the software Fiji ( 89 ). Measurements were exported in Excel (Microsoft) and plotted with GraphPad Prism 10 (GraphPad Software). Figures were assembled using Adobe Illustrator 2025. Data quantifications Dot plots allow the data distribution to be visualized along with the 95% confidence intervals (thick vertical bars) calculated around the median (thin horizontal lines). This representation allows the statistical comparison between different conditions and timepoints because when the vertical bar of one condition does not overlap with one in another condition, the difference between the medians is statistically significant ( P < 0.05). The measurements were first cleaned from outliers using Prism 8 (GraphPad), using ROUT with a cut-off of Q at 2%. Plots were produced using GraphPad Prism 10 (GraphPad Software). Bar plots of signal intensity were generated using ImageJ Fiji ( 89 ) quantification of signal of the raw image (intregrated density), acquired with a BioRad ChemiDoc MP Imaging System (.scn files), after using the background subtraction function of Fiji. Normalization against total substrate or against total kinase was used in case of pull-down from cell lysates or in cases in which there was significant variation of substrate or kinase amount between conditions, and the amount of signal was always calculated as relative signal to the reference condition (set to 1). The final value represents the arithmetic mean between two, three or four replicates, as described in figure legends. Protein conservation was studied and visualised using Jalview ( 90 ). All protein sequences for Figure 2B used in the alignment were obtained from UniProt ( 91 ). Sequence Logo were generated by creating a fasta file of the T-loop region around the phospho-threonine of Aurora A (T288), Aurora B (T232) and PLK1 (T210) from all the species annotated on TreeFam ( 92 ). For PLK1, all the entries annotated as other isoforms of PLK (isoform 2, 3) were removed. The resulting file was loaded on WEBLOGO server from University of Berkeley ( 93 , 94 ). FUNDING This work was supported by the Max Planck Society (A.Mu.), the European Research Council (synergy grant 951430 BIOMECANET to A.Mu.), the Marie-Curie Training Network (DivIDE project number 675737 to A.Mu.), the German Research Foundation (DGF Collaborative Research Centre grant 1430 “Molecular Mechanisms of Cell State Transitions” to A.Mu.), the CANTAR network under the Netzwerke-NRW program (A.Mu.), and an EMBO Long-Term Fellowship ALFT439-2019 (D.C.). AUTHOR CONTRIBUTIONS Conceptualization: A.E.V., A.Mu. Investigation: A.E.V., C.K., D.C., A.Ma., I.R.V., L.O.-L., P.D.R.P Funding acquisition: A.Mu. Project Administration: A.E.V., A.Mu. Resources: M.E.P. Supervision: A.E.V., A.Mu. Validation: A.E.V., A.Mu. Visualization: A.E.V., A.Mu. Writing – original draft: A.E.V., A.Mu. Writing – Review & editing: A.E.V. A.Mu., A.Ma., D.C., M.E.P. COMPETING INTERESTS The authors declare no competing interest. Supplementary Figure Legends Download figure Open in new tab Figure S1. Aurora A binds to its cofactors in vitro A ) Schematic representation of the Aurora A protein cofactors used in the experiment. Bora, TPX2 and CEP192 are N-terminally fused to MBP, as described in more detail in Material and Methods. Aurora A binding sites of the cofactors are indicated with a green box. B ) Analytical size exclusion chromatography of recombinant human Aurora A (5 µM) mixed with equimolar amounts of recombinant human Bora, TPX2 and CEP192 proteins used throughout the study. Download figure Open in new tab Figure S2. PLK1 WT and P1 interface mutants: activation and activity on substrate A ) Predicted Aligned Error (PAE) plot for the Aurora A: Bora 18-280 :PLK1 AlphaFold 2 model shown in Figure 2A . B ) List of interactions detected in the AlphaFold2 model, by using the PDBePisa server ( 95 ). C) Sequence alignment across species of the regions of Bora involved in Aurora A binding (indicated as M1, M2 and M3) and PLK1 binding (P1). Residues selected for mutagenesis and biochemical validation are marked with an asterisk. The extent of sequence conservation is rendered in different colors, ranging from white (no conservation) to darkest orange (full conservation). D) Sequence alignment across species of the regions of PLK1 involved in the binding with the P1 motif of Bora. Residues selected for mutagenesis and biochemical validation are marked with an asterisk. The extent of sequence conservation is rendered in different colors, ranging from white (no conservation) to darkest blue (full conservation). E ) PLK1 WT and mutants time-course activity assay on SA2 1217-1231 (n=2), containing the PLK1 target site S1224. Download figure Open in new tab Figure S3. Binding of Aurora A to Bora P1 mutants A) Phosphorylation of Ndc80 bonsai and H3 nucleosome histone tail by Aurora kinase A T288V was monitored in presence or absence of Bora and the P1 motif mutants (pre-phosphorylated with CDK1). B) Size-exclusion chromatography-based binding assay showing that Bora WT or Bora -F56A-W58A bind Aurora A identically at equimolar concentrations (set to 5 µM). C ) Schematic representation of the reaction protocol used in Figure 3D-F and Figure S3A. D ) Total protein lysate input of the IP experiment shown in Figure 3A . E ) Total protein lysate input of the IP experiment shown in Figure 3B . F ) Control samples of the live-cell imaging experiment shown in Figure 3D . G ) Control samples of the live-cell imaging experiment shown in Figure 3E . Download figure Open in new tab Figure S4. PLK1 WT and K208R activation and activity on substrate A) Sequence alignment across species of the T-loop region around PLK1 Thr210. Alignment was downloaded from the Proviz web server ( 96 ). B ) MBP-PLK1 WT or MBP-PLK1 K208R were assayed in parallel for activity towards an SA2 peptide containing Ser1224. C ) Schematic representation of the reaction protocol used in Figure 4A-B -C. D ) Dead PLK1 WT , PLK1 K208R , PLK1 E206L , PLK1 R207K were assayed in parallel for activation with the same Aurora complexes. A western blot is shown to assess relative phosphorylation of the substrates, quantified with Aurora A:Bora activity on PLK1 K208R as reference (equal to 1.0). The arithmetic mean of two independent experiments is shown. A Coomassie Brilliant Blue stained gel of the same samples was combined to verify equal protein loading between conditions. E ) Phosphorylation of PLK1 WT or PLK1 K208R by increasing amounts of Aurora A:Bora or Aurora B:INCENP. The phosphorylation after 30 minutes at 30°C was visualized by use of phospho-specific antibodies by Western blotting, while the protein content of each sample is shown on Coomassie Blue stained SDS-PAGE gels of the same samples. The experiment was repeated twice. Download figure Open in new tab Figure S5. MAP205 and pBUB1 binding to PLK1 A) Analytical size exclusion chromatography of recombinant human dead PLK1 (5 µM) mixed with equimolar amounts of recombinant MAP205 and BUB1 peptides, also used in the activation assay in Figure 4D . B ) Total protein lysate input of the IP experiment shown in Figure 4A . C ) Total protein lysate input of the IP experiment shown in Figure 4B . D ) Total protein lysate input of the IP experiment shown in Figure 4E . E ) mNeonGreen-PLK1 expression level in the samples in Figure 5C were quantified by recording fluorescence signal intensity during the live-cell imaging experiment. F ) Depletion control for Bora and PLK1 related to the live-cell imaging experiment in Figure 5C . Download figure Open in new tab Figure S6. PLK1 WT and KR dephosphorylation by phosphatases A) PP1 -mediated dephosphorylation of PLK1 WT or PLK1 K208R mutant in a time-course reaction in vitro. Aurora A activation-loop dephosphorylation was also monitored as control. Aurora A and PLK1 blots, together with a Coomassie Blue stained gel were included to verify equal protein loading. B ) λ-PPP-mediated dephosphorylation of PLK1 WT or PLK1 K208R mutant in a time-course reaction in vitro. Aurora A activation-loop dephosphorylation was also monitored as control. Aurora A and PLK1 blots, together with a Coomassie Blue stained gel were included to verify equal protein loading. ACKNOWLEDGMENTS We thank Patricia Stege, Doro Vogt and Tiziano Crocilla for their support in cloning, insect cell, bacterial expression and protein purification. We thank Marius Hedtfeld for sharing the Halo-Aurora B:INCENP reagent; Jan Michael Peters and Gabriele Litos for sharing the antibody against pSA2 1224; Carlos Conde for sharing active His-PP1; Andrew Carter for sharing protocols and reagents related to protein expression in Expi293F cells; the Biotechnology facility of our institute for generating competent cells, providing lambda-phosphatase, and reagents for protein expression and buffer preparation; Stefano Maffini and Nico Schmidt for support with light microscopy; Franca Rossi for helpful comments on the manuscript. We are very grateful to Anaïs Pillan and Lionel Pintard for sharing results in advance of publication. References 1. ↵ E. A. Nigg , Mitotic kinases as regulators of cell division and its checkpoints . Nat Rev Mol Cell Biol 2 , 21 – 32 ( 2001 ). OpenUrl CrossRef PubMed Web of Science 2. ↵ A. T. Saurin , Kinase and Phosphatase Cross-Talk at the Kinetochore . Front Cell Dev Biol 6 , 62 ( 2018 ). 3. ↵ D. L. Brautigan , S. Shenolikar , Protein Serine/Threonine Phosphatases: Keys to Unlocking Regulators and Substrates . Annu Rev Biochem 87 , 921 – 964 ( 2018 ). OpenUrl CrossRef PubMed 4. ↵ R. Bayliss , A. Fry , T. Haq , S. Yeoh , On the molecular mechanisms of mitotic kinase activation . Open Biol 2 , 120136 ( 2012 ). 5. L. N. Johnson , M. E. Noble , D. J. Owen , Active and inactive protein kinases: structural basis for regulation . Cell 85 , 149 – 158 ( 1996 ). OpenUrl CrossRef PubMed Web of Science 6. ↵ M. Huse , J. Kuriyan , The conformational plasticity of protein kinases . Cell 109 , 275 – 282 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 7. ↵ L. Pintard , V. Archambault , A unified view of spatio-temporal control of mitotic entry: Polo kinase as the key . Open Biol 8 , ( 2018 ). 8. ↵ E. Zasadzinska , D. R. Foltz , Orchestrating the Specific Assembly of Centromeric Nucleosomes . Prog Mol Subcell Biol 56 , 165 – 192 ( 2017 ). OpenUrl CrossRef PubMed 9. ↵ V. Archambault , M. Carmena , Polo-like kinase-activating kinases: Aurora A, Aurora B and what else? Cell Cycle 11 , 1490 – 1495 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 10. O. Kelm , M. Wind , W. D. Lehmann , E. A. Nigg , Cell cycle-regulated phosphorylation of the Xenopus polo-like kinase Plx1 . J Biol Chem 277 , 25247 – 25256 ( 2002 ). OpenUrl Abstract / FREE Full Text 11. K. S. Lee , R. L. Erikson , Plk is a functional homolog of Saccharomyces cerevisiae Cdc5, and elevated Plk activity induces multiple septation structures . Mol Cell Biol 17 , 3408 – 3417 ( 1997 ). OpenUrl Abstract / FREE Full Text 12. Y. W. Qian , E. Erikson , J. L. Maller , Mitotic effects of a constitutively active mutant of the Xenopus polo-like kinase Plx1 . Mol Cell Biol 19 , 8625 – 8632 ( 1999 ). OpenUrl Abstract / FREE Full Text 13. Y. J. Jang , S. Ma , Y. Terada , R. L. Erikson , Phosphorylation of threonine 210 and the role of serine 137 in the regulation of mammalian polo-like kinase . J Biol Chem 277 , 44115 – 44120 ( 2002 ). OpenUrl Abstract / FREE Full Text 14. A. L. Lasek , B. M. McPherson , N. G. Trueman , M. E. Burkard , The Functional Significance of Posttranslational Modifications on Polo-Like Kinase 1 Revealed by Chemical Genetic Complementation . PLoS One 11 , e0150225 ( 2016 ). OpenUrl CrossRef PubMed 15. ↵ H. Silva Cascales , K. Burdova , A. Middleton , V. Kuzin , E. Mullers , H. Stoy , L. Baranello , L. Macurek , A. Lindqvist , Cyclin A2 localises in the cytoplasm at the S/G2 transition to activate PLK1 . Life Sci Alliance 4 , ( 2021 ). 16. ↵ L. Gheghiani , D. Loew , B. Lombard , J. Mansfeld , O. Gavet , PLK1 Activation in Late G2 Sets Up Commitment to Mitosis . Cell Rep 19 , 2060 – 2073 ( 2017 ). OpenUrl PubMed 17. ↵ A. Abrieu , T. Brassac , S. Galas , D. Fisher , J. C. Labbe , M. Doree , The Polo-like kinase Plx1 is a component of the MPF amplification loop at the G2/M-phase transition of the cell cycle in Xenopus eggs . J Cell Sci 111 ( Pt 12 ), 1751 – 1757 ( 1998 ). OpenUrl Abstract / FREE Full Text 18. ↵ M. Gobran , A. Z. Politi , L. Welp , J. Jakobi , H. Urlaub , P. Lenart , PLK1 inhibition delays mitotic entry revealing changes to the phosphoproteome of mammalian cells early in division . EMBO J , ( 2025 ). 19. ↵ S. Vigneron , L. Sundermann , J. C. Labbe , L. Pintard , O. Radulescu , A. Castro , T. Lorca , Cyclin A-cdk1-Dependent Phosphorylation of Bora Is the Triggering Factor Promoting Mitotic Entry . Dev Cell 45 , 637 – 650 e637 ( 2018 ). OpenUrl CrossRef PubMed 20. ↵ J. Alexander , D. Lim , B. A. Joughin , B. Hegemann , J. R. Hutchins , T. Ehrenberger , F. Ivins , F. Sessa , O. Hudecz , E. A. Nigg , A. M. Fry , A. Musacchio , P. T. Stukenberg , K. Mechtler , J. M. Peters , S. J. Smerdon , M. B. Yaffe , Spatial exclusivity combined with positive and negative selection of phosphorylation motifs is the basis for context-dependent mitotic signaling . Sci Signal 4 , ra42 ( 2011 ). 21. K. Grosstessner-Hain , B. Hegemann , M. Novatchkova , J. Rameseder , B. A. Joughin , O. Hudecz , E. Roitinger , P. Pichler , N. Kraut , M. B. Yaffe , J. M. Peters , K. Mechtler , Quantitative phospho-proteomics to investigate the polo-like kinase 1-dependent phospho-proteome . Mol Cell Proteomics 10 , M111 008540 ( 2011 ). 22. ↵ A. Santamaria , B. Wang , S. Elowe , R. Malik , F. Zhang , M. Bauer , A. Schmidt , H. H. Sillje , R. Korner , E. A. Nigg , The Plk1-dependent phosphoproteome of the early mitotic spindle . Mol Cell Proteomics 10 , M110 004457 ( 2011 ). 23. ↵ G. Manning , D. B. Whyte , R. Martinez , T. Hunter , S. Sudarsanam , The protein kinase complement of the human genome . Science 298 , 1912 – 1934 ( 2002 ). OpenUrl Abstract / FREE Full Text 24. ↵ A. O. Walter , W. Seghezzi , W. Korver , J. Sheung , E. Lees , The mitotic serine/threonine kinase Aurora2/AIK is regulated by phosphorylation and degradation . Oncogene 19 , 4906 – 4916 ( 2000 ). OpenUrl CrossRef PubMed Web of Science 25. Y. Yasui , T. Urano , A. Kawajiri , K. Nagata , M. Tatsuka , H. Saya , K. Furukawa , T. Takahashi , I. Izawa , M. Inagaki , Autophosphorylation of a newly identified site of Aurora-B is indispensable for cytokinesis . J Biol Chem 279 , 12997 – 13003 ( 2004 ). OpenUrl Abstract / FREE Full Text 26. ↵ L. E. Littlepage , H. Wu , T. Andresson , J. K. Deanehan , L. T. Amundadottir , J. V. Ruderman , Identification of phosphorylated residues that affect the activity of the mitotic kinase Aurora-A . Proc Natl Acad Sci U S A 99 , 15440 – 15445 ( 2002 ). OpenUrl Abstract / FREE Full Text 27. ↵ M. Carmena , X. Pinson , M. Platani , Z. Salloum , Z. Xu , A. Clark , F. Macisaac , H. Ogawa , U. Eggert , D. M. Glover , V. Archambault , W. C. Earnshaw , The chromosomal passenger complex activates Polo kinase at centromeres . PLoS Biol 10 , e1001250 ( 2012 ). OpenUrl CrossRef PubMed 28. ↵ D. Kachaner , X. Pinson , K. B. El Kadhi , K. Normandin , L. Talje , H. Lavoie , G. Lepine , S. Carreno , B. H. Kwok , G. R. Hickson , V. Archambault , Interdomain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis . J Cell Biol 207 , 201 – 211 ( 2014 ). OpenUrl Abstract / FREE Full Text 29. ↵ W. Bruinsma , L. Macurek , R. Freire , A. Lindqvist , R. H. Medema , Bora and Aurora-A continue to activate Plk1 in mitosis . J Cell Sci 127 , 801 – 811 ( 2014 ). OpenUrl Abstract / FREE Full Text 30. ↵ A. Seki , J. A. Coppinger , C. Y. Jang , J. R. Yates , G. Fang , Bora and the kinase Aurora a cooperatively activate the kinase Plk1 and control mitotic entry . Science 320 , 1655 – 1658 ( 2008 ). OpenUrl Abstract / FREE Full Text 31. ↵ L. Macurek , A. Lindqvist , D. Lim , M. A. Lampson , R. Klompmaker , R. Freire , C. Clouin , S. S. Taylor , M. B. Yaffe , R. H. Medema , Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery . Nature 455 , 119 – 123 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 32. ↵ N. Tavernier , Y. Thomas , S. Vigneron , P. Maisonneuve , S. Orlicky , P. Mader , S. G. Regmi , L. Van Hove , N. M. Levinson , G. Gasmi-Seabrook , N. Joly , M. Poteau , G. Velez-Aguilera , O. Gavet , A. Castro , M. Dasso , T. Lorca , F. Sicheri , L. Pintard , Bora phosphorylation substitutes in trans for T-loop phosphorylation in Aurora A to promote mitotic entry . Nat Commun 12 , 1899 ( 2021 ). OpenUrl PubMed 33. W. Bruinsma , M. Aprelia , J. Kool , L. Macurek , A. Lindqvist , R. H. Medema , Spatial Separation of Plk1 Phosphorylation and Activity . Front Oncol 5 , 132 ( 2015 ). 34. ↵ A. Hutterer , D. Berdnik , F. Wirtz-Peitz , M. Zigman , A. Schleiffer , J. A. Knoblich , Mitotic activation of the kinase Aurora-A requires its binding partner Bora . Dev Cell 11 , 147 – 157 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 35. ↵ R. Bayliss , T. Sardon , I. Vernos , E. Conti , Structural basis of Aurora-A activation by TPX2 at the mitotic spindle . Mol Cell 12 , 851 – 862 ( 2003 ). OpenUrl CrossRef PubMed Web of Science 36. ↵ J. Holder , J. A. Miles , M. Batchelor , H. Popple , M. Walko , W. Yeung , N. Kannan , A. J. Wilson , R. Bayliss , F. Gergely , CEP192 localises mitotic Aurora-A activity by priming its interaction with TPX2 . EMBO J 43 , 5381 – 5420 ( 2024 ). OpenUrl PubMed 37. ↵ J. G. Park , H. Jeon , S. Shin , C. Song , H. Lee , N. K. Kim , E. E. Kim , K. Y. Hwang , B. J. Lee , I. G. Lee , Structural basis for CEP192-mediated regulation of centrosomal AURKA . Sci Adv 9 , eadf8582 ( 2023 ). 38. ↵ A. Zorba , V. Buosi , S. Kutter , N. Kern , F. Pontiggia , Y. J. Cho , D. Kern , Molecular mechanism of Aurora A kinase autophosphorylation and its allosteric activation by TPX2 . Elife 3 , e02667 ( 2014 ). OpenUrl CrossRef PubMed 39. ↵ F. Sessa , M. Mapelli , C. Ciferri , C. Tarricone , L. B. Areces , T. R. Schneider , P. T. Stukenberg , A. Musacchio , Mechanism of Aurora B activation by INCENP and inhibition by hesperadin . Mol Cell 18 , 379 – 391 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 40. ↵ J. D. Bishop , J. M. Schumacher , Phosphorylation of the carboxyl terminus of inner centromere protein (INCENP) by the Aurora B Kinase stimulates Aurora B kinase activity . J Biol Chem 277 , 27577 – 27580 ( 2002 ). OpenUrl Abstract / FREE Full Text 41. R. Honda , R. Korner , E. A. Nigg , Exploring the functional interactions between Aurora B, INCENP, and survivin in mitosis . Mol Biol Cell 14 , 3325 – 3341 ( 2003 ). OpenUrl Abstract / FREE Full Text 42. ↵ D. Segura-Pena , O. Hovet , H. Gogoi , J. Dawicki-McKenna , S. M. Hansen Woien , M. Carrer , B. E. Black , M. Cascella , N. Sekulic , The structural basis of the multi-step allosteric activation of Aurora B kinase . Elife 12 , ( 2023 ). 43. ↵ A. E. Elia , P. Rellos , L. F. Haire , J. W. Chao , F. J. Ivins , K. Hoepker , D. Mohammad , L. C. Cantley , S. J. Smerdon , M. B. Yaffe , The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain . Cell 115 , 83 – 95 ( 2003 ). OpenUrl CrossRef PubMed Web of Science 44. A. E. Elia , L. C. Cantley , M. B. Yaffe , Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates . Science 299 , 1228 – 1231 ( 2003 ). OpenUrl Abstract / FREE Full Text 45. ↵ K.-Y. Cheng , E. D. Lowe , J. Sinclair , E. A. Nigg , L. N. Johnson , The crystal structure of the human polo-like kinase-1 polo box domain and its phosho-peptide complex . The EMBO Journal 22 , 5757 – 5768 ( 2003 ). OpenUrl Abstract / FREE Full Text 46. ↵ A. Parrilla , L. Cirillo , Y. Thomas , M. Gotta , L. Pintard , A. Santamaria , Mitotic entry: The interplay between Cdk1, Plk1 and Bora . Cell Cycle 15 , 3177 – 3182 ( 2016 ). OpenUrl CrossRef PubMed 47. ↵ N. Tavernier , A. Noatynska , C. Panbianco , L. Martino , L. Van Hove , F. Schwager , T. Leger , M. Gotta , L. Pintard , Cdk1 phosphorylates SPAT-1/Bora to trigger PLK-1 activation and drive mitotic entry in C. elegans embryos . J Cell Biol 208 , 661 – 669 ( 2015 ). OpenUrl Abstract / FREE Full Text 48. ↵ Y. Thomas , L. Cirillo , C. Panbianco , L. Martino , N. Tavernier , F. Schwager , L. Van Hove , N. Joly , A. Santamaria , L. Pintard , M. Gotta , Cdk1 Phosphorylates SPAT-1/Bora to Promote Plk1 Activation in C. elegans and Human Cells . Cell Rep 15 , 510 – 518 ( 2016 ). OpenUrl PubMed 49. ↵ E. H. Chan , A. Santamaria , H. H. Sillje , E. A. Nigg , Plk1 regulates mitotic Aurora A function through betaTrCP-dependent degradation of hBora . Chromosoma 117 , 457 – 469 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 50. ↵ J. Xu , C. Shen , T. Wang , J. Quan , Structural basis for the inhibition of Polo-like kinase 1 . Nature Structural & Molecular Biology 20 , 1047 – 1053 ( 2013 ). OpenUrl PubMed 51. ↵ A. Seki , J. A. Coppinger , H. Du , C. Y. Jang , J. R. Yates , 3rd , G. Fang , Plk1- and beta-TrCP-dependent degradation of Bora controls mitotic progression . J Cell Biol 181 , 65 – 78 ( 2008 ). OpenUrl Abstract / FREE Full Text 52. ↵ M. Raab , Y. Matthess , C. A. Raab , N. Gutfreund , V. Dotsch , S. Becker , M. Sanhaji , K. Strebhardt , A dimerization-dependent mechanism regulates enzymatic activation and nuclear entry of PLK1 . Oncogene 41 , 372 – 386 ( 2022 ). OpenUrl PubMed 53. ↵ H. Shao , Y. Huang , L. Zhang , K. Yuan , Y. Chu , Z. Dou , C. Jin , M. Garcia-Barrio , X. Liu , X. Yao , Spatiotemporal dynamics of Aurora B-PLK1-MCAK signaling axis orchestrates kinetochore bi-orientation and faithful chromosome segregation . Sci Rep 5 , 12204 ( 2015 ). 54. V. Joukov , J. C. Walter , A. De Nicolo , The Cep192-organized aurora A-Plk1 cascade is essential for centrosome cycle and bipolar spindle assembly . Mol Cell 55 , 578 – 591 ( 2014 ). OpenUrl CrossRef PubMed 55. V. Joukov , A. De Nicolo , A. Rodriguez , J. C. Walter , D. M. Livingston , Centrosomal protein of 192 kDa (Cep192) promotes centrosome-driven spindle assembly by engaging in organelle-specific Aurora A activation . Proc Natl Acad Sci U S A 107 , 21022 – 21027 ( 2010 ). OpenUrl Abstract / FREE Full Text 56. L. Meng , J. E. Park , T. S. Kim , E. H. Lee , S. Y. Park , M. Zhou , J. K. Bang , K. S. Lee , Bimodal Interaction of Mammalian Polo-Like Kinase 1 and a Centrosomal Scaffold, Cep192, in the Regulation of Bipolar Spindle Formation . Mol Cell Biol 35 , 2626 – 2640 ( 2015 ). OpenUrl Abstract / FREE Full Text 57. E. Gallaud , L. Richard-Parpaillon , L. Bataille , A. Pascal , M. Metivier , V. Archambault , R. Giet , The spindle assembly checkpoint and the spatial activation of Polo kinase determine the duration of cell division and prevent tumor formation . PLoS Genet 18 , e1010145 ( 2022 ). OpenUrl CrossRef PubMed 58. M. Ikeda , S. Chiba , K. Ohashi , K. Mizuno , Furry protein promotes aurora A-mediated Polo-like kinase 1 activation . J Biol Chem 287 , 27670 – 27681 ( 2012 ). OpenUrl Abstract / FREE Full Text 59. ↵ V. Joukov , A. De Nicolo , Aurora-PLK1 cascades as key signaling modules in the regulation of mitosis . Sci Signal 11 , ( 2018 ). 60. ↵ J. Jumper , R. Evans , A. Pritzel , T. Green , M. Figurnov , O. Ronneberger , K. Tunyasuvunakool , R. Bates , A. Zidek , A. Potapenko , A. Bridgland , C. Meyer , S. A. A. Kohl , A. J. Ballard , A. Cowie , B. Romera-Paredes , S. Nikolov , R. Jain , J. Adler , T. Back , S. Petersen , D. Reiman , E. Clancy , M. Zielinski , M. Steinegger , M. Pacholska , T. Berghammer , S. Bodenstein , D. Silver , O. Vinyals , A. W. Senior , K. Kavukcuoglu , P. Kohli , D. Hassabis , Highly accurate protein structure prediction with AlphaFold . Nature 596 , 583 – 589 ( 2021 ). OpenUrl CrossRef PubMed 61. ↵ S. G. Zeitlin , R. D. Shelby , K. F. Sullivan , CENP-A is phosphorylated by Aurora B kinase and plays an unexpected role in completion of cytokinesis . J Cell Biol 155 , 1147 – 1157 ( 2001 ). OpenUrl Abstract / FREE Full Text 62. J. Y. Hsu , Z. W. Sun , X. Li , M. Reuben , K. Tatchell , D. K. Bishop , J. M. Grushcow , C. J. Brame , J. A. Caldwell , D. F. Hunt , R. Lin , M. M. Smith , C. D. Allis , Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes . Cell 102 , 279 – 291 ( 2000 ). OpenUrl CrossRef PubMed Web of Science 63. J. G. DeLuca , W. E. Gall , C. Ciferri , D. Cimini , A. Musacchio , E. D. Salmon , Kinetochore microtubule dynamics and attachment stability are regulated by Hec1 . Cell 127 , 969 – 982 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 64. ↵ I. M. Cheeseman , J. S. Chappie , E. M. Wilson-Kubalek , A. Desai , The conserved KMN network constitutes the core microtubule-binding site of the kinetochore . Cell 127 , 983 – 997 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 65. ↵ R. Evans , M. O’Neill , A. Pritzel , N. Antropova , A. Senior , T. Green , A. Žídek , R. Bates , S. Blackwell , J. Yim , O. Ronneberger , S. Bodenstein , M. Zielinski , A. Bridgland , A. Potapenko , A. Cowie , K. Tunyasuvunakool , R. Jain , E. Clancy , P. Kohli , J. Jumper , D. Hassabis , Protein complex prediction with AlphaFold-Multimer . bioRxiv , ( 2022 ). 66. ↵ I. Sumara , E. Vorlaufer , P. T. Stukenberg , O. Kelm , N. Redemann , E. A. Nigg , J. M. Peters , The dissociation of cohesin from chromosomes in prophase is regulated by Polo-like kinase . Mol Cell 9 , 515 – 525 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 67. ↵ O. Feine , E. Hukasova , W. Bruinsma , R. Freire , A. Fainsod , J. Gannon , H. M. Mahbubani , A. Lindqvist , M. Brandeis , Phosphorylation-mediated stabilization of Bora in mitosis coordinates Plx1/Plk1 and Cdk1 oscillations . Cell Cycle 13 , 1727 – 1736 ( 2014 ). OpenUrl PubMed 68. ↵ M. A. van Vugt , A. Bras , R. H. Medema , Polo-like kinase-1 controls recovery from a G2 DNA damage-induced arrest in mammalian cells . Mol Cell 15 , 799 – 811 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 69. ↵ S. Ferrari , O. Marin , M. A. Pagano , F. Meggio , D. Hess , M. El-Shemerly , A. Krystyniak , L. A. Pinna , Aurora-A site specificity: a study with synthetic peptide substrates . Biochem J 390 , 293 – 302 ( 2005 ). OpenUrl Abstract / FREE Full Text 70. A. N. Kettenbach , D. K. Schweppe , B. K. Faherty , D. Pechenick , A. A. Pletnev , S. A. Gerber , Quantitative phosphoproteomics identifies substrates and functional modules of Aurora and Polo-like kinase activities in mitotic cells . Sci Signal 4 , rs5 ( 2011 ). 71. ↵ A. Koch , K. Krug , S. Pengelley , B. Macek , S. Hauf , Mitotic substrates of the kinase aurora with roles in chromatin regulation identified through quantitative phosphoproteomics of fission yeast . Sci Signal 4 , rs6 ( 2011 ). 72. N. Dephoure , C. Zhou , J. Villen , S. A. Beausoleil , C. E. Bakalarski , S. J. Elledge , S. P. Gygi , A quantitative atlas of mitotic phosphorylation . Proc Natl Acad Sci U S A 105 , 10762 – 10767 ( 2008 ). OpenUrl Abstract / FREE Full Text 73. M. Nousiainen , H. H. Sillje , G. Sauer , E. A. Nigg , R. Korner , Phosphoproteome analysis of the human mitotic spindle . Proc Natl Acad Sci U S A 103 , 5391 – 5396 ( 2006 ). OpenUrl Abstract / FREE Full Text 74. ↵ B. Hegemann , J. R. Hutchins , O. Hudecz , M. Novatchkova , J. Rameseder , M. M. Sykora , S. Liu , M. Mazanek , P. Lenart , J. K. Heriche , I. Poser , N. Kraut , A. A. Hyman , M. B. Yaffe , K. Mechtler , J. M. Peters , Systematic phosphorylation analysis of human mitotic protein complexes . Sci Signal 4 , rs12 ( 2011 ). 75. ↵ L. Ren , R. Gasper , E. M. Pesenti , P. Janning , F. Müller , C. Koerner , P. Geue , S. Wohlgemuth , A. Esposito Verza , I. R. Vetter , A. Musacchio , Molecular anatomy of PLK1 master docking motifs . bioRxiv , ( 2025 ). 76. ↵ T. Ansai , L. C. Dupuy , S. Barik , Interactions between a minimal protein serine/threonine phosphatase and its phosphopeptide substrate sequence . J Biol Chem 271 , 24401 – 24407 ( 1996 ). OpenUrl Abstract / FREE Full Text 77. ↵ L. Palmieri , G. Rastelli , alphaC helix displacement as a general approach for allosteric modulation of protein kinases . Drug Discov Today 18 , 407 – 414 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 78. ↵ A. Pillan , P. Ormancey , C. Ben Choug , S. Orlicky , N. Tavernier , L. Van Hove , B. Ossareh-Nazari , N. Joly , F. Sicheri , T. Lorca , L. Pintard , Molecular basis for the activation of Aurora A and Plk1 kinases by Bora during mitotic entry . Submitted , ( 2025 ). 79. ↵ L. Xu , M. Ali , W. Duan , X. Yuan , F. Garba , M. Mullen , B. Sun , I. Poser , H. Duan , J. Lu , R. Tian , Y. Ge , L. Chu , W. Pan , D. Wang , A. Hyman , H. Green , L. Li , Z. Dou , D. Liu , X. Liu , X. Yao , Feedback control of PLK1 by Apolo1 ensures accurate chromosome segregation . Cell Rep 36 , 109343 ( 2021 ). 80. ↵ D. P. Byrne , S. Shrestha , M. Galler , M. Cao , L. A. Daly , A. E. Campbell , C. E. Eyers , E. A. Veal , N. Kannan , P. A. Eyers , Aurora A regulation by reversible cysteine oxidation reveals evolutionarily conserved redox control of Ser/Thr protein kinase activity . Sci Signal 13 , ( 2020 ). 81. G. F. Wang , Q. Dong , Y. Bai , J. Yuan , Q. Xu , C. Cao , X. Liu , Oxidative stress induces mitotic arrest by inhibiting Aurora A-involved mitotic spindle formation . Free Radic Biol Med 103 , 177 – 187 ( 2017 ). OpenUrl CrossRef PubMed 82. ↵ D. C. Lim , V. Joukov , T. J. Rettenmaier , A. Kumagai , W. G. Dunphy , J. A. Wells , M. B. Yaffe , Redox priming promotes Aurora A activation during mitosis . Sci Signal 13 , ( 2020 ). 83. ↵ S. Trowitzsch , C. Bieniossek , Y. Nie , F. Garzoni , I. Berger , New baculovirus expression tools for recombinant protein complex production . J Struct Biol 172 , 45 – 54 ( 2010 ). OpenUrl CrossRef PubMed 84. ↵ P. J. Huis In’t Veld , S. Wohlgemuth , C. Koerner , F. Muller , P. Janning , A. Musacchio , Reconstitution and use of highly active human CDK1:Cyclin-B:CKS1 complexes . Protein Sci 31 , 528 – 537 ( 2022 ). OpenUrl CrossRef PubMed 85. ↵ T. Ikehara , S. Nakashima , J. Nakashima , T. Kinoshita , T. Yasumoto , Efficient production of recombinant PP2A at a low temperature using a baculovirus expression system . Biotechnol Rep (Amst ) 11 , 86 – 89 ( 2016 ). OpenUrl PubMed 86. ↵ C. Ciferri , S. Pasqualato , E. Screpanti , G. Varetti , S. Santaguida , G. Dos Reis , A. Maiolica , J. Polka , J. G. De Luca , P. De Wulf , M. Salek , J. Rappsilber , C. A. Moores , E. D. Salmon , A. Musacchio , Implications for kinetochore-microtubule attachment from the structure of an engineered Ndc80 complex . Cell 133 , 427 – 439 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 87. ↵ K. Walstein , A. Petrovic , D. Pan , B. Hagemeier , D. Vogt , I. R. Vetter , A. Musacchio , Assembly principles and stoichiometry of a complete human kinetochore module . Sci Adv 7 , ( 2021 ). 88. ↵ V. Krenn , A. Wehenkel , X. Li , S. Santaguida , A. Musacchio , Structural analysis reveals features of the spindle checkpoint kinase Bub1-kinetochore subunit Knl1 interaction . J Cell Biol 196 , 451 – 467 ( 2012 ). OpenUrl Abstract / FREE Full Text 89. ↵ J. Schindelin , I. Arganda-Carreras , E. Frise , V. Kaynig , M. Longair , T. Pietzsch , S. Preibisch , C . Rueden , S. Saalfeld , B. Schmid , J. Y. Tinevez , D. J. White , V. Hartenstein , K. Eliceiri , P. Tomancak , A. Cardona , Fiji: an open-source platform for biological-image analysis . Nat Methods 9 , 676 – 682 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 01. ↵ A. M. Waterhouse , J. B. Procter , D. M. Martin , M. Clamp , G. J. Barton , Jalview Version 2--a multiple sequence alignment editor and analysis workbench . Bioinformatics 25 , 1189 - 1191 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 91. ↵ C. UniProt , UniProt: the Universal Protein Knowledgebase in 2023 . Nucleic Acids Res 51 , D523 – D531 ( 2023 ). OpenUrl CrossRef PubMed 92. ↵ H. Li , A. Coghlan , J. Ruan , L. J. Coin , J. K. Heriche , L. Osmotherly , R. Li , T. Liu , Z. Zhang , L. Bolund , G. K. Wong , W. Zheng , P. Dehal , J. Wang , R. Durbin , TreeFam: a curated database of phylogenetic trees of animal gene families . Nucleic Acids Res 34 , D572 – 580 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 93. ↵ T. D. Schneider , R. M. Stephens , Sequence logos: a new way to display consensus sequences . Nucleic Acids Res 18 , 6097 – 6100 ( 1990 ). OpenUrl CrossRef PubMed Web of Science 94. ↵ G. E. Crooks , G. Hon , J. M. Chandonia , S. E. Brenner , WebLogo: a sequence logo generator . Genome Res 14 , 1188 – 1190 ( 2004 ). OpenUrl Abstract / FREE Full Text 95. ↵ E. Krissinel , K. Henrick , Inference of macromolecular assemblies from crystalline state . J Mol Biol 372 , 774 – 797 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 96. ↵ P. Jehl , J. Manguy , D. C. Shields , D. G. Higgins , N. E. Davey , ProViz-a web-based visualization tool to investigate the functional and evolutionary features of protein sequences . Nucleic Acids Res 44 , W11 – 15 ( 2016 ). OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted July 29, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Molecular requirements for PLK1 activation by T-loop phosphorylation Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Molecular requirements for PLK1 activation by T-loop phosphorylation Arianna Esposito Verza , Duccio Conti , Paulo D. Rodrigues Pedroso , Lina Oberste-Lehn , Carolin Koerner , Sabine Wohlgemuth , Artem Mansurkhodzhaev , Ingrid R. Vetter , Marion E. Pesenti , Andrea Musacchio bioRxiv 2025.07.25.666769; doi: https://doi.org/10.1101/2025.07.25.666769 Share This Article: Copy Citation Tools Molecular requirements for PLK1 activation by T-loop phosphorylation Arianna Esposito Verza , Duccio Conti , Paulo D. Rodrigues Pedroso , Lina Oberste-Lehn , Carolin Koerner , Sabine Wohlgemuth , Artem Mansurkhodzhaev , Ingrid R. Vetter , Marion E. Pesenti , Andrea Musacchio bioRxiv 2025.07.25.666769; doi: https://doi.org/10.1101/2025.07.25.666769 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Biochemistry Subject Areas All Articles Animal Behavior and Cognition (7624) Biochemistry (17651) Bioengineering (13871) Bioinformatics (41882) Biophysics (21424) Cancer Biology (18566) Cell Biology (25461) Clinical Trials (138) Developmental Biology (13365) Ecology (19867) Epidemiology (2067) Evolutionary Biology (24290) Genetics (15590) Genomics (22476) Immunology (17714) Microbiology (40331) Molecular Biology (17148) Neuroscience (88483) Paleontology (666) Pathology (2828) Pharmacology and Toxicology (4817) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9815) Zoology (2268)