Autophosphorylation of the CK1 kinase domain regulates enzyme activity and function

CK1 enzymes are conserved, acidophilic serine/threonine kinases with a variety of critical cellular functions; misregulation of CK1 contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Despite this, little is known about how CK1 activity is controlled. Here, we describe a new mechanism of CK1 autoregulation that is conserved in CK1 enzymes from yeast to human – the autophosphorylation of a threonine in the mobile L-EF loop proximal to the active site. Phosphorylation at this site inhibits kinase activity, in contrast to well-characterized T-loop autophosphorylation in other kinase families. Consequently, yeast and human enzymes with phosphoablating mutations at this site are hyperactive. In S. pombe , hyperactive CK1 causes defects in cell growth and morphology at a high level but protection from heat shock at a low level, highlighting the necessity of regulated CK1 function. We propose that phosphorylation on the L-EF loop prevents substrate docking with the kinase domain by shielding the positively charged binding pocket and/or sterically hindering the active site. Due to the strong sequence conservation of this autophosphorylation site and the functional importance of the L-EF loop, which is unique to the CK1 family of kinases, this mechanism is likely to regulate the majority of CK1 enzymes in vivo. Significance Statement Kinases in the CK1 family are important signaling enzymes, and they function in multiple pathways within the same cell. Misregulation of CK1 activity contributes to human disease, including cancer, neurodegenerative disease, and sleep phase disorders, yet the mechanisms that control CK1 activity are not well understood. We have identified a conserved autophosphorylation site in the CK1 kinase domain that inhibits substrate phosphorylation. We hypothesize that by using kinase domain autophosphorylation in combination with other regulatory mechanisms, CK1 enzymes can coordinate the phosphorylation of their substrates in different pathways.