Two SKAP2 modular organizations differently recognize SRC kinases depending on their activation status and localization

Background: The activation of SRC kinases by trans-phosphorylation raises the question of their interplay. This work addresses this question by studying the interactions of SRC kinases with the SRC Kinase Adaptor Phosphoprotein 2 (SKAP2) by combining a luciferase complementation assay and extensive site-directed mutagenesis. Results: : SKAP2 interacts with SRC kinases through two different module organizations that have different affinities for the activated and non-activated kinase conformations. The DIM domain of SKAP2 is necessary and sufficient to bind to most activated SRC kinases. A second SKAP2 module organization for binding to SRC kinases at their steady state is composed of the DIM and SH3 domains as well as the linker located between the PH and the SH3 domains. Classical binding sites of the PH and SH3 domains and the tyrosines located inside linkers modulate these interactions. Analysis of HCK mutants supports this model and shows the role of two residues, Y390 and K7, on its degradation following activation. Conclusion: In this article, we show that SKAP2 have a modular architecture composed of 2 domains and one interdomain to interact with SRC kinases, which binding capacity of each module depends on their localization and activation. This work opens new perspectives on how activation of SRC kinases occurs, which could have new and interesting therapeutic consequences.