Deciphering the reciprocal regulation of the Pyk2-Src activation complex

Abstract

Pyk2 and Src are non-receptor tyrosine kinases that assemble into a complex to promote mutual activation. The Pyk2 FERM domain mediates autoinhibition by direct interaction with the kinase. Src autoinhibition is maintained by intramolecular interactions of the SH3 and SH2 domains with internal ligands, including an inhibitory C-terminal phosphotyrosine. FERM disengagement permits Pyk2 autophosphorylation of the FERM-kinase linker to generate a scaffolding site for Src recruitment. Although Src-mediated phosphorylation of the Pyk2 activation loop is well-established, the mechanism by which complex formation activates Src remains unclear. We reconstituted defined phosphorylation and regulatory states of both kinases, combining site-directed mutagenesis with phosphosite-resolved activity profiling to dissect the reciprocal regulation. Src phosphorylates the Pyk2 activation loop via an ordered, self-primed dual phosphorylation mechanism. Although activated Pyk2 productively phosphorylates the Src activation loop, Pyk2-Src complex formation does not significantly increase activation loop phosphorylation above the rate of Src autophosphorylation alone. Conversely, autoinhibited Src strictly requires Pyk2 scaffolding engagement to productively phosphorylate Pyk2. Pyk2 therefore relieves Src autoinhibition by presenting competing scaffolding sites to disengage intramolecular SH3 and SH2 conformational constraints. The results distinguish activation loop phosphorylation from the conformational competence required for substrate phosphorylation. We propose that autophosphorylated Pyk2 functions principally as a conformational activator and scaffolding platform, opening Src for phosphatase-mediated removal of the inhibitory C-terminal phosphosite and committing both Pyk2 and Src to downstream substrate phosphorylation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/739604v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@b37a05org.highwire.dtl.DTLVardef@11014fdorg.highwire.dtl.DTLVardef@d20527org.highwire.dtl.DTLVardef@50826f_HPS_FORMAT_FIGEXP M_FIG C_FIG