Abstract
c-Src kinase activity is regulated by phosphorylation of Y527 and Y416. Y527 phosphorylation stabilizes a closed conformation, which suppresses kinase activity towards substrates, whereas phosphorylation at Y416 promotes an elevated kinase activity by stabilizing the activation loop in a manner permissive for substrate binding. Here we investigated the correlation of Y416 phosphorylation with c-Src activity when c-Src was locked into the open and closed conformations (by mutations Y527F and Q528E, P529E, G530I respectively). Consistent with prior findings, we found Y416 to be more greatly phosphorylated when c-Src was in an open, active conformation. However, we also observed an appreciable amount of Y416 was phosphorylated when c-Src was in a closed, repressed conformation under conditions by which c-Src was unable to phosphorylate substrate STAT3. The phosphorylation of Y416 in the closed conformation arose by autophosphorylation, since abolishing kinase activity by mutating the ATP binding site (K295M) prevented phosphorylation. Basal Y416 phosphorylation correlated positively with cellular levels of c-Src suggesting autophosphorylation depended on self-association. Using sedimentation velocity analysis on cell lysate with fluorescence detection optics, we confirmed that c-Src forms monomers and dimers, with the open conformation also forming a minor population of larger mass complexes. Collectively, our studies suggest a model by which dimerization of c-Src primes c-Src via Y416 phosphorylation to enable rapid potentiation of activity when Src adopts an open conformation. Once in the open conformation, c-Src can amplify the response by recruiting and phosphorylating substrates such as STAT3 and increasing the extent of autophosphorylation.
Highlights
Introduction cSrc signaling controls many cellular events such as cell growth, proliferation, differentiation, motility and cell adhesion [1]
The greatest extent of Y416 phosphorylation occurred at lower levels of c-Src expression, which is consistent with Y416 phosphorylation occurring cooperatively when c-Src is engaged with ligands or other cellular complexes that are concentration limiting with increasing levels of c-Src
C-Src did not lead to STAT3 phosphorylation, suggesting that kinase activity for autophosphorylation can be decoupled from substrate phosphorylation
Summary
Introduction cSrc signaling controls many cellular events such as cell growth, proliferation, differentiation, motility and cell adhesion [1]. Phosphorylation of Y527 facilitates the formation of the closed conformation by enabling high affinity binding of the SH2 domain to the C-tail. This interaction, as well as binding between the SH3 domain and the SH2-kinase linker, creates a compact structure that represses kinase activity. Open active c-Src can be induced by the mutation Y527F which impairs binding of SH2 and impedes formation of the closed repressed state [5]. Mutating the C-tail at residues Q528E, P529E, G530I, to mimic a high affinity c-Src SH2 ligand induces a constitutively closed state, as reported previously for the Src family member Hck [6]
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