Tyrosine 416 is phosphorylated in the closed, repressed conformation of c-Src.
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.
Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, Australia.
Sevgi
S
Irtegun
Rebecca J
RJ
Wood
Angelique R
AR
Ormsby
Terrence D
TD
Mulhern
Danny M
DM
Hatters
D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D006801:Humans; D010766:Phosphorylation; D017354:Point Mutation; D011487:Protein Conformation; D050796:STAT3 Transcription Factor / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019061:src-Family Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D050796:STAT3 Transcription Factor; D014443:Tyrosine; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human