Csk inhibition of c-Src activity requires both the SH2 and SH3 domains of Src.
The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its carboxy-terminal tail. A kinase that phosphorylates Tyr527, called Csk, has recently been identified. We expressed c-Src in yeast to test the role of the SH2 and SH3 domains of Src in the negative regulation exerted by Tyr527 phosphorylation. Inducible expression of c-Src in Schizosaccharomyces pombe caused cell death. Co-expression of Csk counteracted this effect. Src proteins mutated in either the SH2 or SH3 domain were as lethal as wild type c-Src, but were insensitive to Csk, even though they were substrates for Csk in vivo. Peptide binding experiments revealed that Src proteins with mutant SH3 domains adopted a conformation in which the SH2 domain was not interacting with the tail. These data support the model of an SH2 domain-phosphorylated tail interaction repressing c-Src activity, but expand it to include a role for the SH3 domain. We propose that the SH3 domain contributes to the maintenance of the folded, inactive configuration of the Src molecule by stabilizing the SH2 domain-phosphorylated tail interaction. Moreover, the system we describe here allows for further study of the regulation of tyrosine kinases in a neutral background and in an organism amenable to genetic analysis.
Superti-Furga
G
G
European Molecular Biology Laboratory, Heidelberg, Germany.
Fumagalli
S
S
Koegl
M
M
Courtneidge
S A
SA
Draetta
G
G
D000595:Amino Acid Sequence; D000081247:CSK Tyrosine-Protein Kinase; D003001:Cloning, Molecular; D005804:Genes, Lethal; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases
D010455:Peptides; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases