The carboxy terminus of pp60c-src is a regulatory domain and is involved in complex formation with the middle-T antigen of polyomavirus.
A large number of mutations were introduced into the carboxy-terminal domain of pp60c-src. The level of phosphorylation on Tyr-416 and Tyr-527, the transforming activity (as measured by focus formation on NIH 3T3 cells), kinase activity, and the ability of the mutant pp60c-src to associate with the middle-T antigen of polyomavirus were examined. The results indicate that Tyr-527 is a major carboxy-terminal element responsible for regulating pp60c-src in vivo. A good but not perfect correlation exists between lack of phosphorylation at Tyr-527 and increased phosphorylation at Tyr-416, between elevated phosphorylation on Tyr-416 and activated kinase activity, and between activated kinase activity and transforming activity. Phosphorylation of Tyr-527 was insensitive to the mutation of adjacent residues, indicating that the primary sequence only has a minor role in recognition by kinases or phosphatases which regulate it in vivo. Three mutants which have in common a modified Glu-524 residue were phosphorylated on Tyr-416 and Tyr-527 and were weakly transforming. This suggests that other mechanisms besides complete dephosphorylation of Tyr-527 can lead to increased phosphorylation of Tyr-416 and activation of the transforming activity of pp60c-src. Furthermore, the residues between Asp-518 and Pro-525 were required to form a stable complex with middle-T antigen. The proximity of these sequences to Tyr-527 suggests a model in which middle-T activates pp60c-src by binding directly to this region of the molecular and thereby preventing phosphorylation of Tyr-527. Alternatively, middle-T binding may mediate a conformational change in this region, which in turn induces an alteration in the level of phosphorylation at Tyr-527 and Tyr-416.
Cheng
S H
SH
Laboratory of Cellular Regulation, Integrated Genetics Inc., Framingham, Massachusetts 01701.
Piwnica-Worms
H
H
Harvey
R W
RW
Roberts
T M
TM
Smith
A E
AE
D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D003062:Codon; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010449:Peptide Mapping; D010766:Phosphorylation; D011120:Polyomavirus / Q000235:genetics*; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src)
D000952:Antigens, Polyomavirus Transforming; D003062:Codon; D011518:Proto-Oncogene Proteins; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src)