Structural differences between repressed and derepressed forms of p60c-src.
The kinase activity of p60c-src is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms of p60c-src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60c-src. The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60c-src, the kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus, and novel phosphorylation sites. The results identify parts of p60c-src that change conformation upon kinase activation and suggest functions for the amino-terminal region.
MacAuley
A
A
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Cooper
J A
JA
D004789:Enzyme Activation; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011233:Precipitin Tests; D011402:Pronase; D011487:Protein Conformation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src); D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014157:Transcription Factors / Q000378:metabolism*; D014357:Trypsin; D014443:Tyrosine / Q000378:metabolism
D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D014443:Tyrosine; D011494:Protein Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D014357:Trypsin; D011402:Pronase