Evidence for autoinhibitory regulation of the c-src gene product. A possible interaction between the src homology 2 domain and autophosphorylation site.
In the previous study (Sato, K., Miki, S., Tachibana, H., Hayashi, F., Akiyama, T., and Fukami, Y. (1990) Biochem. Biophys. Res. Commun. 171, 1152-1159), we found a synthetic peptide, termed peptide A, that inhibited the kinase activity of p60v-src. The peptide A sequence corresponds to residues 137 to 157 of p60v-src which are included in the amino-terminal portion of the src homology 2 domain. In this study, we attempted to specify the inhibitory sequence in this domain and to identify its target site. The most potent peptide A derivative was one that corresponds to residues 140 through 157. The target site of peptide A was assumed to reside in the autophosphorylation site of p60v-src, since synthetic peptides containing the sequence Phe424-Pro-Ile-Lys-Trp428 which is present downstream of the autophosphorylated Tyr416 partially counteracted the inhibitory effect of peptide A. An antibody was prepared against one of such target peptides, termed pepY. Cross-linking experiments showed that 125I-labeled peptide A could bind to p60v-src blotted on a membrane, and the binding was blocked by the anti-pepY antibody but not by other anti-p60v-src antibodies. Conversely, immunoblotting of p60v-src with anti-pepY antibody was blocked by the cross-linking of peptide A to p60v-src. To our surprise, anti-pepY antibody did not affect the p60v-src activity. Furthermore, p60c-src was activated 2- to 6-fold by this antibody. These results suggest that the pepY region in the catalytic domain of p60v-src or of p60c-src is not essential for the catalytic activity but rather is involved in the negative regulation of the kinase activity of p60c-src.
Fukami
Y
Y
Laboratory of Molecular Biology, Kobe University, Japan.
Sato
K
K
Ikeda
K
K
Kamisango
K
K
Koizumi
K
K
Matsuno
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic; D004926:Escherichia coli / Q000235:genetics; D016391:Genes, src; D006706:Homeostasis; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D015688:Oncogene Protein pp60(v-src) / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; C098708:peptide A; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src); D016392:Proto-Oncogene Proteins pp60(c-src)