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Selective binding of activated pp60c-src by an immobilized synthetic phosphopeptide modeled on the carboxyl terminus
R R Roussel1, S R Brodeur, D Shalloway
1Department of Biochemistry, University of New Hampshire, Durham 03824.
Abstract:
Phosphorylation of the carboxyl terminus of pp60c-src, the product of the c-src protooncogene, at Tyr-527 suppresses its tyrosine kinase activity and transforming potential. It has been proposed that the phosphorylated carboxyl terminus of pp60c-src inhibits kinase activity by binding to the SH2 (src homology 2) domain. We have synthesized peptides corresponding to the carboxyl-terminal 13 residues of pp60c-src phosphorylated and nonphosphorylated at Tyr-527. A highly transforming mutant, pp60c-src(F527), in which Tyr-527 is mutated to Phe, bound to the phosphorylated peptide immobilized to Affi-Gel 10. Binding of the phosphorylated peptide was abolished by deletion of residues 144-175 in the SH2 domain but not by deletion of residues 93-143, which removes most of the SH3 domain. The phosphorylated peptide also bound to pp60v-src, the transforming protein of Rous sarcoma virus. Only traces of pp60v-src and pp60c-src(F527) bound to the corresponding nonphosphorylated c-src peptide. Normal pp60c-src bound much less efficiently to the phosphorylated peptide than did pp60c-src(F527). A phosphorylated peptide corresponding to the carboxyl terminus of the c-fgr protein also bound to pp60c-src(F527), but with weaker affinity. Furthermore, the phosphorylated synthetic carboxyl-terminal pp60c-src peptide markedly inhibited phosphorylation of pp60c-src(F527) during cytoskeletal kinase assays. These results provide direct evidence for models in which the phosphorylated carboxyl terminus of pp60c-src binds intramolecularly or intermolecularly to the SH2 domain of the c-src protein.
Insights
Phosphorylation of pp60c-src at Tyr-527 suppresses its activity by binding to the SH2 domain. This study provides direct evidence for this interaction, crucial for understanding c-src protein regulation.
Area of Science:
- Molecular Biology
- Cellular Signaling
- Biochemistry
Background:
- The c-src protooncogene product, pp60c-src, is a tyrosine kinase regulated by phosphorylation.
- Phosphorylation at Tyr-527 is known to suppress kinase activity and transforming potential.
- It is hypothesized that this phosphorylated C-terminus interacts with the SH2 domain.
Purpose of the Study:
- To investigate the interaction between the phosphorylated C-terminus of pp60c-src and its SH2 domain.
- To provide direct evidence for the proposed inhibitory mechanism of pp60c-src activity.
Main Methods:
- Synthesis of phosphorylated and nonphosphorylated peptides representing the C-terminus of pp60c-src.
- Affinity binding assays using immobilized peptides and various src protein mutants.
- Enzyme activity assays to assess the effect of peptides on kinase phosphorylation.
Main Results:
- A highly transforming mutant, pp60c-src(F527), bound to the phosphorylated peptide, with binding abolished by SH2 domain deletion.
- The phosphorylated peptide also bound pp60v-src and inhibited pp60c-src(F527) phosphorylation in kinase assays.
- Binding to the nonphosphorylated peptide was minimal, and normal pp60c-src showed less efficient binding than the mutant.
Conclusions:
- Direct evidence supports the model where the phosphorylated C-terminus of pp60c-src binds to the SH2 domain.
- This interaction likely mediates the suppression of tyrosine kinase activity and transforming potential.
- The findings are crucial for understanding the regulation of c-src family kinases.