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Phosphopeptide occupancy and photoaffinity cross-linking of the v-Src SH2 domain attenuates tyrosine kinase activity
P Garcia1, S E Shoelson, J S Drew
1Department of Physiology and Biophysics, School of Medicine State University of New York, Stony Brook 11794.
Abstract:
Phosphorylation of c-Src at carboxyl-terminal Tyr-527 suppresses tyrosine kinase activity and transforming potential, presumably by facilitating the intramolecular interaction of the C terminus of Src with its SH2 domain. In addition, it has been shown previously that occupancy of the c-Src SH2 domain with a phosphopeptide stimulates c-Src kinase catalytic activity. We have performed analogous studies with v-Src, the transforming protein from Rous sarcoma virus, which has extensive homology with c-Src. v-Src lacks an autoregulatory phosphorylation site, and its kinase domain is constitutively active. Phosphopeptides corresponding to the sequences surrounding c-Src Tyr-527 and a Tyr-Glu-Glu-Ile motif from the hamster polyoma virus middle T antigen inhibit tyrosine kinase activity of baculovirus-expressed v-Src 2- and 4-fold, respectively. To determine the mechanism of this regulation, the Tyr-527 phosphopeptide was substituted with the photoactive amino acid p-benzoylphenylalanine at the adjacent positions (N- and C-terminal) to phosphotyrosine. These peptides photoinactivate the v-Src tyrosine kinase 5-fold in a time- and concentration-dependent manner. Furthermore, the peptides cross-link an isolated Src SH2 domain with similar rates and specificity. These data indicate that occupancy of the v-Src SH2 domain induces a conformational change that is transmitted to the kinase domain and attenuates tyrosine kinase activity.
Insights
v-Src tyrosine kinase activity is regulated by its SH2 domain. Occupying the SH2 domain with specific phosphopeptides inhibits v-Src kinase activity, revealing a conformational change mechanism.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- c-Src tyrosine kinase activity is regulated by phosphorylation at Tyr-527, promoting intramolecular interaction with the SH2 domain.
- v-Src, a viral homolog of c-Src, lacks this regulatory site and exhibits constitutive kinase activity.
Purpose of the Study:
- To investigate the regulation of v-Src tyrosine kinase activity by its SH2 domain.
- To elucidate the mechanism by which phosphopeptides modulate v-Src kinase activity.
Main Methods:
- v-Src kinase activity was assessed in the presence of phosphopeptides corresponding to c-Src Tyr-527 and a polyoma virus middle T antigen motif.
- Photoactive amino acid substitution was used to probe SH2 domain interactions with v-Src.
- Cross-linking studies were performed with isolated Src SH2 domains.
Main Results:
- Phosphopeptides inhibited baculovirus-expressed v-Src tyrosine kinase activity 2- to 4-fold.
- Photoactive peptides induced time- and concentration-dependent photoinactivation of v-Src kinase (5-fold).
- These peptides cross-linked with isolated Src SH2 domains, indicating specific binding.
Conclusions:
- Occupancy of the v-Src SH2 domain by phosphopeptides induces a conformational change.
- This conformational change is transmitted to the kinase domain, leading to attenuated tyrosine kinase activity.
- The SH2 domain is a critical regulator of v-Src kinase activity.