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Suppression of c-Src activity by C-terminal Src kinase involves the c-Src SH2 and SH3 domains: analysis with
S M Murphy1, M Bergman, D O Morgan
1Department of Physiology, University of California, San Francisco 94143-0444.
Abstract:
The kinase activity of c-Src is normally repressed in vertebrate cells by extensive phosphorylation of Y-527. C-terminal Src kinase (CSK) is a candidate for the enzyme that catalyzes this phosphorylation. We have used budding yeast to study the regulation of c-Src activity by CSK in intact cells. Expression of c-Src in Saccharomyces cerevisiae, which lacks endogenous c-Src and Y-527 kinases, induces a kinase-dependent growth inhibition. Coexpression of CSK in these cells results in phosphorylation of c-Src on Y-527 and suppression of the c-Src phenotype. CSK does not fully suppress the activity of c-Src mutants lacking portions of the SH2 or SH3 domains, even though these mutant proteins are phosphorylated on Y-527 by CSK both in vivo and in vitro. These results suggest that both the SH2 and SH3 domains of c-Src are required for the suppression of c-Src activity by Y-527 phosphorylation.
Insights
C-terminal Src kinase (CSK) represses c-Src activity by phosphorylating Y-527. Budding yeast studies show CSK requires c-Src
Area of Science:
- Molecular Biology
- Cell Signaling
- Yeast Genetics
Background:
- c-Src kinase activity is regulated by phosphorylation at Y-527 in vertebrate cells.
- C-terminal Src kinase (CSK) is the likely enzyme responsible for Y-527 phosphorylation.
- Understanding CSK regulation of c-Src is crucial for cell signaling research.
Purpose of the Study:
- To investigate the role of CSK in regulating c-Src kinase activity in vivo.
- To determine the necessity of c-Src's SH2 and SH3 domains for CSK-mediated repression.
- To utilize Saccharomyces cerevisiae as a model system for studying c-Src regulation.
Main Methods:
- Expression of c-Src in Saccharomyces cerevisiae to induce kinase-dependent growth inhibition.
- Coexpression of CSK to observe its effect on c-Src activity and Y-527 phosphorylation.
- In vivo and in vitro analysis of c-Src mutants lacking SH2 or SH3 domains.
Main Results:
- Expression of c-Src in yeast caused growth inhibition, dependent on its kinase activity.
- Coexpression of CSK phosphorylated c-Src on Y-527 and suppressed the growth inhibition phenotype.
- CSK failed to fully suppress c-Src mutants lacking SH2 or SH3 domains, despite Y-527 phosphorylation.
Conclusions:
- CSK effectively represses c-Src kinase activity through Y-527 phosphorylation in a cellular context.
- Both the SH2 and SH3 domains of c-Src are essential for CSK-mediated repression of its activity.
- Budding yeast serves as a viable model for dissecting kinase regulation mechanisms.