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Tyrosine phosphorylation of G protein alpha subunits by pp60c-src
W P Hausdorff1, J A Pitcher, D K Luttrell
1Howard Hughes Medical Institute, Department of Medicine, Duke University Medical Center, Durham, NC 27710.
Abstract:
A number of lines of evidence suggest that cross-talk exists between the cellular signal transduction pathways involving tyrosine phosphorylation catalyzed by members of the pp60c-src kinase family and those mediated by guanine nucleotide regulatory proteins (G proteins). In this study, we explore the possibility that direct interactions between pp60c-src and G proteins may occur with functional consequences. Preparations of pp60c-src isolated by immunoprecipitation phosphorylate on tyrosine residues the purified G-protein alpha subunits (G alpha) of several heterotrimeric G proteins. Phosphorylation is highly dependent on G-protein conformation, and G alpha(GDP) uncomplexed by beta gamma subunits appears to be the preferred substrate. In functional studies, phosphorylation of stimulatory G alpha (G alpha s) modestly increases the rate of binding of guanosine 5'-[gamma-[35S]thio]triphosphate to Gs as well as the receptor-stimulated steady-state rate of GTP hydrolysis by Gs. Heterotrimeric G proteins may represent a previously unappreciated class of potential substrates for pp60c-src.
Insights
pp60c-src kinase directly phosphorylates guanine nucleotide regulatory proteins (G proteins). This interaction influences G protein function, suggesting G proteins are novel substrates for pp60c-src.
Area of Science:
- Cellular signaling
- Biochemistry
- Molecular biology
Background:
- Cross-talk between tyrosine phosphorylation (pp60c-src kinase family) and G protein pathways is suggested.
- Direct interactions between pp60c-src and G proteins with functional consequences are explored.
Purpose of the Study:
- To investigate direct interactions between pp60c-src and G proteins.
- To determine if G proteins are substrates for pp60c-src and assess functional consequences.
Main Methods:
- Immunoprecipitation of pp60c-src.
- In vitro phosphorylation assays using purified G-protein alpha subunits (G alpha).
- Functional assays measuring guanosine 5'-[gamma-[35S]thio]triphosphate binding and GTP hydrolysis.
Main Results:
- pp60c-src phosphorylates tyrosine residues on purified G alpha subunits of heterotrimeric G proteins.
- Phosphorylation is conformation-dependent, with G alpha(GDP) being a preferred substrate.
- Phosphorylation of stimulatory G alpha (G alpha s) enhances guanosine 5'-[gamma-[35S]thio]triphosphate binding and receptor-stimulated GTP hydrolysis.
Conclusions:
- Heterotrimeric G proteins are potential substrates for pp60c-src.
- Direct interaction and phosphorylation of G proteins by pp60c-src may represent a novel regulatory mechanism in cellular signaling.