Physical and functional interaction between Hck tyrosine kinase and guanine nucleotide exchange factor C3G results in apoptosis, which is independent of C3G catalytic domain.
The hematopoietic cell kinase Hck is a Src family tyrosine kinase expressed in cells of myelomonocytic lineage, B lymphocytes, and embryonic stem cells. To study its role in signaling pathways we used the Hck-SH3 domain in protein interaction cloning and identified C3G, the guanine nucleotide exchange factor for Rap1 and ... R-Ras, as a protein that associated with Hck. This interaction was direct and was mediated partly through the proline-rich region of C3G. C3G could be co-immunoprecipitated with Hck from Cos-1 cells transfected with Hck and C3G. C3G was phosphorylated on tyrosine 504 in cells when coexpressed with Hck but not with a catalytically inactive mutant of Hck. Phosphorylation of endogenous C3G at Tyr-504 was increased by treatment of human myelomonocytic THP-1 cells with mercuric chloride, which is known to activate Hck tyrosine kinase specifically. Coexpression of Hck with C3G induced a high level of apoptosis in many cell lines by 30-42 h of transfection. Induction of apoptosis was not dependent on Tyr-504 phosphorylation or the catalytic domain of C3G but required the catalytic activity of Hck. Using dominant negative constructs of caspases we found that caspase-1, -8, and -9 are involved in this apoptotic pathway. These results suggest that C3G and Hck interact physically and functionally in vivo to activate kinase-dependent and caspase-mediated apoptosis, which is independent of catalytic domain of C3G.
Mesh Terms:
Animals, Apoptosis, Blotting, Western, COS Cells, Caspase 1, Caspase 8, Caspase 9, Caspases, Catalysis, Catalytic Domain, Cell Line, Cell Line, Tumor, Electrophoresis, Polyacrylamide Gel, Genes, Dominant, Genetic Vectors, Glutathione Transferase, Guanine Nucleotide-Releasing Factor 2, Hela Cells, Humans, Mercuric Chloride, Microscopy, Fluorescence, Phosphorylation, Precipitin Tests, Protein Binding, Protein-Tyrosine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-hck, Signal Transduction, Time Factors, Transfection, Tyrosine, rap1 GTP-Binding Proteins, src Homology Domains
Animals, Apoptosis, Blotting, Western, COS Cells, Caspase 1, Caspase 8, Caspase 9, Caspases, Catalysis, Catalytic Domain, Cell Line, Cell Line, Tumor, Electrophoresis, Polyacrylamide Gel, Genes, Dominant, Genetic Vectors, Glutathione Transferase, Guanine Nucleotide-Releasing Factor 2, Hela Cells, Humans, Mercuric Chloride, Microscopy, Fluorescence, Phosphorylation, Precipitin Tests, Protein Binding, Protein-Tyrosine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-hck, Signal Transduction, Time Factors, Transfection, Tyrosine, rap1 GTP-Binding Proteins, src Homology Domains
J. Biol. Chem.
Date: Dec. 26, 2003
PubMed ID: 14551197
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