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.
Shivakrupa
R
R
Centre for Cellular and Molecular Biology, Hyderabad 500 007, India.
Radha
Vegesna
V
Sudhakar
Ch
Ch
Swarup
Ghanshyam
G
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D019556:COS Cells; D020170:Caspase 1 / Q000378:metabolism; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D002460:Cell Line; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D005799:Genes, Dominant; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D020735:Guanine Nucleotide-Releasing Factor 2 / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008627:Mercuric Chloride / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051572:Proto-Oncogene Proteins c-hck; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism; D018909:src Homology Domains
D020735:Guanine Nucleotide-Releasing Factor 2; D011518:Proto-Oncogene Proteins; D014443:Tyrosine; D008627:Mercuric Chloride; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D020170:Caspase 1; D020827:rap1 GTP-Binding Proteins