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.The Src family tyrosine kinases play an important role in linking signals received by transmembrane receptors and a variety of intracellular pathways, thereby regulating diverse cellular responses such as proliferation, differentiation, and cell death (1, 2). This is achieved through their non-catalytic sequences, which enable multiple interactions with cellular proteins, and through the kinase domain, which phosphorylates substrates to alter their activity, change their subcellular location, and effect their intermolecular interactions. The hematopoietic cell kinase (Hck) 1 is a Src family member that is expressed in cells of myelomonocytic lineage, B lymphocytes, and embryonic stem cells with higher levels in differentiated cells, suggesting a role for this enzyme in signaling pathways of mature hematopoietic cells (3-5). Hck is activated by agents that induce macrophage differentiation and in response to cytokines such as interleukin-3, granulocyte-macrophage colony stimulating factor, and leukemia inhibitory factor. It is also involved in cytokine production in macrophages in response to lipopolysaccharide and viral infection (6 -9).Structurally, Hck is similar to other members of the Src family in that it has a catalytic domain at the C terminus that is preceded by a 100-amino acid SH2 domain and a 50-amino acid SH3 domain. The SH2 and SH3 domains are protein interaction modules that mediate either intramolecular or intermolecular associations. SH2 domains bind to phosphorylated tyrosine residues in a specific amino acid context, whereas SH3 domain interacts with polyproline tracts in polypept...