Synergistic growth of stem cell factor and granulocyte macrophage colony-stimulating factor involves kinase-dependent and -independent contributions from c-Kit

Stem cell factor (SCF) binds and activates the receptor tyrosine kinase c-Kit, and this interaction is critical for normal hematopoiesis. SCF also synergizes with a variety of growth factors, including those binding members of the cytokine receptor superfamily. The mechanisms mediating this synergy remain to be defined. The present study investigates both structural and biochemical cross-talk between c-Kit and the receptor for granulocyte macrophage colony-stimulating factor (GM-CSF). We have found that c-Kit forms a complex with the beta-chain of the GM-CSF receptor, and this interaction involves the first part of the c-Kit kinase domain. Although inhibition of c-Kit kinase activity completely blocked SCF-induced proliferation, there was still greater than additive growth induced by SCF in combination with GM-CSF. In contrast, an inhibitory antibody against the extracellular domain of c-Kit (K-27) completely inhibited growth in response to SCF alone or in combination with GM-CSF. These results support a kinase-independent component of the synergistic growth induced by SCF and GM-CSF that may relate to interaction of these receptors. It is also clear that a significant part of the synergistic growth is dependent of c-Kit kinase activity. Although synergistic increases in phosphorylation of c-Kit and the beta-chain of the GM-CSF receptor were not observed, SCF and GM-CSF in combination prolonged the duration of Erk1/2 phosphorylation in a phosphatidylinositol 3-kinase-dependent manner. Consistent with these findings, phosphatidylinositol 3-kinase is synergistically activated by SCF and GM-CSF together. Hence, c-Kit makes both kinase-independent and -dependent contributions to the proliferative synergy induced by SCF in combination with GM-CSF.     

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.     

Phosphatidylinositol 3-kinase and Src family kinases are required for phosphorylation and membrane recruitment of Dok-1 in c-Kit signaling

Dok-1 is an adaptor protein that is a substrate for Bcr-Abl and other tyrosine protein kinases. The presence of pleckstrin homology and phosphotyrosine binding domains as well as multiple tyrosine phosphorylation sites suggests that Dok-1 is involved in protein-protein and/or protein-lipid interactions. Here we show that stimulation of Mo7 hematopoietic cells with c-Kit ligand (KL) induces phosphatidylinositol (PI) 3-kinase-dependent tyrosine phosphorylation and membrane recruitment of Dok-1. Addition of the K-Ras membrane-targeting motif to Dok-1 generated a constitutively membrane-bound Dok-1 protein whose tyrosine phosphorylation was independent of PI 3-kinase. Membrane localization of Dok-1 was required for its ability to function as a negative regulator of cell proliferation. Additional experiments revealed that Dok-1 associated with the juxtamembrane region and C-terminal tail of c-Kit. Lyn promoted phosphorylation of c-Kit and association of c-Kit and Dok-1. Both Lyn and Tec were capable of phosphorylating Dok-1. However, the use of primary bone marrow mast cells from normal and Lyn-deficient mice demonstrated that Lyn is required for KL-dependent Dok-1 tyrosine phosphorylation. Taken together, these data indicate that activation of PI 3-kinase by KL promotes binding of the Dok pleckstrin homology domain and Dok-1 recruitment to the plasma membrane where Dok-1 is phosphorylated by Src and/or Tec family kinases.     

Lyn contributes to regulation of multiple Kit-dependent signaling pathways in murine bone marrow mast cells

SCF induces autophosphorylation of Kit and activates a variety of signaling components including Jnks, Erks, PI 3 Kinase, the JAK-Stat pathway and members of the Src family. Previously we showed that Lyn is activated at multiple points during SCF-induced cell cycle progression and contributes to SCF-mediated growth, chemotaxis and internalization of Kit. However, the Kit-dependent biochemical events that require Lyn are unknown. In this study, we used Lyn-deficient bone marrow mast cells (BMMC) to examine the contribution of this Src family member to tyrosine phosphorylation of Kit and SCF-induced activation of Jnks, Akt, Stat3 and Erks. Although surface expression of Kit was increased in Lyn-deficient BMMC, SCF-induced phosphorylation and growth was reduced compared to wild-type BMMC. Downstream of Kit, SCF-induced activation of Jnks was markedly reduced in Lyn-deficient BMMC. Further, Lyn was required for SCF-induced tyrosine phosphorylation of Stat3. Interestingly, Kit was constitutively associated with PI 3 Kinase in Lyn-deficient BMMC and this correlated with constitutive phosphorylation of Akt. This was in marked contrast to wild-type BMMC, where both these events were induced by SCF. These data indicate that in BMMC, Lyn contributes to SCF-induced phosphorylation of Kit, as well as phosphorylation of Jnks and Stat3. In contrast, Lyn may negatively regulate the PI 3 Kinase/Akt pathway. The opposing effects of Lyn on these signaling pathways may explain the pleiotropic effects ascribed to this Src family member in the literature.