Stem cell factor induces phosphatidylinositol 3'-kinase-dependent Lyn/Tec/Dok-1 complex formation in hematopoietic cells

Stem cell factor (SCF) has an important role in the proliferation, differentiation, survival, and migration of hematopoietic cells. SCF exerts its effects by binding to cKit, a receptor with intrinsic tyrosine kinase activity. Activation of phosphatidylinositol 3'-kinase (PI3-K) by cKit was previously shown to contribute to many SCF-induced cellular responses. Therefore, PI3-K-dependent signaling pathways activated by SCF were investigated. The PI3-K-dependent activation and phosphorylation of the tyrosine kinase Tec and the adapter molecule p62Dok-1 are reported. The study shows that Tec and Dok-1 form a stable complex with Lyn and 2 unidentified phosphoproteins of 56 and 140 kd. Both the Tec homology and the SH2 domain of Tec were identified as being required for the interaction with Dok-1, whereas 2 domains in Dok-1 appeared to mediate the association with Tec. In addition, Tec and Lyn were shown to phosphorylate Dok-1, whereas phosphorylated Dok-1 was demonstrated to bind to the SH2 domains of several signaling molecules activated by SCF, including Abl, CrkL, SHIP, and PLCgamma-1, but not those of Vav and Shc. These findings suggest that p62Dok-1 may function as an important scaffold molecule in cKit-mediated signaling

Tyrosine kinase receptor RON functions downstream of the erythropoietin

Erythropoietin (EPO) is required for cell survival during differentiation and for progenitor expansion during stress erythropoiesis. Although signaling pathways may couple directly to docking sites on the EPO receptor (EpoR), additional docking molecules expand the signaling platform of the receptor. We studied the roles of the docking molecules Grb2-associated binder-1 (Gab1) and Gab2 in EPO-induced signal transduction and erythropoiesis. Inhibitors of phosphatidylinositide 3-kinase and Src kinases suppressed EPO-dependent phosphorylation of Gab2. In contrast, Gab1 activation depends on recruitment and phosphorylation by the tyrosine kinase receptor RON, with which it is constitutively associated. RON activation induces the phosphorylation of Gab1, mitogen-activated protein kinase (MAPK), and protein kinase B (PKB) but not of signal transducer and activator of transcription 5 (Stat5). RON activation was sufficient to replace EPO in progenitor expansion but not in differentiation. In conclusion, we elucidated a novel mechanism specifically involved in the expansion of erythroblasts involving RON as a downstream target of the Epo

Protein kinase C alpha controls erythropoietin receptor signaling.

Protein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We analyzed the effect of PKC inhibitors with distinct modes of action on EpoR signaling in primary human erythroblasts and in a recently established murine erythroid cell line. Active PKC appeared essential for Epo-induced phosphorylation of the Epo receptor itself, STAT5, Gab1, Erk1/2, AKT, and other downstream targets. Under the same conditions, stem cell factor-induced signal transduction was not impaired. LY294002, a specific inhibitor of phosphoinositol 3-kinase, also suppressed Epo-induced signal transduction, which could be partially relieved by activators of PKC. PKC inhibitors or LY294002 did not affect membrane expression of the EpoR, the association of JAK2 with the EpoR, or the in vitro kinase activity of JAK2. The data suggest that PKC controls EpoR signaling instead of being a downstream effector. PKC and phosphoinositol 3-kinase may act in concert to regulate association of the EpoR complex such that it is responsive to ligand stimulation. Reduced PKC-activity inhibited Epo-dependent differentiation, although it did not effect Epo-dependent "renewal divisions" induced in the presence of Epo, stem cell factor, and dexamethasone