Functional and Biochemical Consequences of Abrogating the Activation of Multiple Diverse Early Signaling Pathways in Kit

Abstract

Kit receptor tyrosine kinase and erythropoietin receptor (Epo-R) cooperate in regulating blood cell development. Mice that lack the expression of Kit or Epo-R die in utero of severe anemia. Stimulation of Kit by its ligand, stem cell factor activates several distinct early signaling pathways, including phospholipase C␥, phosphatidylinositol 3-kinase, Src kinase, Grb2, and Grb7. The role of these pathways in Kit-induced growth, proliferation, or cooperation with Epo-R is not known. We demonstrate that inactivation of any one of these early signaling pathways in Kit significantly impairs growth and proliferation. However, inactivation of the Src pathway demonstrated the most profound defect. Combined stimulation with Epo also resulted in impaired cooperation between Src-defective Kit mutant and Epo-R and, to a lesser extent, with Kit mutants defective in the activation of phosphatidylinositol 3-kinase or Grb2. The impaired cooperation between the Src-defective Kit mutant and Epo-R was associated with reduced transphosphorylation of Epo-R and expression of c-Myc. Remarkably, restoration of only the Src pathway in a Kit receptor defective in the activation of all early signaling pathways demonstrated a 50% correction in proliferation in response to Kit stimulation and completely restored the cooperation with Epo-R. These data demonstrate an essential role for Src pathway in regulating growth, proliferation, and cooperation with Epo-R downstream from Kit. Receptor tyrosine kinases (RTKs)1 trigger multitude of cellular events, including proliferation, survival, differentiation, and migration. In response to ligand-induced stimulation, RTKs undergo dimerization and autophosphorylation on several distinct cytoplasmic tyrosine residues (1-4). These phosphorylated tyrosine residues become binding sites for a variety of Src homology 2 domain-containing enzymes and adaptor proteins such as phospholipase C␥ (PLC-␥), phosphatidylinositol 3-kinase p85 subunit (PI 3-kinase), Ras GTPase-activating protein, SHP2 phosphatase, Src kinases, Grb2, Grb7, and Shc (5). In this manner, the phosphorylated tyrosine residues initiate signal transduction via several distinct early signaling pathways. A major unresolved question in the field of RTK signaling is whether these diverse signaling pathways result in redundant or nonredundant biological functions. Recent studies utilizing the platelet-derived growth factor (PDGF) RTK have begun to address some of these issues in nonhematopoietic cells (6 -8). However, relatively little is known about the biological consequence(s) of activation of diverse signaling pathways by RTKs in hematopoietic cells.In hematopoietic cells, the RTK Kit plays an essential role in regulating proliferation, survival, differentiation, and migration of stem and progenitor cells (9 -11). The proto-oncogene Kit encodes the receptor for stem cell factor (SCF) and belongs to the type III receptor tyrosine kinase subfamily (9 -11). This family of cytokine receptor includes the macrophage colonystimulating factor (M-CSF) r...

Synergistic Growth of Stem Cell Factor and Granulocyte Macrophage Colony-stimulating Factor Involves Kinase-dependent and -independent Contributions from c-Kit

“…Further, Src family kinases may play a role in this process (37). In contrast, we find no evidence for trans-phosphorylation between c-Kit and the GM-CSF receptor (Figs.…”

Stem Cell Factor Synergistically Enhances Thrombopoietin‐Induced STAT5 Signaling in Megakaryocyte Progenitors through JAK2 and Src Kinase

“…A physical association between c-kit and Epo-R has been reported, and stimulation of cells with SCF has been shown to transphosphorylate Epo-R [21][22][23]. It has recently been demonstrated that SCF-induced activation of the Src kinase pathway plays an essential role in the synergistic action with Epo [20]. Furthermore, Kapur and Zang [24] have shown that SCF and Epo can synergistically regulate erythroid progenitor survival by a mechanism in which SCF maintains protein expression of the Epo-R, STAT5, and Bcl-X L .…”

“…As a result, downstream signaling cascades are initiated, including the signal transducer and activator of transcription (STAT) pathway, the phosphatidyl inositol 3-kinase (PI3K) pathway, and the extracellular-regulated kinase (ERK) pathway [15][16][17][18]. The receptor for SCF, c-kit, is a member of the tyrosine kinase family of receptors and undergoes autophosphorylation upon binding SCF, resulting in activation of multiple signaling proteins such as PI3K, Src kinases, Shc, Grb2, Grb7, and Ras [19,20].…”

Kit transduced signals counteract erythroid maturation by MAPK-dependent modulation of erythropoietin signaling and apoptosis induction in mouse fetal liver

“…[5][6][7] However, Kit has been attributed a primary role in proliferation, 8,9 whereas EpoR has its main role in mediating differentiation and survival. …”

“…3,4 The roles of Kit and EpoR in erythropoiesis are partially overlapping and signal integration after co-stimulation results in proliferative synergy and enhanced survival. [5][6][7] However, Kit has been attributed a primary role in proliferation, 8,9 whereas EpoR has its main role in mediating differentiation and survival. 10 In terms of intracellular signaling, stimulation of either Kit or EpoR can activate the phosphoinositide 3-kinase (PI3K) and the mitogen-activated protein kinase (MAPK) pathways.…”