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

Abstract

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 ␤-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 ␤-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.The mechanisms mediating hematopoiesis are not fully understood but require interactions between a wide array of cell surface receptors with growth factors and cytokines. Two receptor families important for hematopoiesis are the cytokine receptor superfamily and the receptor tyrosine kinase superfamily. Ligands for the cytokine receptors include granulocyte macrophage colony-stimulating factor (GM-CSF), 1 interleukin-3 (IL-3), and erythropoietin (Epo). The receptor for human GM-CSF is comprised of a GM-CSF-specific ␣-chain and a ␤-chain shared with the receptors for IL-3 and IL-5 (1, 2). Upon GM-CSF stimulation the ␣-and ␤-chains cluster together, bringing associated cytoplasmic tyrosine kinases close together resulting in activation and subsequent tyrosine phosphorylation of the ␤-chain. Stem cell factor (SCF) binds to the receptor tyrosine kinase c-Kit leading to its homodimerization and autophosphorylation (3). Phosphorylated tyrosine residues, in cKit and the ␤-chain, function as docking sites for signal transduction molecules with SH2 domains. The SH2 domain is a stretch of ϳ100 amino acids that binds phosphorylated tyrosine residues in an amino acid sequence context-dependent manner (4).Previous studies have suggested that c-Kit may play a role in responses mediated by IL-3 ...

Complex interactions in EML cell stimulation by stem cell factor and IL-3

“…IgG(5ug/ml) Ab-1(5ug/ml) IL-3(2ng/ml) SCF(50ng/ml) The receptor c-kit has been known for some time to interact with certain other tyrosine kinase receptors including EpoR, GM-CSF receptor (GM-CSFR), and IL-7 receptor, and this physical interaction may be associated with cross-phosphorylation of c-kit by erythropoietin (EPO) or other cytokines (24)(25)(26)(27). The physiologic significance of this cross-phosphorylation has been questioned previously.…”

“…EML cells were deprived of SCF and horse serum for 4 h, and 293T cells were deprived of serum for 6 h. This deprivation period was followed by the addition of SCF (50 ng/mL) or IL-3 (4 ng/mL) or the combination of SCF and IL-3. The cells were induced for the indicated times and then were collected and washed with PBS buffer and lysed at 1 × 10 7 cells/mL with ice-cold lysis buffer A (lysis buffer for total cell protein analysis) or lysed at 1 × 10 7 cells/mL with ice-cold lysis buffer B (lysis buffer for immunoprecipitation) as described previously (24). For immunoprecipitation, the lysates were cleared by centrifugation before antibody application.…”

Testis tissue explantation cures spermatogenic failure in c-Kit ligand mutant mice

“…Previous studies reported that KITL has synergistic effects with other cytokines. The effect of KITL on hematopoietic cell lineages to promote their proliferation, differentiation, or survival can be augmented synergistically by combination with other cytokines such as colony stimulating factor-1, -2, or -3 (CSF-1, CSF-2, CSF-3), interleukin-3, and erythropoietin (EPO) (13)(14)(15)(16)(17). We added those cytokines to the medium to elucidate their effects on spermatogenesis.…”

KIT associated intracellular tyrosines play an essential role in EpoR co-signaling

“…To this end, recent studies have suggested that the physical interaction between KIT and other cytokine receptors could contribute to the cooperation by bringing the two receptors in physical proximity, thereby inducing receptor trans-phosphorylation and activation of downstream substrates. Although, this scenario has been studied to some extent utilizing the KIT/EpoR system, in other receptor systems, including the GM-CSF/SCF system, no evidence for trans-phosphorylation of KIT or the GM-CSF receptor has been reported, however studies have shown the formation of a complex between KIT and the GM-CSF receptor does take place in this system [30]. Thus, the precise role of intracellular tyrosines in KIT mediated co-signaling with cytokine receptors is still poorly understood.…”

“…To this end, combined stimulation of cells with SCF and GM-CSF results in sustained activation of Mek1/2 and Erk1/2 compared to either cytokine alone [30]. Sustained activation of Erk1/2 is PI-3Kinase dependent in both the SCF/GM-CSF system as well as the SCF/Epo system [29,30].…”