Critical roles of c-Kit tyrosine residues 567 and 719 in stem cell factor–induced chemotaxis: contribution of src family kinase and PI3-kinase on calcium mobilization and cell migration

Abstract

Introduction c-Kit is a receptor tyrosine kinase (RTK), which constitutes a type III RTK subfamily with the receptors for platelet-derived growth factor (PDGF), colony-stimulating factor 1 (CSF-1), and flt-3 ligand. 1,2 The type III RTKs are characterized by an extracellular domain with 5 immunoglobulinlike domains and a cytoplasmic domain consisting of a kinase domain that is interrupted by a kinase insert. c-Kit (KIT) and its ligand stem cell factor (SCF) play an important role in hematopoiesis, melanogenesis, and gametogenesis, 3 as has been clearly shown by loss of function mutations of c-kit gene. In addition, c-kit gene product has been associated with various forms of neoplasms. Activating mutants of KIT, either in the juxtamembrane domain or the catalytic domain, were identified as the cause for transformation of hematopoietic stem cells, mast cells, and gastrointestinal stromal cells. [4][5][6][7][8][9][10] Thus, KIT/SCF has pleiotropic functions such as proliferation, survival, differentiation, and transformation. In this report, we focus on SCF/KITmediated cell migration, which is also a characteristic function of SCF in hematopoietic stem cells and mast cells, [11][12][13] and has critical roles in immunity, metastasis, and development.On ligand stimulation, KIT receptors dimerize, activate its intrinsic tyrosine kinase, and autophosphorylate. The phosphorylated KIT receptor generates binding sites for SH2 domaincontaining proteins, which include proteins of the p21Ras-mitogenactivated protein kinase (MAPK) pathway, 14 the p85 subunit of phosphatidylinositol 3Ј kinase (PI3K), 15 phospholipase C-␥ 1 , the Grb2 adaptor protein, 16 the Src family kinases (SFKs), 17 Cbl, CRKL, 19 SHP1, and SHP2. 20 Those proteins are subsequently activated or phosphorylated and further transduce signaling cascades that lead to various cellular responses. However, little is known about which signaling is essential for SCF-mediated migration. Recently, a few reports indicated that Lyn or p38 MAPK plays an important role, 21,22 but no comprehensive investigation has been done in which the tyrosine residue of KIT is involved in signal transduction, which is required for cell migration. In this study, we have converted all possible tyrosine (Y) residues on KIT cytoplasmic domain to phenylalanine (F) and introduced these YF substitute mutants on 293T cells or murine interleukin 3 (IL-3)-dependent BAF3 cells. We used these cell lines to elucidate signaling cascades that are important for SCF-mediated cell Supported in part by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology, the Japanese Ministry of Health, Labor and Welfare, and the Japan Society for Promotion of Science.Reprints: Yuzuru Kanakura, Department of Hematology and Oncology, Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan; e-mail: kanakura@bldon.med.osaka-u.ac.jp.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate thi...

Suppressor of Cytokine Signaling 6 Associates with KIT and Regulates KIT Receptor Signaling

“…The MAPK pathways ERK1/2 and p38 were in part activated through Tyr-567 in the juxtamembrane membrane region of KIT (37). SRC and SHP2, two putative Tyr-567 interactors, are likely to be upstream activators of MAPKs in KIT signaling (37,43).…”

“…SCF induced chemotaxis of hematopoietic cells (37). We have examined the effect of ectopic expression SOCS6 on cell migration using an in vitro two-chamber assay.…”

The interplay between Src family kinases and receptor tyrosine kinases

“…For example, SFK association with SCF-R is required for migration in response to ligand (Ueda et al, 2002;Hong et al, 2004), and PP2 inhibits VEGF-induced chemotaxis and migration (Abu-Ghazaleh et al, 2001). The adhesive and motile responses promoted by members of the family of Eph receptors are also mediated by SFKs (Bruckner and Klein, 1998;Palmer et al, 2002;Vindis et al, 2003).…”

Impaired Long-Term Expansion and Self-Renewal Potential of Pediatric Acute Myeloid Leukemia–Initiating Cells By PTK787/ZK 222584

“…Interestingly, pAKT was downregulated in all tested pediatric AML samples. It is known that AKT is a downstream target of multiple tyrosine kinase receptors, including VEGF receptors, c-KIT, c-FMS, and PDGFRb (22)(23)(24). Therefore, pAKT might be the main downstream effect of PTK/ZK.…”

“…The downstream effects of VEGF are mainly executed by VEGFR2 binding, resulting in increased AML cell survival and proliferation via downstream signaling pathways such as via mitogen-activated protein kinase (MAPK) or the phosphatidylinositol 3-kinase (PI3K) pathway (20,21). However, these downstream signaling pathways can also be activated by various other RTKs including c-KIT and PDGFR-b (22)(23)(24).…”