Kozlowski M, Larose L, Lee F, Le DM, Rottapel R, Siminovitch KASHP-1 binds and negatively modulates the c-Kit receptor by interaction with tyrosine 569 in the c-Kit juxtamembrane domain. Mol Cell Biol 18:2089-2099

Health Canada Life Sciences and the University of Ottawa, Canada.
Molecular and Cellular Biology (Impact Factor: 4.78). 04/1998; 18(4):2089-99. DOI: 10.1128/MCB.18.4.2089
Source: PubMed


The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad spectrum of growth factor- and cytokine-driven mitogenic signaling pathways. Included among these is the cascade of intracellular events evoked by stem cell factor binding to c-Kit, a tyrosine kinase receptor which associates with and is dephosphorylated by SHP-1. Using a series of glutathione
-transferase (GST) fusion proteins containing either tyrosine-phosphorylated segments of the c-Kit cytosolic region or the SH2 domains of SHP-1, we have shown that SHP-1 interacts with c-Kit by binding selectively to the phosphorylated c-Kit juxtamembrane region and that the association of c-Kit with the larger of the two SHP-1 isoforms may be mediated through either the N-terminal or C-terminal SHP-1 SH2 domain. The results of binding assays with mutagenized GST-Kit juxtamembrane fusion proteins and competitive inhibition assays with phosphopeptides encompassing each c-Kit juxtamembrane region identified the tyrosine residue at position 569 as the major site for binding of SHP-1 to c-Kit and suggested that tyrosine 567 contributes to, but is not required for, this interaction. By analysis of Ba/F3 cells retrovirally transduced to express c-Kit receptors, phenylalanine substitution of c-Kit tyrosine residue 569 was shown to be associated with disruption of c-Kit–SHP-1 binding and induction of hyperproliferative responses to stem cell factor. Although phenylalanine substitution of c-Kit tyrosine residue 567 in the Ba/F3–c-Kit cells did not alter SHP-1 binding to c-Kit, the capacity of a second c-Kit-binding tyrosine phosphatase, SHP-2, to associate with c-Kit was markedly reduced, and the cells again showed hyperproliferative responses to stem cell factor. These data therefore identify SHP-1 binding to tyrosine 569 on c-Kit as an interaction pivotal to SHP-1 inhibitory effects on c-Kit signaling, but they indicate as well that cytosolic protein tyrosine phosphatases other than SHP-1 may also negatively regulate the coupling of c-Kit engagement to proliferation.

Download full-text


Available from: Louise Larose, Jan 05, 2015
  • Source
    • "Moreover, there are several known substrates of SHP-1 in different cell types, notably in hematopoietic cells [32,46-48]. For example, JAK2 kinase and STAT5 in erythropoietic cells [46], c-KIT kinase in hematopoietic cells [47] and nerve growth factor receptor TrkA in neuron cells [48]. However, there is limited data showing SHP-1 substrates other than p-STAT3 in breast cancer cells. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Signal transducers and activators of transcription 3 (STAT3) signaling is constitutively activated in various cancers including breast cancer, and has emerged as a novel potential anti-cancer target. STAT3 has been demonstrated to be a target of sorafenib, and a protein tyrosine phosphatase Src homology 2-domain containing tyrosine phosphatase 1 (SHP-1) has been demonstrated to downregulate p-STAT3 via its phosphatase activity. Here, we tested the efficacy of two sorafenib analogues, SC-1 and SC-43, in breast cancer cells and examined the drug mechanism. Breast cancer cell lines were used for in vitro studies. Cell viability was examined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was examined by flow cytometry and western blot. Signal transduction pathways in cells were assessed by western blot. In vivo efficacy of sorafenib, SC-1 and SC-43 were tested in xenografted nude mice. SC-1 and SC-43 induced more potent apoptosis than sorafenib, in association with downregulation of p-STAT3 and its downstream proteins cyclin D1 and survivin in a dose-dependent manner in breast cancer cell lines (HCC-1937, MDA-MB-468, MDA-MB-231, MDA-MB-453, SK-BR3, MCF-7). Overexpression of STAT3 in MDA-MB-468 cells protected cells from apoptosis induced by sorafenib, SC-1, and SC-43. Moreover, SC-1 and SC-43 upregulated SHP-1 activity to a greater extent than sorafenib as measured by in vitro phosphatase assays. Knockdown of SHP-1 by siRNA reduced apoptosis induced by SC-1 and SC-43. Importantly, SC-1 and SC-43 showed more efficacious antitumor activity and p-STAT3 downregulation than sorafenib in MDA-MB-468 xenograft tumors. Novel sorafenib analogues SC-1 and SC-43 induce apoptosis through SHP-1 dependent STAT3 inactivation and demonstrate greater potency than sorafenib in human breast cancer cells.
    Full-text · Article · Aug 2013 · Breast cancer research: BCR
  • Source
    • "SHP2 is known mainly as a positive regulator of the Ras-Erk pathway. Another protein tyrosine phosphatase, SHP1, binds to Tyr570 [57]. Thus a number of positive as well as negative signals emanate from the juxtamembrane region. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The development of melanocytes is regulated by the tyrosine kinase receptor c-KIT and the basic-helix-loop-helix-leucine zipper transcription factor Mitf. These essential melanocyte survival regulators are also well known oncogenic factors in malignant melanoma. Despite their importance, not much is known about the regulatory mechanisms and signaling pathways involved. In this study, we therefore sought to identify the signaling pathways and mechanisms involved in c-KIT mediated regulation of Mitf. We report that c-KIT stimulation leads to the activation of Mitf specifically through the c-KIT phosphorylation sites Y721 (PI3 kinase binding site), Y568 and Y570 (Src binding site). Our study not only confirms the involvement of Ras-Erk signaling pathway in the activation of Mitf, but also establishes that Src kinase binding to Y568 and Y570 of c-KIT is required. Using specific inhibitors we observe and verify that c-KIT induced activation of Mitf is dependent on PI3-, Akt-, Src-, p38- or Mek kinases. Moreover, the proliferative effect of c-KIT is dependent on Mitf in HEK293T cells. In contrast, c-KIT Y568F and Y721F mutants are less effective in driving cell proliferation, compared to wild type c-KIT. Our results reveal novel mechanisms by which c-KIT signaling regulates Mitf, with implications for understanding both melanocyte development and melanoma.
    Full-text · Article · Aug 2011 · PLoS ONE
  • Source
    • "The KIT gene encodes a 145 kDa transmembrane protein that is a member of the type III receptor tyrosine kinase (RTK) family (Yarden et al., 1987), regulates cell survival, proliferation or differentiation (Schlessinger, 2000), and participates in normal mechanisms of hematopoiesis, melanogenesis, and gametogenesis. KIT protein expression is modulated by a variety of mechanisms including microRNAs (miRNAs) (Felli et al., 2005) and/or proteolytic degradation (Masson et al., 2006) and is subjected to covalent posttranslational modifications, which influence its tyrosine kinase activity through interaction with a variety of factors including KIT ligand (also known as stem cell factor), tyrosine phosphatases (Kozlowski et al., 1998), protein kinase C, and calcium ionophores (Miyazawa et al., 1994; Yee et al., 1993). KIT is overexpressed and/or mutated in several human neoplasms, including gastrointestinal stromal tumors (GISTs), germ cell tumors, and hematologic malignancies (Ikeda et al., 1991). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The biologic and clinical significance of KIT overexpression that associates with KIT gain-of-function mutations occurring in subsets of acute myeloid leukemia (AML) (i.e., core binding factor AML) is unknown. Here, we show that KIT mutations lead to MYC-dependent miR-29b repression and increased levels of the miR-29b target Sp1 in KIT-driven leukemia. Sp1 enhances its own expression by participating in a NFkappaB/HDAC complex that further represses miR-29b transcription. Upregulated Sp1 then binds NFkappaB and transactivates KIT. Therefore, activated KIT ultimately induces its own transcription. Our results provide evidence that the mechanisms of Sp1/NFkappaB/HDAC/miR-29b-dependent KIT overexpression contribute to leukemia growth and can be successfully targeted by pharmacological disruption of the Sp1/NFkappaB/HDAC complex or synthetic miR-29b treatment in KIT-driven AML.
    Full-text · Article · Apr 2010 · Cancer cell
Show more