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Regulation of stem cell factor receptor signaling by Cbl family proteins (Cbl-b/c-Cbl)

Shan Zeng,

Zhiheng Xu,

Stan Lipkowitz, and

Jack B. Longley





From the Departments of Dermatology and Pathology, College of Physicians and Surgeons, Columbia University, New York, NY; and Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD.







Abstract

Activation of the KIT receptor tyrosine kinase contributes to the pathogenesis of several human diseases, but the mechanisms regulating KIT signaling have not been fully characterized. Here, we show that stem cell factor (SCF), the ligand for KIT, in...



Introduction

KIT, the stem cell factor (SCF) receptor, is a receptor protein tyrosine kinase (RTK) that is primarily expressed on mast cells, hematopoietic stem cells, germ cells, melanocytes, and the interstitial cells of Cajal. 1, 2 Studies in white-spotting an...



Ubiquitination and subsequent degradation of proteins has been implicated as a key mechanism regulating duration and intensity of many intracellular signals, and it has been suggested that activated KIT is degraded through the polyubiquitination-depe...



Cbl family members are newly established as components of the ubiquitin ligation machinery involved in the degradation of phosphorylated proteins. 8 In vitro assays have shown that Cbl proteins can function as E3 ubiquitin (Ub) ligases, and Cbl prote...



We hypothesized that Cbl proteins may mediate the ubiquitination and degradation of activated KIT. In this study, we provide evidence that SCF stimulation induces KIT binding and phosphorylation of Cbl proteins. Furthermore, by using Cbl-b or c-Cbl a...



Materials and methods

Materials 



Antibodies against KIT (C-19), c-Cbl (C-20), and Cbl-b (G-1) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antihemagglutinin (anti-HA) and anti-myc antisera were purchased from Clontech (Palo Alto, CA). Antiphosphotyrosine (4G10) and ...



cDNA constructs in mammalian expression vectors 



All cDNA constructs of Cbl-b or c-Cbl in expression vector (pCEFL) have been described previously. 11 The full length of cDNAs of human wild-type c-KIT in pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA) has been previously described. 17 Substit...



Cell culture 



MC9 murine mast cells were kind gifts from Dr E. W. Gelfand (National Jewish Medical Center, Denver, CO) and were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 50 µM 2-mercapto-ethanol, 0.1 mM nonessential amino acids, 1.0 mM so...



Cell stimulation and transient transfection 



The 293 cells were transfected with various constructs by calcium phosphate method as described. 18 After 20 hours, transfected cells were subjected to stimulation. For SCF stimulation, cells were starved with 0.5% FCS for 4 to 6 hours, then stimulat...



Coimmunoprecipitation and Western immunoblotting 



Coimmunoprecipitatation and Western immunoblotting were performed as previously described. 18 



Results

Both Cbl-b and c-Cbl can induce ubiquitination and degradation of KIT