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. 1996 Dec 10;93(25):14665-9.
doi: 10.1073/pnas.93.25.14665.

Oncogenic Mutation in the Kit Receptor Tyrosine Kinase Alters Substrate Specificity and Induces Degradation of the Protein Tyrosine Phosphatase SHP-1

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Free PMC article

Oncogenic Mutation in the Kit Receptor Tyrosine Kinase Alters Substrate Specificity and Induces Degradation of the Protein Tyrosine Phosphatase SHP-1

X Piao et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Activating mutations in the Kit receptor tyrosine kinase have been identified in both rodent and human mast cell leukemia. One activating Kit mutation substitutes a valine for aspartic acid at codon 816 (D816V) and is frequently observed in human mastocytosis. Mutation at the equivalent position in the murine c-kit gene, involving a substitution of tyrosine for aspartic acid (D814Y), has been described in the mouse mastocytoma cell line P815. We have investigated the mechanism of oncogenic activation by this mutation. Expression of this mutant Kit receptor tyrosine kinase in a mast cell line led to the selective tyrosine phosphorylation of a 130-kDa protein and the degradation, through the ubiquitin-dependent proteolytic pathway, of a 65-kDa phosphoprotein. The 65-kDa protein was identified as the src homology domain 2 (SH2)-containing protein tyrosine phosphatase SHP-1, a negative regulator of signaling by Kit and other hematopoietic receptors, and the protein product of the murine motheaten locus. This mutation also altered the sites of receptor autophosphorylation and peptide substrate selectivity. Thus, this mutation activates the oncogenic potential of Kit by a novel mechanism involving an alteration in Kit substrate recognition and the degradation of SHP-1, an attenuator of the Kit signaling pathway.

Figures

Figure 1
Figure 1
Reduced SHP-1 protein in mast cells expressing the mutant KDY. (A) Anti-phosphotyrosine blots of whole cell lysates obtained from control and Steel-factor-stimulated mast cells, IC2/Kit, and IC2/KDY cells. The small arrows indicate the positions of the mature (upper arrow) and immature (lower arrow) forms of p145Kit, the open arrow indicates the phosphorylated p130 protein, and the large solid arrow indicates the 65-kDa protein. (B) Detection of SHP-1 protein in cells expressing Kit or KDY. (Left) Immunoblot of SHP-1 with C-19 antibody to SHP-1 on total cell lysates. (Right) SHP-1 was immunoprecipitated with the C-19 anti-SHP-1 antibodies from control and Steel-factor-stimulated IC2/Kit and IC2/KDY cells. Western blots of the immunoprecipitates were probed with anti-SHP-1 antiserum directed against the SH2 domain of SHP-1 (17). (C) Expression of SHP-1 transcripts in cells expressing Kit or KDY. Total cellular RNA was separated by gel electrophoresis, and the blot was hybridized to cDNA probe for SHP-1 and mouse ribosomal protein L32.
Figure 2
Figure 2
(A) Rapid turnover of SHP-1 in cells expressing KDY. After a 10-min pulse with [35S]methionine, 35S-labeled SHP-1 was chased for 15 min. The vertical bar indicates the multiple protein bands larger than 65 kDa immunoprecipitated by anti-SHP-1 antibody C-19. (B) Ubiquitination of SHP-1 in cells expressing KDY. SHP-1 immunoprecipitates from control and Steel-factor-stimulated IC2/Kit and IC2/KDY cells were examined by immunoblot analysis with an antibody to Ub.
Figure 3
Figure 3
Tryptic phosphopeptide mapping of the Kit and KDY receptors. 32P-labeled Kit and KDY receptors isolated from control and Steel-factor-stimulated cells were digested with trypsin and peptides were resolved by separation in two dimensions on TLC plates. The results are summarized in a schematic phosphopeptide map. The phosphopeptides present in the unstimulated wt Kit receptor, which all contain phosphoserine, are shown as open circles (peptides 1 to 7); phosphopeptides that appear in the wt Kit receptor after stimulation with Steel factor, representing presumptive autophosphorylation sites, are shown as solid circles (peptides a to d).
Figure 4
Figure 4
Substrate specificity of the wt Kit and mutant KDY receptors. Receptors were immunoprecipitated from IC2/Kit and IC2/KDY cells, respectively, with rabbit anti-Kit antiserum and used to phosphorylate the EGFR optimal substrate (EGFS) AEEEEYFELVAKKKK (10 mM), Abl optimal substrate (ABLS) EAIYAAPFAKKKFEAKKK (10 mM), or Src optimal substrate (SRCS) AEEEIYGEFEAKKK (10 mM).

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