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Mol Cell Biol, April 1998, p. 2089-2099, Vol. 18, No. 4
Health Canada Life Sciences and the
University of Ottawa,
Received 17 July 1997/Returned for modification 1 September
1997/Accepted 22 December 1997
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 S-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.
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
SHP-1 Binds and Negatively Modulates the c-Kit
Receptor by Interaction with Tyrosine 569 in the c-Kit
Juxtamembrane Domain
*
Corresponding author. Mailing address: Life Sciences
Division, Tunney's Pasture, Ottawa K1A 0L2, Canada. Phone: (613)
941-6594. Fax: (613) 941-8933. E-mail: Maya_Kozlowski{at}hc-sc.gc.ca.
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