SHP-1 BINDS AND NEGATIVELY MODULATES THE C-KIT RECEPTOR BY INTERACTION WITH TYROSINE-569 IN THE C-KIT JUXTAMEMBRANE DOMAIN

The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad spectrum of growth factor-and cytokine-dr iven 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 dephosphorylat ed by SHP-1, Using a series of glutathione S-transferase (GST) fusion proteins containing either tyrosine-phosphorylated segments of the c-K it cytosolic region or the SH2 domains of SHP-1, we have shown that SH P-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 competitiv e inhibition assays with phosphopeptides encompassing each c-Kit juxta membrane 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 anal ysis of Ba/F3 cells retrovirally transduced to express c-Kit receptors , phenylalanine substitution of c-Kit tyrosine residue 569 was shown t o be associated with disruption of c-Kit-SHP-1 binding and induction o f hyperproliferative responses to stem cell factor. Although phenylala nine substitution of c-Kit tyrosine residue 567 in the Ba/F3-c-Kit cel ls did mot alter SHP-1 binding to c-Kit, the capacity of a second c-Ki t-binding tyrosine phosphatase, SHP-2, to associate with c-Kit was mar kedly reduced, and the cells again showed hyperproliferative responses to stem cell factor, These data therefore identify SHP-1 binding to t yrosine 569 on c-Kit as an interaction pivotal to SHP-1 inhibitory eff ects on c-Kit signaling, but they indicate as well that cytosolic prot ein tyrosine phosphatases other than SHP-1 may also negatively regulat e the coupling of c-Kit engagement to proliferation.