Recent data derived from biochemical and genetic studies of SHP-1 interactions with the c-Kit PTK receptor have revealed that SHP-1 negatively regulates c-Kit signaling and thereby mitigates the signaling events linking c-Kit engagement to hemopoietic cell proliferation and differentiation (25
). We have investigated the structural basis for SHP-1 binding to and consequent inhibitory influence on c-Kit and report here that SHP-1 binds to a specific tyrosine-containing peptide sequence within the juxtamembrane region of c-Kit. Our data indicate that both SHP-1 SH2 domains participate in its interaction with c-Kit and identify the tyrosine residue at position 569 within the phosphorylated c-Kit juxtamembrane region as the major SHP-1 binding site on c-Kit. Mutation of Tyr569
not only abrogates SHP-1 interaction with c-Kit but also results in enhanced c-Kit signaling in response to SCF stimulation. Therefore, binding of SHP-1 to Tyr569
appears to be critical to the capacity of SHP-1 to both associate with c-Kit and negatively modulate the signaling pathways coupling the activated receptor to cellular responses.
The data reported here confirm previous observations indicating that tyrosine-phosphorylated c-Kit interacts with the SHP-1 N-terminal, but not C-terminal, SH2 domain (54
). Binding of the alternatively spliced C-terminal SHP-1 SH2 domain (SH2-C′) to phosphorylated c-Kit in vitro was also demonstrated in the current study and appeared to be equivalent to the interaction detected between c-Kit and the SHP-1 N- and C-terminal SH2 domains together (Fig. B). Thus, c-Kit association with SHP-1 can be mediated through either the SH2-N, SH2-C′, or tandem SH2-N and -C (or -C′) domains. These data provide the first direct evidence that the ligand binding properties of the SHP-1 C and C′ SH2 domains and, by extension, the two SHP-1 isoforms may be distinct, a conclusion consistent with structural data on SH2 domains indicating that the 39-amino-acid SH2 domain segment distinguishing the SHP-1 isoforms maps within a region forming EFβ strands implicated in defining SH2 domain peptide-binding specificity (2
). Along similar lines, the SHP-1 SH2-N and SH2-C domains also diverge in terms of their binding specificities, SHP-1 association with the activated erythropoietin, CD22, and IL-3β receptors being mediated through the N-terminal SHP-1 SH2 domain (23
), while its interaction with the natural killer inhibitory and FcγRIIβ receptors is mediated by the C-terminal SH2 domain (3
). Such divergence in terms of the peptide-binding specificities of its individual SH2 domains gives SHP-1 the potential to associate with and modulate a broad array of signaling effectors and, accordingly, to assume pivotal roles in regulating many facets of hemopoietic and epithelial cell behavior.
In the current study, the tyrosine residue at position 569 in the c-Kit juxtamembrane region was identified as the critical site for SHP-1 association with activated c-Kit receptors. This tyrosine, in turn, is flanked by Tyr and Val residues at the −2 and −1 positions, respectively, followed by Ile, Asp, and Pro at the +1, +2, and +3 positions, respectively. By contrast, previous studies of the binding motifs for SHP-1 interactions with the CD22 and FcγRIIB receptors on B lymphocytes and KIR, the p58 receptor on natural killer cells, have identified the sequence Val/IleX[pTyr]XXLeu as a consensus motif for association of SHP-1 with each of these receptors. Engagement of the latter receptors and their coincident recruitment of SHP-1 inhibit activation through the B-cell antigen (in the cases of FcγRIIB and CD22) or CD16 (in the case of p58) receptors, and accordingly, this conserved motif has been designated the immunoreceptor tyrosine-based inhibitory motif (6
). The SHP-1 N-terminal SH2 domain has also been shown to select the peptide sequence [pTyr]-hydrophobic-X-hydrophobic from a degenerate phosphopeptide library (43
). This finding is consistent with the identification here of [pTyr]IsoAspPro as the SHP-1 binding site on c-Kit as well as data identifying [pTyr]ThrIsoLeu as the SHP-1 binding site on the erythropoietin receptor (19
). Together these data indicate the capacity for SHP-1 to recognize phosphotyrosines in a multiplicity of amino acid contexts, a property not solely attributable to structural differences between the SHP-1 N- and C-terminal SH2 domains, as receptors such as the erythropoietin and CD22 receptors both bind SHP-1 via its N-terminal SH2 domain despite the differences in their SHP-1 binding site sequences.
In contrast to SHP-1, the SHP-2 PTP was shown to bind c-Kit by interacting with a tyrosine residue (Tyr567
) within the sequence [pTyr]ValTyrIle, a motif which matches the peptide sequence ([pTyr]Val/IleX Val/Ile) that the SHP-2 N-terminal SH2 domain preferentially selects from a degenerate peptide library (44
). Importantly, phenylalanine replacement of this tyrosine in GST–c-Kit–JUX fusion proteins was associated not only with disruption of SHP-2 binding, but also with some reduction in SHP-1 binding to the phosphorylated fusion protein (Fig. ). By contrast, Phe replacement of Tyr569
had no effect on the capacity of either GST–c-Kit–JUX fusion proteins (Fig. B) or c-Kit receptors expressed in Ba/F3 cells (Fig. B) to associate with SHP-2. Thus, unlike Tyr569
, which is required for SHP-1 but not involved in SHP-2 interactions with c-Kit, Tyr567
may play both an essential role in SHP-2 binding and a facilitory role in SHP-1 binding to phosphorylated c-Kit. In this context, it is possible that SHP-1 and SHP-2 compete for binding to the latter site on the c-Kit juxtamembrane region.
The involvement of Tyr567
in SHP-2 and, potentially, SHP-1 binding to c-Kit is of particular interest in view of previous data showing that deletion of the comparable tyrosine and juxtaposed valine residues (Tyr568
) substantially enhances the mitogenic and transforming properties of the feline c-Kit receptor and also represents one of the mutations which distinguishes the wild-type receptor from the oncogenic counterpart, v-Kit (13
). Although a similar link between Tyr569
mutation and c-Kit transforming capacity has not been described, the finding that SCF-induced proliferation of Ba/F3-Kit cells is enhanced by either Tyr569
→Phe or Tyr567
→Phe substitutions of the c-Kit receptors on these cells indicates that SHP-1 and SHP-2 can independently exert negative regulatory effects on c-Kit signaling and, by extension, that mutations of c-Kit which reduce or abrogate its binding to SHP-1 or SHP-2 can engender enhanced mitogenic and potentially oncogenic c-Kit activity. This apparent overlap in the effects of SHP-1 and SHP-2 on c-Kit signaling suggests that in at least some cell lineages c-Kit signaling may be unaffected by loss of function of one of these PTPs and thus provides a molecular explanation for the cell lineage-dependent effects of SHP-1 on c-Kit function observed in Wv
/motheaten mice (25
While our data provide evidence for the capacity of both SHP-1 and SHP-2 to negatively regulate c-Kit signaling, the mechanisms whereby this inhibitory influence is realized remain to be defined. For example, although c-Kit has been identified as an SHP-1 substrate in vitro, it is unclear whether SHP-1 or SHP-2 directly dephosphorylates c-Kit in vivo and/or elicits dephosphorylation of the receptor indirectly by dephosphorylating and inhibiting cytosolic PTKs that act on c-Kit. Downregulation of c-Kit signaling by these PTPs may also reflect the dephosphorylation of signaling effectors involved in downstream transduction of the ligand-binding activation signal, a paradigm recently demonstrated with respect to SHP-1 interactions with the erythropoietin receptor (17
). In the latter example, the negative influence of SHP-1 on receptor signaling has been linked to SHP-1-mediated dephosphorylation of the cytosolic JAK2 PTK (17
). As JAK2 has also been shown to associate with and modulate the activated c-Kit receptor (51
), it is possible that SHP-1 dephosphorylation of JAK2 either impairs c-Kit phosphorylation following ligand engagement or, by analogy with the erythropoietin receptor, interferes with JAK2-mediated activation and recruitment of signaling effectors required to evoke a cellular response. In addition to these possibilities, previous data indicating that the association of Src family tyrosine kinases with the c-Kit related platelet-derived growth factor receptor is mediated through Tyr579
, sites which represent homologs of Tyr567
on c-Kit (27
), also suggest that SHP-1 and/or SHP-2 inhibitory effects on c-Kit signaling may reflect the capacity of these PTPs to compete with and displace Src PTKs. Resolution of these issues should elucidate the molecular mechanisms whereby c-Kit signaling is regulated and translated to particular biological outcomes.