SHP-1 binds and negatively modulates the c-Kit receptor by interaction with tyrosine 569 in the c-Kit juxtamembrane domain
SHP-1 binds and negatively modulates the c-Kit receptor by interaction with tyrosine 569 in the c-Kit juxtamembrane domain
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DOI：
10.1128/mcb.18.4.2089
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发表时间：
1998-04-01
影响因子：
5.3
通讯作者：
Siminovitch, KA
中科院分区：
文献类型：
--
作者：
Kozlowski, M;Larose, L;Siminovitch, KA
含有 SH2 结构域的 SHP-1 酪氨酸磷酸酶已被证明可以负调节广泛的生长因子和细胞因子驱动的有丝分裂信号通路，其中包括干细胞因子与 c-Kit 结合引起的细胞内事件级联，c-Kit 是一种酪氨酸激酶受体，与 SHP-1 相关并被 SHP-1 去磷酸化，使用一系列谷胱甘肽 S-转移酶 (GST) 融合蛋白，其中包含通过研究 c-Kit 胞质区域的酪氨酸磷酸化片段或 SHP-1 的 SH2 结构域，我们发现 SHP-1 通过选择性结合磷酸化的 c-Kit 近膜区域与 c-Kit 相互作用，并且 c-Kit 与两个 SHP-1 亚型中较大的一个亚型的关联可能是通过 N 端或 C 端 SHP-1 SH2 结构域介导的。使用诱变的 GST-Kit 近膜融合蛋白进行的结合测定以及使用包含每个 c-Kit 近膜区域的磷酸肽进行的竞争性抑制测定的结果，确定了第 569 位的酪氨酸残基是 SHP-1 与 c-Kit 结合的主要位点，并表明酪氨酸 567 对这种相互作用有贡献，但不是必需的。通过对逆转录病毒转导表达 c-Kit 受体的 Ba/F3 细胞进行分析，发现 c-Kit 酪氨酸残基 569 的苯丙氨酸取代与 c-Kit-SHP-1 结合的破坏和干细胞因子过度增殖反应的诱导有关。尽管 Ba/F3-c-Kit 细胞中 c-Kit 酪氨酸残基 567 的苯丙氨酸取代并未改变 SHP-1 与 c-Kit 的结合，但第二种 c-Kit 结合酪氨酸磷酸酶 SHP-2 与 c-Kit 结合的能力显着降低，并且细胞再次表现出对干细胞因子的过度增殖反应。因此，这些数据确定 SHP-1 与 c-Kit 上的酪氨酸 569 结合作为 SHP-1 对 c-Kit 信号传导抑制作用的关键相互作用，但它们也表明除 SHP-1 之外的胞质蛋白酪氨酸磷酸酶也可能负向调节 c-Kit 参与与增殖的耦合。
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 mot 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.