Induced direct binding of the adapter protein Nck to the GTPase-activating protein-associated protein p62 by epidermal growth factor.
Induced direct binding of the adapter protein Nck to the GTPase-activating protein-associated protein p62 by epidermal growth factor.
复制标题
通过表皮生长因子诱导接头蛋白 Nck 与 GTP 酶激活蛋白相关蛋白 p62 直接结合。
DOI：
10.1038/sj.onc.1201351
复制
发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Li,W
中科院分区：
文献类型：
--
作者：
Tang,J;Feng,GS;Li,W
SH3-SH3-SH3-SH2 接头蛋白 Nck 将受体酪氨酸激酶（例如 EGF 和 PDGF 受体）与下游信号传导途径连接，其中涉及 p21 cdc42/rac 激活的激酶级联、Sos 激活的 Ras 信号传导和人 Wiskott-Aldrich 综合征蛋白 (WASp) 介导的肌动蛋白细胞骨架变化。在 EGF 刺激的细胞中，Nck 与许多磷酸酪氨酸蛋白（包括 EGF 受体）共免疫沉淀（Li 等人，1992 Mol. Cell. Biol. 12：5824–2833）。为了鉴定与 Nck 直接相互作用的磷酸酪氨酸蛋白并将其与间接相关蛋白区分开来，在测试与 Nck 的结合之前，通过加热和 SDS 解离细胞裂解物中预先存在的磷酸酪氨酸蛋白复合物。我们发现 Nck 不直接与 EGF 受体结合，而是通过其 SH2 结构域与 62 kDa 磷酸酪氨酸蛋白结合。我们提供的证据表明，Nck 结合的 p62 与先前鉴定的 GTP 酶激活蛋白 (GAP) 相关的磷酸酪氨酸蛋白 p62 相关。(1) Nck 结合和 GAP 结合的 p62 蛋白在 SDS-PAGE 中相互共同迁移。(2) 来自 Nck 和 GAP 的 SH2 结构域在体外竞争与 p62 的结合。(3) 纯化的 GST-Nck-SH2 直接与GAP 相关 p62。在这些条件下，PLCγ、PI-3 激酶、SHC 和 Grb2 的 SH2 结构域不结合 p62。(4) Nck 和 GAP 相关 p62 蛋白的胰蛋白酶磷酸肽图谱是相同的。然而，Nck 和 GAP 不会相互共免疫沉淀，并且显然与不同的 p62 库结合。这项研究表明，GAP 相关的 p62 充当 SH2 结构域对接蛋白，并介导 Nck 和 EGF 受体之间的相互作用，以响应 EGF 刺激。
The SH3-SH3-SH3-SH2 adapter protein Nck links receptor tyrosine kinases, such as EGF and PDGF receptors, to downstream signaling pathways, among which p21 cdc42/rac-activated kinase cascade, Sos-activated Ras signaling and the human Wiskott-Aldrich Syndrome protein (WASp)-mediated actin cytoskeleton changes, have been implicated. In EGF stimulated cells, Nck co-immunoprecipitates with a number of phosphotyrosine proteins including the EGF receptor (Li et al., 1992 Mol. Cell. Biol. 12: 5824–2833). To identify the phosphotyrosine protein (s) that directly interacts with Nck and to distinguish it from indirectly associated proteins, pre-existing phosphoytrosine protein complexes in the cell lysate were dissociated by heat and SDS prior to the test for binding to Nck. We found that Nck does not directly bind to EGF receptor, instead it binds via its SH2 domain to a 62 kDa phosphotyrosine protein. We present evidence demonstrating that the Nck-bound p62 is related to the previously identified GTPase-activating protein (GAP)-associated phosphotyrosine protein p62.(1) The Nck-bound and the GAP-bound p62 proteins co-migrate with each other in SDS–PAGE.(2) SH2 domains from Nck and GAP compete for binding to p62 in vitro.(3) Purified GST-Nck-SH2 binds directly to the GAP-associated p62. Under these conditions, SH2 domains from PLCγ, PI-3 kinase, SHC, and Grb2 did not bind p62.(4) Tryptic phosphopeptide maps of the Nck-and the GAP-associated p62 proteins are identical. However, Nck and GAP do not co-immunoprecipitate with each other and apparently bind to different pools of p62. This study suggests that the GAP-associated p62 acts as an SH2 domain docking protein and mediates the interaction between Nck and EGF receptor in response to EGF stimulation.