Protein-tyrosine phosphatase 1B complexes with the insulin receptor in vivo and is tyrosine-phosphorylated in the presence of insulin
Protein-tyrosine phosphatase 1B complexes with the insulin receptor in vivo and is tyrosine-phosphorylated in the presence of insulin
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DOI：
10.1074/jbc.272.3.1639
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发表时间：
1997-01-17
影响因子：
4.8
通讯作者：
Kusari, J
中科院分区：
文献类型：
--
作者：
Bandyopadhyay, D;Kusari, A;Kusari, J
响应于胰岛素，蛋白酪氨酸磷酸酶1B（PTP 1B）使95-和160-180-kDa酪氨酸磷酸化（PY）蛋白去磷酸化（Kenner，K.一、Anyanwu，E.，Olefsky，J. M.，和Kusari，J.（1996）J.Biol.Chem.271，19810-19816）。为了表征这些蛋白质，对表达催化失活PTPase 1B（CS）的对照和胰岛素处理细胞的裂解物进行免疫吸附，随后使用磷酸酪氨酸、PTPase 1B和胰岛素受体（IR）抗体的各种组合进行免疫印迹。抗PTSD 1B抗体从胰岛素刺激的细胞中共沉淀95 kDa PY蛋白，随后鉴定为IR β亚基。类似地，抗IR抗体从胰岛素处理的细胞中共沉淀50-kDa PY-PTAR 1B蛋白。为了鉴定响应于胰岛素而磷酸化的PTP 1B酪氨酸（Tyr）残基，用苯丙氨酸替换三个候选位点（Tyr（66）、Tyr（152）和Tyr（153））。替换Tyr（66）或Tyr（152）和Tyr（153）显著降低胰岛素刺激的PTAR 1B磷酸酪氨酸含量，以及其与IR的关联。使用突变IR的研究表明，IR自磷酸化是PTAR 1B-IR相互作用所必需的。这些结果表明，PTAR 1B复合物与自身磷酸化的胰岛素受体在完整的细胞中，直接或涉及其他蛋白质的复合物内。这种相互作用需要在受体和PTP 1B内的多个酪氨酸磷酸化位点。
In response to insulin, protein tyrosine phosphatase 1B (PTPase 1B) dephosphorylates 95- and 160-180-kDa tyrosine phosphorylated (PY) proteins (Kenner, K. A., Anyanwu, E., Olefsky, J. M., and Kusari, J. (1996) J. Biol. Chem. 271, 19810-19816). To characterize these proteins, lysates from control and insulin-treated cells expressing catalytically inactive PTPase 1B (CS) were immunoadsorbed and subsequently immunoblotted using various combinations of phosphotyrosine, PTPase 1B, and insulin receptor (IR) antibodies. Anti-PTPase 1B antibodies coprecipitated a 95-kDa PY protein from insulin-stimulated cells, subsequently identified as the IR beta-subunit. Similarly, anti-IR antibodies coprecipitated the 50-kDa PY-PTPase 1B protein from insulin-treated cells. To identify PTPase 1B tyrosine (Tyr) residues that are phosphorylated in response to insulin, three candidate sites (Tyr(66), Tyr(152), and Tyr(153)) were replaced with phenylalanine. Replacing Tyr(66) or Tyr(152) and Tyr(153) significantly reduced insulin-stimulated PTPase 1B phosphotyrosine content, as well as its association with the IR. Studies using mutant IRs demonstrated that IR autophosphorylation is necessary for the PTPase 1B-IR interaction. These results suggest that PTPase 1B complexes with the autophosphorylated insulin receptor in intact cells, either directly or within a complex involving additional proteins. The interaction requires multiple tyrosine phosphorylation sites within both the receptor and PTPase 1B.