Bandyopadhyay D, Kusari A, Kenner K A, Liu F, Chernoff J, Gustafson T A, Kusari J
Department of Physiology, Tulane University Medical Center, New Orleans, Louisiana 70112-2699, USA.
J Biol Chem. 1997 Jan 17;272(3):1639-45. doi: 10.1074/jbc.272.3.1639.
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 (Tyr66, Tyr152, and Tyr153) were replaced with phenylalanine. Replacing Tyr66 or Tyr152 and Tyr153 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.
作为对胰岛素的反应，蛋白酪氨酸磷酸酶1B（PTPase 1B）使95 kDa和160 - 180 kDa的酪氨酸磷酸化（PY）蛋白去磷酸化（肯纳，K.A.，阿尼扬武，E.，奥莱夫斯基，J.M.，和库萨里，J.（1996年）《生物化学杂志》271卷，19810 - 19816页）。为了鉴定这些蛋白，对表达无催化活性的PTPase 1B（CS）的对照细胞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分析。抗PTPase 1B抗体从胰岛素刺激的细胞中共沉淀出一种95 kDa的PY蛋白，随后鉴定为IRβ亚基。同样，抗IR抗体从胰岛素处理的细胞中共沉淀出50 kDa的PY - PTPase 1B蛋白。为了鉴定响应胰岛素而被磷酸化的PTPase 1B酪氨酸（Tyr）残基，将三个候选位点（Tyr66、Tyr152和Tyr153）替换为苯丙氨酸。替换Tyr66或Tyr152和Tyr153显著降低了胰岛素刺激的PTPase 1B磷酸酪氨酸含量及其与IR的结合。使用突变IR的研究表明，IR自身磷酸化对于PTPase 1B - IR相互作用是必需的。这些结果表明，在完整细胞中，PTPase 1B与自身磷酸化的胰岛素受体形成复合物，要么直接形成，要么在涉及其他蛋白的复合物中形成。这种相互作用需要受体和PTPase 1B内的多个酪氨酸磷酸化位点。
