INSULIN-STIMULATED PHOSPHORYLATION OF RECOMBINANT PP120/HA4, AN ENDOGENOUS SUBSTRATE OF THE INSULIN-RECEPTOR TYROSINE KINASE
INSULIN-STIMULATED PHOSPHORYLATION OF RECOMBINANT PP120/HA4, AN ENDOGENOUS SUBSTRATE OF THE INSULIN-RECEPTOR TYROSINE KINASE
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DOI：
10.1021/bi00029a009
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发表时间：
1995-07-25
期刊：
影响因子：
2.9
通讯作者：
TAYLOR, SI
中科院分区：
文献类型：
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作者：
NAJJAR, SM;PHILIPPE, N;TAYLOR, SI
胰岛素与其受体的α亚基结合刺激受体酪氨酸激酶磷酸化β亚基和几种内源性蛋白底物，包括pp120/HA4，一种肝脏特异性的M(r) 120,000的质膜糖蛋白。通过对大鼠肝脏pp1201HA4氨基酸序列的分析，揭示了细胞质域酪氨酸磷酸化的两个潜在位点（Tyr（488）和Tyr(513)）以及潜在的camp依赖性蛋白激酶磷酸化位点（Ser(503)）。为了确定这些位点中的哪一个在胰岛素作用下被磷酸化，每个氨基酸残基都被位点定向诱变改变了。然后在NIH 3T3细胞中稳定转染表达突变cdna。两个突变（Phe（488）和Ala(503)）破坏了胰岛素诱导的pp120/HA4磷酸化，表明pp120/HA4经历了多位点磷酸化。似乎Tyr（488）可能被胰岛素受体激酶磷酸化，而Ser（513）的磷酸化可能有助于酪氨酸磷酸化的调节。由于pp120/HA4被认为与Ca2+/Mg2+依赖性外atp酶活性有关，我们确定了胰岛素诱导的磷酸化对该酶活性的影响。在共表达胰岛素受体和pp120/HA4的NIH 3T3细胞中，胰岛素导致外泌atpase活性增加2倍。此外，pp120/HA4磷酸化位点的消除损害了胰岛素刺激外atp酶活性的能力。这些数据表明pp120/HA4的酪氨酸磷酸化可能调节Ca2+/Mg2+依赖性外atp酶活性。
Insulin binding to the alpha-subunit of its receptor stimulates the receptor tyrosine kinase to phosphorylate the beta-subunit and several endogenous protein substrates, including pp120/HA4, a liver-specific plasma membrane glycoprotein of M(r) 120 000. Analysis of the deduced amino acid sequence of rat liver pp1201HA4 revealed two potential sites for tyrosine phosphorylation in the cytoplasmic domain (Tyr(488) and Tyr(513)), as well as a potential cAMP-dependent protein kinase phosphorylation site (Ser(503)). To determine which of these sites is phosphorylated in response to insulin, each of these amino acid residues was altered by site-directed mutagenesis. Mutant cDNAs were then expressed by stable transfection in NIH 3T3 cells. Two mutations (Phe(488) and Ala(503)) impaired insulin-induced phosphorylation of pp120/HA4, suggesting that pp120/HA4 undergoes multisite phosphorylation. It seems likely that Tyr(488) is phosphorylated by the insulin receptor kinase, and phosphorylation of Ser(513) may contribute to the regulation of tyrosine phosphorylation. Since pp120/HA4 is believed to be associated with a Ca2+/Mg2+-dependent ecto-ATPase activity, we determined the effects of insulin-induced phosphorylation on this enzymatic activity. In NIH 3T3 cells co-expressing the insulin receptor and pp120/HA4, insulin caused a 2-fold increase in ecto-ATPase activity. Moreover, elimination of the phosphorylation sites of pp120/HA4 impaired the ability of insulin to stimulate the ecto-ATPase activity. These data suggest that tyrosine phosphorylation of pp120/HA4 may regulate Ca2+/Mg2+-dependent ecto-ATPase activity.