Cis-autophosphorylation of juxtamembrane tyrosines in the insulin receptor kinase domain.
Cis-autophosphorylation of juxtamembrane tyrosines in the insulin receptor kinase domain.
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胰岛素受体激酶结构域中近膜酪氨酸的顺式自磷酸化。
DOI：
10.1021/bi970170x
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Kohanski,RA
中科院分区：
文献类型：
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作者：
Cann,AD;Kohanski,RA
受体酪氨酸激酶经历配体诱导的二聚化，促进激酶结构域反式自磷酸化。然而，由于共价α2β2全聚结构，胰岛素受体的激酶结构域有效地二聚。这一事实使得确定全息内自磷酸化的分子机制变得困难，但有证据表明，在没有和存在胰岛素的情况下，顺式和反式自磷酸化都存在。在这里，我们利用人胰岛素受体的细胞质激酶结构域（CKD），证明了近膜（JM）亚结构域的自磷酸化遵循酸性反应途径。在6 nM ~ 3 μM范围内，JM的自磷酸化与CKD浓度无关，并进行了动力学表征：在75 μM ATP [5 mM Mn(CH3CO2)2]下观察到半饱和（KATP），最大速率为0.24 mol PO4(mol of CKD)-1min-1。在另外两个自磷酸化亚域中，由位点定向诱变产生的Phe对Tyr的成对替换改变了JM自磷酸化的动力学，但没有改变酸反应的途径。Tyr1328、1334 - Phe（在羧基末端亚域）使JM自磷酸化效率提高了2倍，而Tyr1162、1163 - Phe（在激活环亚域）使JM自磷酸化效率提高了约38倍，主要是由于katp降低了23倍。这些发现证明了ATP在CKD诱导自磷酸化的基础状态结合，以及胰岛素受体激酶亚域之间新的基础状态调节相互作用。基于这些结果和该激酶保守催化核心的晶体结构[Hubbard， S. R.,et al. (1994)Nature 372, 746]，提出了一个模型，该模型将jmcis反应和激活环-抑制/反式反应与胰岛素受体自磷酸化的复杂动力学相协调[Kohanski， R. a . (1993)Biochemistry 32,5766]。
Receptor tyrosine kinases undergo ligand-induced dimerization that promotes kinase domaintrans-autophosphorylation. However, the kinase domains of the insulin receptor are effectively dimerized because of the covalent α2β2holomeric structure. This fact has made it difficult to determine the molecular mechanism of intraholomeric autophosphorylation, but there is evidence for bothcis- andtrans-autophosphorylation in the absence and presence of insulin. Here, using the cytoplasmic kinase domain (CKD) of the human insulin receptor, we demonstrate that autophosphorylation in the juxtamembrane (JM) subdomain follows acis-reaction pathway. JM autophosphorylation was independent of CKD concentration over the range 6 nM−3 μM and was characterized kinetically: Half-saturation (KATP) was observed at 75 μM ATP [5 mM Mn(CH3CO2)2] with a maximal rate of 0.24 mol of PO4(mol of CKD)-1min-1. Pairwise substitutions of Phe for Tyr in the other two autophosphorylation subdomains, generated by site-directed mutagenesis, altered the kinetics of JM autophosphorylation but did not change the pathway from acis-reaction. Tyr1328,1334to Phe (in the carboxy-terminal subdomain) yielded <2-fold increase in the efficiency of JM autophosphorylation, whereas Tyr1162,1163to Phe (in the activation loop subdomain) yielded ≈38-fold increased efficiency of JM autophosphorylation, due predominantly to a 23-fold decreasedKATP. These findings demonstrate basal state binding of ATP to the CKD leading tocis-autophosphorylation and novel basal state regulatory interactions among the subdomains of the insulin receptor kinase. On the basis of these results and the crystal structure of the conserved catalytic core of this kinase [Hubbard, S. R.,et al. (1994)Nature 372, 746], a model is proposed which reconciles the JMcis-reaction and the activation loopcis-inhibition/trans-reaction with the complex kinetics of insulin receptor autophosphorylation [Kohanski, R. A. (1993)Biochemistry 32, 5766].