Kinetic properties of the insulin receptor tyrosine protein kinase: activation through an insulin-stimulated tyrosine-specific, intramolecular autophosphorylation.
Kinetic properties of the insulin receptor tyrosine protein kinase: activation through an insulin-stimulated tyrosine-specific, intramolecular autophosphorylation.
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胰岛素受体酪氨酸蛋白激酶的动力学特性：通过胰岛素刺激的酪氨酸特异性分子内自磷酸化激活。
DOI：
10.1016/0003-9861(86)90098-6
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发表时间：
1986
影响因子：
3.9
通讯作者：
Avruch,J
中科院分区：
文献类型：
--
作者：
Kwok,YC;Nemenoff,RA;Powers,AC;Avruch,J
胰岛素受体是一种胰岛素激活的酪氨酸特异性蛋白激酶。先前的研究表明，在Mr 95,000上的酪氨酸残基的自磷酸化与蛋白激酶活性对外源蛋白底物的激活有关。我们采用了高度纯化的胰岛素受体，固定在胰岛素琼脂糖凝胶或洗脱在一个积极的形式，以确定金属/ATP激酶激活的要求，受体自磷酸化激活的关系，和动力学特性的自磷酸化，激活受体激酶。预先将固定的受体与2 mmATP、10 mmMg（或10 mmMn）孵育，然后除去这些反应物，用于消除随后测量的组蛋白2b（酪氨酸）磷酸化速率的向上曲线。与未修饰的固定化受体相比，这种处理也显著增加了组蛋白2b磷酸化的速率，这是在本身最小化进一步激活的条件下估计的。与MgATP或MnATP预孵育获得的受体组蛋白2b（酪氨酸）激酶的最大活化程度是相同的，然而，MnATP的受体的亲和力约为MgATP的10倍。对于自磷酸化/自活化和组蛋白2b酪氨酸激酶活性，观察到MnATP受体的较高亲和力（KmMnATP ~ 0.01 mm;KmMgATP ~ 0.1 mm）。自磷酸化/自活化本身不会显著改变对MeATP（或蛋白质底物，如先前报道的）的表观亲和力，但会增加Vmax。受体组蛋白2b（酪氨酸）激酶的激活是由于Mr 95，000（β）亚基的酪氨酸特异性自磷酸化;因此，总32 P掺入β亚基的程度与激酶激活的程度精确相关，无论是随着时间的推移还是在各种Me 2 +ATP浓度下。用弹性蛋白酶和胰蛋白酶顺序处理自磷酸化受体，产生一个单一的、基本上带电荷的32 P-肽，Mr< 2000。从胰岛素-琼脂糖凝胶洗脱后，比较未磷酸化和完全磷酸化受体的功能特性。两种形式的受体的胰岛素结合特性是不可区分的;激酶特性差异很大;而未磷酸化受体的组蛋白2b活性在基础状态下很低，并且被胰岛素激活10倍，完全自磷酸化的受体在基础状态下表现出最大的组蛋白2b激酶，并且不受胰岛素添加的影响。总之，胰岛素在受体蛋白激酶功能激活中的作用主要（可能完全）是通过胰岛素刺激的、分子内的、酪氨酸特异性的受体β亚基在Mr < 2000的肽段上的自磷酸化。反过来，自磷酸化激活酪氨酸蛋白激酶，而不改变受体对Me 2 +ATP或蛋白质底物的亲和力，并使激酶具有组成性活性和胰岛素依赖性。
The insulin receptor is an insulin-activated, tyrosine-specific protein kinase. Previous studies have shown that autophosphorylation of tyrosine residues on theMr95,000 is associated with an activation of the protein kinase activity toward exogenous protein substrates. We have employed the highly purified insulin receptor, immobilized on insulin-Sepharose or eluted in an active form, to define the metal/ATP requirements for kinase activation, the relationship of receptor autophosphorylation to activation, and the kinetic properties of the autophosphorylated, activated receptor kinase. Prior incubation of the immobilized receptor with 2 mmATP, 10 mmMg (or 10 mmMn), followed by removal of these reactants, served to abolish the upward curvilinearity in the rate of histone 2b (tyrosine) phosphorylation measured subsequently. This treatment also markedly increased the rate of histone 2b phosphorylation as compared to that observed with the unmodified, immobilized receptor, as estimated under conditions that per se minimized further activation. The extents of maximal activation of receptor histone 2b (tyrosine) kinase obtained on preincubation with MgATP or MnATP are identical; however, the affinity of the receptor for MnATP is approximately 10-fold higher than that for MgATP. The higher affinity of the receptor for MnATP is observed for both autophosphorylation/autoactivation and histone 2b tyrosine kinase activity (KmMnATP ~ 0.01 mm;KmMgATP ~ 0.1 mm). Autophosphorylation/autoactivation per se does not significantly alter the apparent affinity for MeATP (or protein substrate, as previously reported) but increasesVmax. Activation of receptor histone 2b (tyrosine) kinase is due to tyrosine-specific autophosphorylation of theMr95,000 (β) subunit; thus the extent of total32P incorporation into the β subunit correlates precisely with the extent of kinase activation, both over time and at a wide variety of Me2+ATP concentrations. Sequential treatment of the autophosphorylated receptor with elastase and trypsin yields a single, basically charged32P-peptide,Mr< 2000. The functional properties of the unphosphorylated and fully phosphorylated receptor were compared after elution from insulin-Sepharose. The insulin binding characteristics of the two forms of the receptor were indistinguishable; the kinase properties differed greatly; whereas the histone 2b activity of the unphosphorylated receptor was low in the basal state, and activated 10-fold by insulin, the fully autophosphorylated receptor exhibits maximal histone 2b kinase in the basal state and is unaffected by insulin addition. In conclusion, the role of insulin in the activation of receptor protein kinase function is mediately largely (and probably entirely) through the insulin-stimulated, intramolecular, tyrosine-specific autophos-phorylation of the receptor β subunit on a peptide segment ofMr< 2000. In turn, autophosphorylation activates the tyrosine protein kinase without altering the affinity of the receptor for the Me2+ATP or protein substrates and renders the kinase constitutively active and insulin independent.