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首页> 外文期刊>Biochemistry >A single substitution of the insulin receptor kinase inhibits serine autophosphorylation in vitro: evidence for an interaction between the C-terminus and the activation loop.
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A single substitution of the insulin receptor kinase inhibits serine autophosphorylation in vitro: evidence for an interaction between the C-terminus and the activation loop.

机译：胰岛素受体激酶的单取代在体外抑制丝氨酸自磷酸化：C末端和激活环之间相互作用的证据。

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摘要

We examined the effects of mutations of tyrosine and serine autophosphorylation sites on the dual specificity of the insulin receptor kinase (IRKD) in vitro using autophosphorylation and substrate phosphorylation and phosphopeptide mapping. For comparable studies, the recombinant kinases were overexpressed in the baculovirus system, purified, and analyzed. The phosphate incorporation into the enzymes was in the range of 3-4.5 mol/mol, and initial velocities of autophosphorylation were reduced up to 2-fold. However, the mutation Y1151F in the activation loop inhibited phosphate incorporation in the C-terminal serine residues 1275 and 1309, due to a 10-fold decrease of the initial velocity of serine autophosphorylation. Although the K(M) and V(MAX) values of this mutant were only slightly altered in substrate phosphorylation reactions using a recombinant C-terminal insulin receptor peptide (K(M): Y1151F, 9.9 +/- 0.4 microM; IRKD, 6.1 +/- 0.2 microM; V(MAX): Y1151F, 72 +/- 4 nmol min(-)(1) mg(-)(1); IRKD, 117 +/- 6 nmol min(-)(1) mg(-)(1)), diminished phosphate incorporation into serine residues of the peptide was observed. In contrast, the phosphorylation of a recombinant IRS-1 fragment, which was shown to be phosphorylated markedly on serine residues by IRKD, was not affected by any kinase mutation. These results underline that IRKD is a kinase with dual specificity. The substrate specificity toward C-terminal serine phosphorylation sites can be modified by a single amino acid substitution in the activation loop, whereas the specificity toward IRS-1 is not affected, suggesting that the C-terminus and the activation loop interact.
机译：我们检查了酪氨酸和丝氨酸自磷酸化位点的突变对胰岛素受体激酶（IRKD）双重特异性的体外影响，方法是使用自磷酸化，底物磷酸化和磷酸肽作图。为了进行可比的研究，重组激酶在杆状病毒系统中过表达，纯化和分析。磷酸盐掺入酶的范围为3-4.5 mol / mol，自磷酸化的初始速度降低了2倍。但是，由于丝氨酸自磷酸化的起始速度降低了10倍，激活环中的突变Y1151F抑制了磷酸在C端丝氨酸残基1275和1309中的掺入。虽然此突变体的K（M）和V（MAX）值在使用重组C端胰岛素受体肽的底物磷酸化反应中仅稍有改变（K（M）：Y1151F，9.9 +/- 0.4 microM; IRKD，6.1 +/- 0.2 microM; V（MAX）：Y1151F，72 +/- 4 nmol min（-）（1）mg; IRKD，117 +/- 6 nmol min（-）（1）mg （-）（1）），观察到磷酸盐掺入到肽的丝氨酸残基中的减少。相反，重组IRS-1片段的磷酸化（不受IRKD的影响在丝氨酸残基上被显着磷酸化）不受任何激酶突变的影响。这些结果强调IRKD是具有双重特异性的激酶。可以通过激活环中的单个氨基酸取代来修饰对C端丝氨酸磷酸化位点的底物特异性，而对IRS-1的特异性则不受影响，表明C末端和激活环相互作用。

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