Activation of a GST-tagged AKT2/PKBβ
Activation of a GST-tagged AKT2/PKBβ
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DOI：
10.1016/j.bbagen.2005.04.002
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发表时间：
2005-10-10
影响因子：
3
通讯作者：
Al-Hasani, H
中科院分区：
文献类型：
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作者：
Baer, K;Lisinski, I;Al-Hasani, H
AKT蛋白激酶是细胞生长、细胞存活和代谢胰岛素作用的关键调节因子。然而，体内活化AKT的机制，可能涉及激酶的膜募集、寡聚化和多重磷酸化事件，尚不完全清楚。在本研究中，我们通过杆状病毒表达系统表达和纯化了人蛋白激酶AKT2 （Delta PH-AKT2）二聚体gst融合蛋白（毫克量）。用磷酸酶抑制剂冈田酸（OA）处理病毒感染的昆虫细胞，导致两个调节磷酸化位点Thr（309）和Ser（474）磷酸化，并激活激酶。同样，通过重组PDK1将Thr（309）磷酸化或将Thr（309）和Ser（474）突变为酸性残基，使该激酶具有组成性活性。然而，尽管我们的AKT2的特异性活性比之前的报道增加了15倍，gst介导的二聚化本身并没有导致激酶的激活。虽然诱变和磷酸化都导致酶的周转数量增加，但只有后者导致外源底物Crosstide的表观K值显著降低（20倍），这表明这种广泛使用的诱变仅部分模拟磷酸化。GST-AKT2的动力学分析表明，该激酶激活环中Thr（309）的磷酸化是观察到的k降低和随后酶的催化效率（k(cat)/ k -m）提高150倍的主要原因。高活性的AKT2构建体用于体外自磷酸化反应，其中无活性的AKT2激酶作为底物。事实上，我们发现证据表明AKT2有轻微的自磷酸化活性，但两个调控位点，Thr（309）或Ser（474）中的任何一个都没有显著的自磷酸化。(c) 2005 Elsevier B.V.版权所有
The protein kinase AKT is a key regulator for cell growth, cell survival and metabolic insulin action. However, the mechanism of activation of AKT in vivo, which presumably involves membrane recruitment of the kinase, oligomerization, and multiple phosphorylation events, is not fully understood. In the present study, we have expressed and purified dimeric GST-fusion proteins of human protein kinase AKT2 (Delta PH-AKT2) in milligram quantities via the baculovirus expression system. Treatment of virus-infected insect, cells with the phosphatase inhibitor okadaic acid (OA) led to phosphorylation of the two regulatory phosphorylation sites, Thr(309) and Ser(474), and to activation of the kinase. Likewise, phosphorylation of Thr(309) in vitro by recombinant PDK1 or mutation of Thr(309) and Ser(474) to acidic residues rendered the kinase constitutively active. However, even though the specific activity of our AKT2 was increased 15-fold compared to previous reports, GST-mediated dimerization alone did not lead to an activation of the kinase. Whereas both mutagenesis and phosphorylation led to an increase in the turnover number of the enzyme, only the latter resulted in a marked reduction (20-fold) of the apparent K,, value for the exogenous substrate Crosstide, indicating that this widely used mutagenesis only partially mimics phosphorylation. Kinetic analysis of GST-AKT2 demonstrates that phosphorylation of Thr(309) in the activation loop of the kinase is largely responsible for the observed reduction in K. and for a subsequent 150-fold increase in the catalytic efficiency (k(cat)/K-m) of the enzyme. Highly active AKT2 constructs were used in autophosphorylation reactions in vitro, where inactive AKT2 kinases served as substrates. As a matter of fact, we found evidence for a minor autophosphorylation activity of AKT2 but no significant autophosphorylation of any of the two regulatory sites, Thr(309) or Ser(474). (c) 2005 Elsevier B.V. All rights reserved.