Hill Michelle M, Feng Jianhua, Hemmings Brian A
Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058, Basel, Switzerland.
Curr Biol. 2002 Jul 23;12(14):1251-5. doi: 10.1016/s0960-9822(02)00973-9.
Protein kinase B (PKB/Akt) has been well established as an important signaling intermediate, and its deregulation has been implicated in the development of human cancer and diabetes (reviewed in). Full activation of PKB requires phosphorylation on residues Thr308 and Ser473. While the Thr308 kinase, named 3-phosphoinositide-dependent kinase-1 (PDK1), has been extensively characterized (reviewed in ), the identity of the Ser473 kinase remains unclear. We have focused our study on the plasma membrane (PM) fraction because membrane localization is sufficient to activate PKB, and this suggests that PKB upstream kinases are constitutively active at the membrane. Here, we report the identification of a constitutively active PKB Ser473 kinase activity enriched in buoyant, detergent-insoluble plasma membrane rafts that are distinct from the cytosolic distribution of PKB and PDK1. This Ser473 kinase activity was released from the membrane by high salt, and gel filtration analysis showed that the kinase responsible is present in a large complex of >500 kDa. Two major phosphoproteins and integrin-linked kinase (ILK) were detected in partially purified PKB Ser473 kinase preparations. In contrast to previous observations, however, ILK immunoprecipitates did not retain Ser473 kinase activity. Thus, we have identified a novel raft-associated PKB Ser473 kinase, implicating a role for lipid rafts in PKB signaling.
蛋白激酶B（PKB/Akt）已被确认为一种重要的信号转导中间体，其失调与人类癌症和糖尿病的发生发展有关（相关综述见……）。PKB的完全激活需要苏氨酸308（Thr308）和丝氨酸473（Ser473）位点的磷酸化。虽然已对名为3-磷酸肌醇依赖性激酶-1（PDK1）的Thr308激酶进行了广泛研究（相关综述见……），但Ser473激酶的身份仍不清楚。我们将研究重点放在质膜（PM）部分，因为膜定位足以激活PKB，这表明PKB上游激酶在膜上持续处于活性状态。在此，我们报告鉴定出一种组成型活性PKB Ser473激酶活性，其在漂浮的、去污剂不溶性质膜微区中富集，这些微区与PKB和PDK1的胞质分布不同。这种Ser473激酶活性可通过高盐从膜上释放，凝胶过滤分析表明负责该激酶的成分存在于一个大于500 kDa的大复合物中。在部分纯化的PKB Ser473激酶制剂中检测到两种主要的磷酸化蛋白和整合素连接激酶（ILK）。然而，与之前的观察结果不同，ILK免疫沉淀复合物不保留Ser473激酶活性。因此，我们鉴定出一种新的与微区相关的PKB Ser473激酶，这表明脂筏在PKB信号传导中发挥作用。
