A method to identify serine kinase substrates - Akt phosphorylates a novel adipocyte protein with a Rab GTPase-activating protein (GAP) domain
A method to identify serine kinase substrates - Akt phosphorylates a novel adipocyte protein with a Rab GTPase-activating protein (GAP) domain
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DOI：
10.1074/jbc.c200198200
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发表时间：
2002-06-21
影响因子：
4.8
通讯作者：
Lienhard, GE
中科院分区：
文献类型：
--
作者：
Kane, S;Sano, H;Lienhard, GE
本研究描述了一种鉴定特定丝氨酸激酶底物的方法。通过免疫沉淀，使用对激酶产生的磷酸化底物具有特异性的抗体分离磷酸化底物，并通过肽的串联质谱法鉴定分离的蛋白质。该方法被应用于鉴定蛋白激酶Akt的底物，该蛋白激酶Akt特异性磷酸化RXRXYS/T基序。用胰岛素处理3 T3-L1脂肪细胞以激活Akt，并通过与针对该基序的磷酸化形式的抗体的免疫沉淀分离推定的Akt底物蛋白。这导致了一种新的160 kDa的Akt底物的鉴定。Akt的160-kDa底物被命名为AS 160，具有Rab GAP结构域。重组AS 160被证明是Akt的底物，并且通过质谱和突变鉴定了两个磷酸化位点，均位于RXRXXS/T基序中。胰岛素处理的脂肪细胞引起AS 160从低密度微粒体重新分配到胞质溶胶。
This study describes a method for the identification of the substrates of specific serine kinases. An antibody specific for the phosphomotif generated by the kinase is used to isolate phosphorylated substrates by immunoprecipitation, and the isolated proteins are identified by tandem mass spectrometry of peptides. This method was applied to the identification of substrates for the protein kinase Akt, which specifically phosphorylates the RXRXYS/T motif. 3T3-L1 adipocytes were treated with insulin to activate Akt, and the putative Akt substrate proteins were isolated by immunoprecipitation with an antibody against the phospho form of this motif. This led to the identification of a novel 160-kDa substrate for Akt. The 160-kDa substrate for Akt, which was designated AS160, has a Rab GAP domain. Recombinant AS160 was shown to be a substrate for Akt, and two sites of phosphorylation, both in RXRXXS/T motifs, were identified by mass spectrometry and mutation. Insulin treatment of adipocytes caused AS160 to redistribute from the low density microsomes to the cytosol.