Biochemical characterization of the phosphatase domain of the tumor suppressor PH domain leucine-rich repeat protein phosphatase.
Biochemical characterization of the phosphatase domain of the tumor suppressor PH domain leucine-rich repeat protein phosphatase.
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DOI：
10.1021/bi500428j
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发表时间：
2014-06-24
期刊：
影响因子：
2.9
通讯作者：
Newton AC
中科院分区：
文献类型：
--
作者：
Sierecki E;Newton AC
PH结构域富亮氨酸重复蛋白磷酸酶（PHLPP）直接使Akt和蛋白激酶C去磷酸化和失活，因此是两个关键信号传导途径（磷脂酰肌醇3-激酶和二酰基甘油信号传导途径）的药理学干预的主要靶标。在这里，我们报告的第一个生化表征的磷酸酶结构域的PHLPP家族成员。人PHLPP 1和PHLPP 2磷酸酶结构域的表达和纯化从细菌或昆虫细胞和它们的活性相比，从哺乳动物细胞免疫沉淀的全长蛋白质。生物化学分析表明，PHLPP磷酸酶结构域有效地脱磷酸化合成和肽底物，其活性由金属和亲脂性化合物调节，并且其具有相对较高的热稳定性。PHLPP 2的突变分析揭示了一个不寻常的活性位点结构相比，典型的PP 2C磷酸酶的结构，并确定关键的酸性残基（天冬氨酸806，谷氨酸989，和天冬氨酸1024）和庞大的芳香族残基（苯丙氨酸783和苯丙氨酸808），其突变损害的活动。与独特的活性位点结构一致，我们鉴定了区分PHLPP 2和PP 2C α的抑制剂。这些数据确立PHLPP作为具有独特活性位点结构的磷酸酶PP 2C家族的成员。
PH domain leucine-rich repeat protein phosphatase (PHLPP) directly dephosphorylates and inactivates Akt and protein kinase C and is therefore a prime target for pharmacological intervention of two key signaling pathways, the phosphatidylinositol 3-kinase and diacylglycerol signaling pathways. Here we report on the first biochemical characterization of the phosphatase domain of a PHLPP family member. The human PHLPP1 and PHLPP2 phosphatase domains were expressed and purified from bacteria or insect cells and their activities compared to that of full-length proteins immunoprecipitated from mammalian cells. Biochemical analyses reveal that the PHLPP phosphatase domain effectively dephosphorylates synthetic and peptidic substrates, that its activity is modulated by metals and lipophilic compounds, and that it has relatively high thermal stability. Mutational analysis of PHLPP2 reveals an unusual active site architecture compared to the canonical architecture of PP2C phosphatases and identifies key acidic residues (Asp 806, Glu 989, and Asp 1024) and bulky aromatic residues (Phe 783 and Phe 808) whose mutation impairs activity. Consistent with a unique active site architecture, we identify inhibitors that discriminate between PHLPP2 and PP2Cα. These data establish PHLPP as a member of the PP2C family of phosphatases with a unique active site architecture.
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影响因子：
20.1
作者：
Miyamoto S;Purcell NH;Smith JM;Gao T;Whittaker R;Huang K;Castillo R;Glembotski CC;Sussman MA;Newton AC;Brown JH
通讯作者：
Brown JH
影响因子：
4.6
作者：
Krieglstein, Josef;Hufnagel, Birgit;Klumpp, Susanne
通讯作者：
Klumpp, Susanne
影响因子：
50.3
作者：
Chen M;Pratt CP;Zeeman ME;Schultz N;Taylor BS;O'Neill A;Castillo-Martin M;Nowak DG;Naguib A;Grace DM;Murn J;Navin N;Atwal GS;Sander C;Gerald WL;Cordon-Cardo C;Newton AC;Carver BS;Trotman LC
通讯作者：
Trotman LC
影响因子：
3.5
作者：
Klumpp, S;Selke, D;Hermesmeier, J
通讯作者：
Hermesmeier, J
关闭 AKT：PHLPP 作为药物靶点。
DOI：
10.1146/annurev-pharmtox-011112-140338
发表时间：
2014
影响因子：
12.5
作者：
Newton AC;Trotman LC
通讯作者：
Trotman LC