Deactylase inhibitors disrupt cellular complexes containing protein phosphatases and deacetylases
Deactylase inhibitors disrupt cellular complexes containing protein phosphatases and deacetylases
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DOI：
10.1074/jbc.m310997200
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发表时间：
2004-02-27
影响因子：
4.8
通讯作者：
Shenolikar, S
中科院分区：
文献类型：
--
作者：
Brush, MH;Guardiola, A;Shenolikar, S
亲和分离的蛋白丝氨酸/苏氨酸磷酸酶的固定化磷酸酶抑制剂微囊藻毒素-LR确定组蛋白脱乙酰酶1（HDAC 1），HDAC 6，和HDAC 10作为新的组件的细胞磷酸酶复合物。其他HDAC，特别是HDAC 2、-3、-4和-5，从这样的复合物中排除。体外生物化学研究表明，重组HDAC 6，而不是HDAC 4，直接绑定到蛋白磷酸酶（PP）1催化亚基。HDAC 6和PP 2A（另一种主要的蛋白磷酸酶）之间没有观察到相关性。PP 1结合定位于HDAC 6中的第二催化结构域和相邻的C-末端序列，并且用胰蛋白酶抑制剂A（TSA）处理细胞破坏内源性HDAC6.PP1复合物。与HDAC 6抑制微管蛋白脱乙酰酶活性一致，TSA增强细胞微管蛋白乙酰化，并且乙酰化微管蛋白存在于TSA处理的细胞的PP 1复合物中。Trapoxin B（一种弱HDAC 6抑制剂）和calyculin A（一种细胞渗透性磷酸酶抑制剂）对HDAC 6.PP 1复合物的稳定性或微管蛋白乙酰化没有影响。使HDAC 6失活的突变阻止其掺入细胞PP 1复合物，并表明当结合在一起时，两种酶都是活性的。有趣的是，TSA破坏了所有分析的细胞HDAC磷酸酶复合物。这项研究为HDAC抑制剂引起细胞蛋白磷酸化和乙酰化协调变化的机制提供了新的见解，并表明这些蛋白质修饰在多个亚细胞位点的变化可能有助于HDAC抑制剂抑制细胞生长和转化的已知能力。
Affinity isolation of protein serine/threonine phosphatases on the immobilized phosphatase inhibitor microcystin-LR identified histone deacetylase 1(HDAC1), HDAC6, and HDAC10 as novel components of cellular phosphatase complexes. Other HDACs, specifically HDAC2, -3, -4, and -5, were excluded from such complexes. In vitro biochemical studies showed that recombinant HDAC6, but not HDAC4, bound directly to the protein phosphatase ( PP) 1 catalytic subunit. No association was observed between HDAC6 and PP2A, another major protein phosphatase. PP1 binding was mapped to the second catalytic domain and adjacent C-terminal sequences in HDAC6, and treatment of cells with trichostatin A (TSA) disrupted endogenous HDAC6.PP1 complexes. Consistent with the inhibition of tubulin deactylase activity of HDAC6, TSA enhanced cellular tubulin acetylation, and acetylated tubulin was present in the PP1 complexes from TSA-treated cells. Trapoxin B, a weak HDAC6 inhibitor, and calyculin A, a cell-permeable phosphatase inhibitor, had no effect on the stability of the HDAC6.PP1 complexes or on tubulin acetylation. Mutations that inactivated HDAC6 prevented its incorporation into cellular PP1 complexes and suggested that when bound together both enzymes were active. Interestingly, TSA disrupted all the cellular HDAC.phosphatase complexes analyzed. This study provided new insight into the mechanism by which HDAC inhibitors elicited coordinate changes in cellular protein phosphorylation and acetylation and suggested that changes in these protein modifications at multiple subcellular sites may contribute to the known ability of HDAC inhibitors to suppress cell growth and transformation.