Phosphorylation and cell cycle-dependent regulation of Na+/H+ exchanger regulatory factor-1 by Cdc2 kinase
Phosphorylation and cell cycle-dependent regulation of Na+/H+ exchanger regulatory factor-1 by Cdc2 kinase
复制标题
DOI：
10.1074/jbc.m106859200
复制
发表时间：
2001-11-09
影响因子：
4.8
通讯作者：
Hall, RA
中科院分区：
文献类型：
--
作者：
He, JQ;Lau, AG;Hall, RA
Na+/H+交换调节因子（NHERF）-1是已知与各种受体、通道、细胞骨架元件和胞质信号蛋白结合的含有PDZ结构域的衔接蛋白。我们在这里报告，NHERF-1的磷酸化状态是深刻的细胞周期调节：在HeLa细胞中的NHERF-1是在有丝分裂期过度磷酸化，在细胞周期的其他点磷酸化少得多。这种NHERF-1的有丝分裂相依赖性磷酸化可以被roscovitine阻断，这与细胞周期蛋白依赖性激酶的磷酸化一致。用纯化的NHERF-1融合蛋白和纯化的激酶进行的体外研究表明，NHERF-1被细胞周期蛋白依赖性激酶Cdc 2强烈磷酸化。相比之下，NHERF-1相对于NHERF-2根本不被Cdc 2磷酸化。NHERF-1具有两个丝氨酸（Ser（279）和Ser（301）），其符合Cdc 2磷酸化优选的SPX（K/R）基序。这些丝氨酸的突变减少Cdc 2介导的磷酸化NHERF-1在体外，和突变的两个残基一起完全废除Cdc 2介导的磷酸化。当S279 A/S301 A NHERF-1突变体在细胞中表达时，它未能表现出在野生型NHERF-1中观察到的有丝分裂相依赖性磷酸化。将Ser（279）和Ser（301）突变为天冬氨酸以模拟NHERF-1的Cdc 2磷酸化，导致NHERF-1突变体在体外寡聚化能力显著受损。类似地，来自有丝分裂期HeLa细胞裂解物的内源性NHERF-1相对于来自间期HeLa细胞裂解物的内源性NHERF-1表现出显著降低的寡聚化能力。有丝分裂期NHERF-1还表现出与Pint结合的能力，Pint是一种含有WW结构域的肽基脯氨酰异构酶，在间期裂解物中不能检测到与NHERF-1结合。NHERF-1与Pint的结合促进了NHERF-1的去磷酸化，如在细胞Pin 1活性被选择性抑制剂胡桃醌阻断的实验中所示。这些数据表明，细胞NHERF-1在有丝分裂期被Cdc 2在Ser（279）和Ser（301）处磷酸化，并且这种磷酸化调节NHERF-1寡聚化和与Pin 1的缔合。
Na+/H+ exchanger regulatory factor (NHERF)-1 is a PDZ domain-containing adaptor protein known to bind to various receptors, channels, cytoskeletal elements, and cytoplasmic signaling proteins. We report here that the phosphorylation state of NHERF-1 is profoundly regulated by the cell cycle: NHERF-1 in HeLa cells is hyperphosphorylated in mitosis phase and much less phosphorylated at other points of the cell cycle. This mitosis phase-dependent phosphorylation of NHERF-1 could be blocked by roscovitine, consistent with phosphorylation by cyclin-dependent kinases. In vitro studies with purified NHERF-1 fusion proteins and purified kinases revealed that NHERF-1 was robustly phosphorylated by the cyclin-dependent kinase Cdc2. In contrast, the NHERF-1 relative NHERF-2 was not phosphorylated at all by Cdc2. NHERF-1 possesses two serines (Ser(279) and Ser(301)) that conform to the SPX(K/R) motif preferred for phosphorylation by Cdc2. Mutation of either of these serines reduced Cdc2-mediated phosphorylation of NHERF-1 in vitro, and mutation of both residues together completely abolished Cdc2-mediated phosphorylation. When the S279A/S301A NHERF-1 mutant was expressed in cells, it failed to exhibit the mitosis phase-dependent phosphorylation observed with wild-type NHERF-1. Mutation of both Ser(279) and Ser(301) to aspartate, to mimic Cdc2 phosphorylation of NHERF-1, resulted in a NHERF-1 mutant with a markedly impaired ability to oligomerize in vitro. Similarly, endogenous NHERF-1 from lysates of mitosis phase HeLa cells exhibited a markedly reduced ability to oligomerize relative to endogenous NHERF-1 from lysates of interphase HeLa cells. Mitosis phase NHERF-1 furthermore exhibited the ability to associate with Pint, a WW domain-containing peptidylprolyl isomerase that does not detectably bind to NHERF-1 in interphase lysates. The association of NHERF-1 with Pint facilitated dephosphorylation of NHERF-1, as shown in experiments in which cellular Pin1 activity was blocked by the selective inhibitor juglone. These data reveal that cellular NHERF-1 is phosphorylated during mitosis phase by Cdc2 at Ser(279) and Ser(301) and that this phosphorylation regulates NHERF-1 oligomerization and association with Pin1.