Nuclear localization and chromatin targets of p21-activated kinase 1
Nuclear localization and chromatin targets of p21-activated kinase 1
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DOI：
10.1074/jbc.m412607200
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发表时间：
2005-05-06
影响因子：
4.8
通讯作者：
Kumar, R
中科院分区：
文献类型：
--
作者：
Singh, RR;Song, CY;Kumar, R
Pak 1（p21-activated kinase 1）是一种保守的哺乳动物信号激酶，是小GTP酶Rac 1和Cdc 42以及生长因子信号的下游效应子。到目前为止，研究的主要焦点一直是Pak 1的胞质功能，它是细胞骨架重组的重要调节剂，因此在细胞存活，迁移和侵袭中起着重要作用。在这份报告中，我们证明了核定位的Pak 1刺激表皮生长因子。在Pak 1的N-末端结构域中鉴定了三个核定位信号（NLS）。通过突变分析，阐明了每个NLS的重要性。所有三个NLS的突变消除了Pak 1的核定位。Pak 1作为融合蛋白与Gal 4-DNA结合域和Gal 4-荧光素酶活性的表达表明，Pak 1可能会增加转录。为了确定核Pak 1的潜在靶点，我们使用了基于Pak 1特异性染色质免疫沉淀的筛选试验，并确定了一系列与Pak 1相互作用的靶染色质，包括磷酸果糖激酶-肌肉亚型（PFK-M）和活化T细胞核因子（NFAT 1）基因。Pak 1与PFK-M的上游增强子序列和启动子相关，参与PFK-M表达的刺激。它还与NFAT 1基因的一部分及其上游区域相关，导致NFAT 1表达的抑制。这些研究提供了Pak 1可以以积极和消极的方式影响其假定的染色质靶点表达的原理证据。总之，这些发现第一次定义了Pak 1的NLS，其与染色质的关联以及由此产生的转录调节，从而为进一步寻找核Pak 1功能和识别推定的Pak 1相互作用核蛋白开辟了新途径。
Pak1 (p21-activated kinase 1), a conserved, mammalian signaling kinase, is a downstream effector of small GTPases Rac1 and Cdc42 and of growth factor signaling. Until now, a major focus of study has been on the cytosolic functions of Pak1, where it is an important modulator of cytoskeletal reorganization, consequently playing a major role in cell survival, migration, and invasion. In this report, we demonstrate the nuclear localization of Pak1 upon stimulation by epidermal growth factor. Three nuclear localization signals (NLSs) were identified in the N-terminal domain of Pak1. With mutational analysis, the importance of each NLS was elucidated. Mutation of all three NLSs eliminated the nuclear localization of Pak1. Expression of Pak1 as a fusion protein with Gal4-DNA binding domain and Gal4-luciferase activity showed that Pak1 might increase transcription. To identify the potential targets of nuclear Pak1, we used a Pak1-specific chromatin immunoprecipitation-based screening assay and identified a series of Pak1-interacting target chromatins, including phosphofructokinase-muscle isoform (PFK-M) and nuclear factor of activated T-cell (NFAT1) genes. Pak1 associated with the upstream enhancer sequence and promoter of PFK-M and was involved in the stimulation of the PFK-M expression. It also associated with a portion of the NFAT1 gene and its upstream region, leading to the repression of NFAT1 expression. These investigations provide proof-of-principle evidence that Pak1 could influence the expression of its putative chromatin targets in both a positive and a negative manner. Together, for the first time, these findings defined the NLSs of the Pak1, its association with chromatin, and the resulting modulation of transcription, thus opening new avenues to further the search for nuclear Pak1 functions and identify putative Pak1-interacting nuclear proteins.