Phosphorylation-dependent regulation of nuclear localization and functions of integrin-linked kinase
Phosphorylation-dependent regulation of nuclear localization and functions of integrin-linked kinase
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DOI：
10.1073/pnas.0701999104
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发表时间：
2007-04-17
影响因子：
11.1
通讯作者：
Kumar, Rakesh
中科院分区：
文献类型：
--
作者：
Acconcia, Filippo;Barnes, Christopher J.;Kumar, Rakesh
整合素连接激酶（ILK）是一种磷酸化蛋白，调节与p21活化激酶1 （PAK1）调控重叠的生理过程。在这里，我们报道了PAK1磷酸化ILK在ILK介导的信号传导和细胞内易位中的可能作用。我们发现PAK1在体外和体内磷酸化ILK的苏氨酸173和丝氨酸246位点。PAK1的缺失降低了体内内源性ILK磷酸化水平。ILK上PAK1磷酸化位点突变为丙氨酸可降低细胞活力和细胞增殖。人类细胞的生化分离、共聚焦显微镜和染色质相互作用分析表明，ILK主要定位于细胞质中，但也存在于细胞核中。转染点突变体ILK-T173A、ILK-S246A或ILK-T173A的MCF-7细胞；S246A （ILK- dm）改变了ILK的定位。PAK1的选择性缺失显著增加了ILK的核和焦点积累，进一步证明了PAK1在ILK易位中的作用。我们还确定了ILK的功能性核定位序列和核输出序列基序，描绘了ILK在维持正常核完整性方面明显不可或缺的作用，并确定了ILK与CNKSR3基因染色质的调控区域相互作用以负向调节其表达。综上所述，这些结果表明ILK是PAK1的底物，在细胞核和细胞质之间进行磷酸化- clependent穿梭，并与基因调控染色质相互作用。
Integrin-linked kinase (ILK) is a phosphorylated protein that regulates physiological processes that overlap with those regulated by p21-activated kinase 1 (PAK1). Here we report the possible role of ILK phosphorylation by PAK1 in ILK-mediated signaling and intracellular translocation. We found that PAK1 phosphorylates ILK at threonine-173 and serine-246 in vitro and in vivo. Depletion of PAK1 decreased the levels of endogenous ILK phosphorylation in vivo. Mutation of PAK1 phosphorylation sites on ILK to alanine reduced cell motility and cell proliferation. Biochemical fractionation, confocal microscopy, and chromatin-interaction analyses of human cells revealed that ILK localizes predominantly in the cytoplasm but also resides in the nucleus. Transfection of MCF-7 cells with point mutants ILK-T173A, ILK-S246A, or ILK-T173A; S246A (ILK-DM) altered ILK localization. Selective depletion of PAK1 dramatically increased the nuclear and focal point accumulation of ILK, further demonstrating a role for PAK1 in ILK translocation. We also identified functional nuclear localization sequence and nuclear export sequence motifs in ILK, delineated an apparently integral role for ILK in maintaining normal nuclear integrity, and established that ILK interacts with the regulatory region of the CNKSR3 gene chromatin to negatively modulate its expression. Together, these results suggest that ILK is a PAK1 substrate, undergoes phosphorylation-clepenclent shuttling between the cell nucleus and cytoplasm, and interacts with gene-regulatory chromatin.