Lightcap Christine M, Kari Gabor, Arias-Romero Luis E, Chernoff Jonathan, Rodeck Ulrich, Williams John C
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA.
PLoS One. 2009 Jun 26;4(6):e6025. doi: 10.1371/journal.pone.0006025.
Pak1 (p21 activated kinase 1) is a serine/threonine kinase implicated in regulation of cell motility and survival and in malignant transformation of mammary epithelial cells. In addition, the dynein light chain, LC8, has been described to cooperate with Pak1 in malignant transformation of breast cancer cells. Pak1 itself may aid breast cancer development by phosphorylating nuclear proteins, including estrogen receptor alpha. Recently, we showed that the LC8 binding site on Pak1 is adjacent to the nuclear localization sequence (NLS) required for Pak1 nuclear import. Here, we demonstrate that the LC8-Pak1 interaction is necessary for epidermal growth factor (EGF)-induced nuclear import of Pak1 in MCF-7 cells, and that this event is contingent upon LC8-mediated Pak1 dimerization. In contrast, Pak2, which lacks an LC8 binding site but contains a nuclear localization sequence identical to that in Pak1, remains cytoplasmic upon EGF stimulation of MCF-7 cells. Furthermore, we show that severe developmental defects in zebrafish embryos caused by morpholino injections targeting Pak are partially rescued by co-injection of wild-type human Pak1, but not by co-injection of mutant Pak1 mRNA disrupting either the LC8 binding or the NLS site. Collectively, these results suggest that LC8 facilitates nuclear import of Pak1 and that this function is indispensable during vertebrate development.
Pak1（p21激活激酶1）是一种丝氨酸/苏氨酸激酶，参与细胞运动和存活的调节以及乳腺上皮细胞的恶性转化。此外，动力蛋白轻链LC8已被描述为在乳腺癌细胞的恶性转化中与Pak1协同作用。Pak1自身可能通过磷酸化包括雌激素受体α在内的核蛋白来促进乳腺癌的发展。最近，我们发现Pak1上的LC8结合位点与Pak1核输入所需的核定位序列（NLS）相邻。在此，我们证明LC8-Pak1相互作用对于表皮生长因子（EGF）诱导的Pak1在MCF-7细胞中的核输入是必需的，并且这一事件取决于LC8介导的Pak1二聚化。相比之下，Pak2缺乏LC8结合位点但含有与Pak1相同的核定位序列，在MCF-7细胞受到EGF刺激后仍保留在细胞质中。此外，我们表明，通过注射针对Pak的吗啉代寡核苷酸导致斑马鱼胚胎出现严重发育缺陷，共注射野生型人Pak1可部分挽救该缺陷，但共注射破坏LC8结合或NLS位点的突变型Pak1 mRNA则不能挽救。总体而言，这些结果表明LC8促进Pak1的核输入，并且该功能在脊椎动物发育过程中是不可或缺的。
