Vadlamudi R K, Adam L, Wang R A, Mandal M, Nguyen D, Sahin A, Chernoff J, Hung M C, Kumar R
University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
J Biol Chem. 2000 Nov 17;275(46):36238-44. doi: 10.1074/jbc.M002138200.
Stimulation of growth factor signaling has been implicated in the development of invasive phenotypes and the activation of p21-activated kinase (Pak1) in human breast cancer cells (Adam, L., Vadlamudi, R., Kondapaka, S. B., Chernoff, J., Mendelsohn, J., and Kumar, R. (1998) J. Biol. Chem. 273, 28238-28246; Adam, L., Vadlamudi, R., Mandal, M., Chernoff, J., and Kumar, R. (2000) J. Biol. Chem. 275, 12041-12050). To study the role of Pak1 in the regulation of motility and growth of breast epithelial cells, we developed human epithelial MCF-7 clones that overexpressed the kinase-active T423E Pak1 mutant under an inducible tetracycline promoter or that stably expressed the kinase-active H83L,H86L Pak1 mutant, which is deficient in small GTPase binding sites. The expression of both T423E and H83L,H86L Pak1 mutants in breast epithelial cells was accompanied by increased cell motility without any apparent effect on the growth rate of cells. The T423E Pak1 mutant was primarily localized to filopodia, and the H83L,H86L Pak1 mutant was primarily localized to ruffles. Cells expressing T423E Pak1 exhibited a regulatable stimulation of mitogen-activated protein kinase and Jun N-terminal kinase activities. The expression of kinase-active Pak1 mutants significantly stimulated anchorage-independent growth of cells in soft agar in a preferential mitogen-activated protein kinase-sensitive manner. In addition, regulatable expression of kinase-active Pak1 resulted in an abnormal organization of mitotic spindles characterized by appearance of multiple spindle orientations. We also provide evidence to suggest a close correlation between the status of Pak1 kinase activity and base-line invasiveness of human breast cancer cells and breast tumor grades. This study is the first demonstration of Pak1 regulation of anchorage-independent growth, potential Pak1 regulation of invasiveness, and abnormal organization of mitotic spindles of human epithelial breast cancer cells.
生长因子信号的刺激与人类乳腺癌细胞侵袭性表型的发展以及p21激活激酶（Pak1）的激活有关（亚当，L.，瓦德拉穆迪，R.，孔达帕卡，S.B.，切尔诺夫，J.，门德尔松，J.，和库马尔，R.（1998年）《生物化学杂志》273卷，28238 - 28246页；亚当，L.，瓦德拉穆迪，R.，曼达尔，M.，切尔诺夫，J.，和库马尔，R.（2000年）《生物化学杂志》275卷，12041 - 12050页）。为了研究Pak1在调节乳腺上皮细胞运动性和生长中的作用，我们构建了人上皮MCF - 7克隆，这些克隆在可诱导的四环素启动子控制下过表达激酶活性的T423E Pak1突变体，或者稳定表达激酶活性的H83L、H86L Pak1突变体，该突变体缺乏小GTP酶结合位点。乳腺上皮细胞中T423E和H83L、H86L Pak1突变体的表达都伴随着细胞运动性增加，而对细胞生长速率没有任何明显影响。T423E Pak1突变体主要定位于丝状伪足，H83L、H86L Pak1突变体主要定位于褶皱。表达T423E Pak1的细胞表现出对丝裂原激活蛋白激酶和Jun N末端激酶活性的可调节刺激。激酶活性Pak1突变体的表达以优先的丝裂原激活蛋白激酶敏感方式显著刺激细胞在软琼脂中不依赖贴壁的生长。此外，激酶活性Pak1的可调节表达导致有丝分裂纺锤体的异常组织，其特征是出现多个纺锤体方向。我们还提供证据表明Pak1激酶活性状态与人类乳腺癌细胞的基线侵袭性和乳腺肿瘤分级之间密切相关。这项研究首次证明了Pak1对人上皮乳腺癌细胞不依赖贴壁生长的调节、对侵袭性的潜在调节以及有丝分裂纺锤体的异常组织。
