Hammer Alan, Rider Leah, Oladimeji Peter, Cook Leslie, Li Quanwen, Mattingly Raymond R, Diakonova Maria
Department of Biological Sciences, University of Toledo, Toledo, OH 43606-3390, USA.
Mol Endocrinol. 2013 Mar;27(3):455-65. doi: 10.1210/me.2012-1291. Epub 2013 Jan 22.
The p21-activated serine-threonine kinase (PAK1) is activated by small GTPase-dependent and -independent mechanisms and regulates cell motility. Both PAK1 and the hormone prolactin (PRL) have been implicated in breast cancer by numerous studies. We have previously shown that the PRL-activated tyrosine kinase JAK2 (Janus tyrosine kinase 2) phosphorylates PAK1 in vivo and identified tyrosines (Tyr) 153, 201, and 285 in the PAK1 molecule as sites of JAK2 tyrosyl phosphorylation. Here, we have used human breast cancer T47D cells stably overexpressing PAK1 wild type or PAK1 Y3F mutant in which Tyr(s) 153, 201, and 285 were mutated to phenylalanines to demonstrate that phosphorylation of these three tyrosines are required for maximal PRL-dependent ruffling. In addition, phosphorylation of these three tyrosines is required for increased migration of T47D cells in response to PRL as assessed by two independent motility assays. Finally, we show that PAK1 phosphorylates serine (Ser) 2152 of the actin-binding protein filamin A to a greater extent when PAK1 is tyrosyl phosphorylated by JAK2. Down-regulation of PAK1 or filamin A abolishes the effect of PRL on cell migration. Thus, our data presented here bring some insight into the mechanism of PRL-stimulated motility of breast cancer cells.
p21激活的丝氨酸-苏氨酸激酶（PAK1）可通过小GTP酶依赖性和非依赖性机制被激活，并调节细胞运动。众多研究表明，PAK1和催乳素（PRL）均与乳腺癌有关。我们之前已经证明，PRL激活的酪氨酸激酶JAK2（Janus酪氨酸激酶2）在体内使PAK1磷酸化，并确定PAK1分子中的酪氨酸（Tyr）153、201和285为JAK2酪氨酸磷酸化位点。在此，我们使用稳定过表达野生型PAK1或PAK1 Y3F突变体（其中Tyr153、201和285突变为苯丙氨酸）的人乳腺癌T47D细胞，以证明这三个酪氨酸的磷酸化是PRL依赖性最大程度皱襞形成所必需的。此外，通过两种独立的运动分析评估，这三个酪氨酸的磷酸化是T47D细胞响应PRL而增加迁移所必需的。最后，我们表明，当PAK1被JAK2酪氨酸磷酸化时，PAK1对肌动蛋白结合蛋白细丝蛋白A的丝氨酸（Ser）2152的磷酸化程度更高。PAK1或细丝蛋白A的下调消除了PRL对细胞迁移的影响。因此，我们在此展示的数据为PRL刺激乳腺癌细胞运动的机制提供了一些见解。
