Oladimeji Peter, Diakonova Maria
The Department of Biological Sciences, University of Toledo, 2801 W. Bancroft Street, Toledo, OH 43606-3390, USA.
Biochem Biophys Res Commun. 2016 Apr 22;473(1):206-211. doi: 10.1016/j.bbrc.2016.03.079. Epub 2016 Mar 19.
Tyrosyl phosphorylation of the p21-activated serine-threonine kinase 1 (PAK1) has an essential role in regulating PAK1 functions in breast cancer cells. We previously demonstrated that PAK1 serves as a common node for estrogen (E2)- and prolactin (PRL)-dependent pathways. We hypothesize herein that intracellular localization of PAK1 is affected by PRL and E2 treatments differently. We demonstrate by immunocytochemical analysis that PAK1 nuclear translocation is ligand-dependent: only PRL but not E2 stimulated PAK1 nuclear translocation. Tyrosyl phosphorylation of PAK1 is essential for this nuclear translocation because phospho-tyrosyl-deficient PAK1 Y3F mutant is retained in the cytoplasm in response to PRL. We confirmed these data by Western blot analysis of subcellular fractions. In 30 min of PRL treatment, only 48% of pTyr-PAK1 is retained in the cytoplasm of PAK1 WT clone while 52% re-distributes into the nucleus and pTyr-PAK1 shuttles back to the cytoplasm by 60 min of PRL treatment. In contrast, PAK1 Y3F is retained in the cytoplasm. E2 treatment causes nuclear translocation of neither PAK1 WT nor PAK1 Y3F. Finally, we show by an in vitro kinase assay that PRL but not E2 stimulates PAK1 kinase activity in the nuclear fraction. Thus, PAK1 nuclear translocation is ligand-dependent: PRL activates PAK1 and induces translocation of activated pTyr-PAK1 into nucleus while E2 activates pTyr-PAK1 only in the cytoplasm.
p21激活的丝氨酸-苏氨酸激酶1（PAK1）的酪氨酸磷酸化在调节PAK1在乳腺癌细胞中的功能方面起着至关重要的作用。我们之前证明PAK1是雌激素（E2）和催乳素（PRL）依赖性信号通路的共同节点。我们在此假设，PAK1的细胞内定位受PRL和E2处理的影响不同。我们通过免疫细胞化学分析证明，PAK1的核转位是配体依赖性的：只有PRL而非E2刺激PAK1的核转位。PAK1的酪氨酸磷酸化对于这种核转位至关重要，因为磷酸酪氨酸缺陷型PAK1 Y3F突变体在受到PRL刺激时保留在细胞质中。我们通过亚细胞组分的蛋白质印迹分析证实了这些数据。在PRL处理30分钟时，只有48%的磷酸化酪氨酸PAK1（pTyr-PAK1）保留在PAK1野生型克隆的细胞质中，而52%重新分布到细胞核中，并且在PRL处理60分钟时pTyr-PAK1穿梭回到细胞质中。相比之下，PAK1 Y3F保留在细胞质中。E2处理既不会导致PAK1野生型也不会导致PAK1 Y3F发生核转位。最后，我们通过体外激酶测定表明，PRL而非E激活细胞核组分中的PAK1激酶活性。因此，PAK1的核转位是配体依赖性的：PRL激活PAK1并诱导活化的pTyr-PAK1转位到细胞核中，而E2仅在细胞质中激活pTyr-PAK1。
