Park Shin-Young, Li Huchun, Avraham Shalom
Division of Experimental Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02215, USA.
Cell Signal. 2007 Feb;19(2):289-300. doi: 10.1016/j.cellsig.2006.07.005. Epub 2006 Aug 30.
The protein tyrosine kinase RAFTK, also termed Pyk2, is a member of the focal adhesion kinase (FAK) subfamily. In this report, we show the role of RAFTK in neuroendocrine PC12 cells upon epidermal growth factor (EGF) stimulation. Following EGF treatment, we observed that RAFTK was tyrosine-phosphorylated in a time- and dose-dependent manner, while FAK was constitutively phosphorylated and primarily regulated by cell adhesion. Moreover, we found that RAFTK associated with the phosphorylated EGF receptor (EGFR) upon EGF stimulation. RAFTK phosphorylation was mediated primarily through PLCgamma-IP3-Ca(2+) signaling and partially through PI3-Kinase. Furthermore, overexpression of PRNK, a specific dominant-negative construct of RAFTK, was sufficient to block EGF-induced cell spreading and movement. Paxillin, a key modulator of the actin cytoskeleton and an RAFTK substrate, was also phosphorylated following EGF treatment. EGF induced a dynamic reorganization of RAFTK and paxillin at neuronal adhesion sites, with the specific localization of paxillin at the inner juxtaposition of RAFTK. Additionally, we observed that RAFTK associated with the scaffold protein c-Cbl and mediated its phosphorylation. Our data demonstrate that while FAK mediated cell adhesion, RAFTK was localized at the cytoplasm where it mediated inside-out signaling through intracellular Ca(2+), thus leading to cell spreading and movement upon EGF stimulation.
蛋白酪氨酸激酶RAFTK，也称为Pyk2，是粘着斑激酶（FAK）亚家族的成员。在本报告中，我们展示了RAFTK在表皮生长因子（EGF）刺激下在神经内分泌PC12细胞中的作用。用EGF处理后，我们观察到RAFTK以时间和剂量依赖性方式发生酪氨酸磷酸化，而FAK则组成性磷酸化并主要受细胞粘附调节。此外，我们发现RAFTK在EGF刺激下与磷酸化的表皮生长因子受体（EGFR）相关联。RAFTK磷酸化主要通过PLCγ-IP3-Ca（2+）信号传导介导，部分通过PI3激酶介导。此外，RAFTK的特异性显性负性构建体PRNK的过表达足以阻断EGF诱导的细胞铺展和运动。桩蛋白是肌动蛋白细胞骨架的关键调节因子和RAFTK底物，在EGF处理后也发生了磷酸化。EGF诱导RAFTK和桩蛋白在神经元粘附位点发生动态重组，桩蛋白特异性定位于RAFTK的内侧并列位置。此外，我们观察到RAFTK与支架蛋白c-Cbl相关联并介导其磷酸化。我们的数据表明，虽然FAK介导细胞粘附，但RAFTK定位于细胞质中，在那里它通过细胞内Ca（2+）介导外向内信号传导，从而导致EGF刺激后细胞铺展和运动。
