RAFTK/Pyk2 regulates EGF-induced PC12 cell spreading and movement.
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.
Park
Shin-Young
SY
Division of Experimental Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02215, USA.
Li
Huchun
H
Avraham
Shalom
S
D000818:Animals; D020013:Calcium Signaling; D002448:Cell Adhesion; D002465:Cell Movement; D003593:Cytoplasm / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism* / Q000502:physiology; D008954:Models, Biological; D016716:PC12 Cells; D051419:Paxillin / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism; D051381:Rats; D025801:Ubiquitin / Q000378:metabolism
D051419:Paxillin; D025801:Ubiquitin; D004815:Epidermal Growth Factor; D050721:Proto-Oncogene Proteins c-cbl; D019869:Phosphatidylinositol 3-Kinases; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; C496052:Ptk2 protein, rat; D011493:Protein Kinase C