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RAFTK/Pyk2 regulates EGF-induced PC12 cell spreading and movement
Shin-Young Park1, Huchun Li, Shalom Avraham
1Division of Experimental Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02215, USA.
Cellular Signalling
|September 2, 2006
Summary
Epidermal growth factor (EGF) stimulates protein tyrosine kinase RAFTK (Pyk2) in PC12 cells, driving cell spreading and movement via intracellular calcium signaling, distinct from cell adhesion regulated by FAK.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Protein tyrosine kinases play crucial roles in cellular signaling pathways.
- Focal adhesion kinase (FAK) and RAFTK (Pyk2) are key regulators of cell adhesion and migration.
- Epidermal growth factor (EGF) is a potent mitogen that triggers various cellular responses.
Purpose of the Study:
- To elucidate the specific role of RAFTK (Pyk2) in neuroendocrine PC12 cells following EGF stimulation.
- To differentiate the signaling pathways of RAFTK (Pyk2) and FAK in response to EGF and cell adhesion.
- To investigate the downstream effectors and cellular consequences of RAFTK (Pyk2) activation by EGF.
Main Methods:
- Time- and dose-dependent analysis of RAFTK (Pyk2) tyrosine phosphorylation upon EGF treatment.
- Co-immunoprecipitation assays to study protein-protein interactions (RAFTK-EGFR, RAFTK-c-Cbl).
- Western blotting to detect phosphorylated proteins (e.g., paxillin) and dominant-negative construct effects.
- Cell spreading and movement assays, and immunofluorescence microscopy for protein localization.
Main Results:
- EGF stimulation induced time- and dose-dependent tyrosine phosphorylation of RAFTK (Pyk2), while FAK phosphorylation was adhesion-dependent.
- RAFTK (Pyk2) associated with the phosphorylated EGF receptor (EGFR) and its phosphorylation was mediated by PLCgamma-IP3-Ca(2+) and PI3-Kinase signaling.
- Overexpression of a dominant-negative RAFTK construct inhibited EGF-induced cell spreading and movement.
- EGF treatment led to paxillin phosphorylation and dynamic reorganization of RAFTK and paxillin at adhesion sites.
- RAFTK (Pyk2) associated with and mediated phosphorylation of the scaffold protein c-Cbl.
Conclusions:
- RAFTK (Pyk2) plays a critical role in EGF-induced cell spreading and movement in PC12 cells, distinct from FAK's role in cell adhesion.
- RAFTK (Pyk2) mediates inside-out signaling through intracellular calcium, triggered by EGF receptor activation.
- The RAFTK (Pyk2)-paxillin-c-Cbl axis is involved in cytoskeletal dynamics and cell migration upon EGF stimulation.
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