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RAFTK/Pyk2 tyrosine kinase mediates the association of p190 RhoGAP with RasGAP and is involved in breast cancer cell
S Zrihan-Licht1, Y Fu, J Settleman
1Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, Massachusetts, MA 02115, USA.
Abstract:
Focal adhesions and actin cytoskeleton are involved in cell growth, shape and movement and in tumor invasion. Mitogen-induced changes in actin cytoskeleton are accompanied by changes in the tyrosine phosphorylation of several focal adhesion proteins. In this study, we have investigated the role of RAFTK, a cytoplasmic tyrosine kinase related to focal adhesion kinase (FAK), in heregulin-mediated signal transduction in breast cancer cells. Stimulation of T47D cells with heregulin (HRG) induced the tyrosine phosphorylation of RAFTK and the formation of a multiprotein complex. Analyses of the members of the HRG-stimulated complex revealed that RAFTK is associated with p190 RhoGAP (p190), RasGAP and ErbB-2, and plays an essential role in mediating the tyrosine phosphorylation of p190 by Src. Mutation of the Src binding site within RAFTK (402) abolished the phosphorylation of p190. In addition, upon HRG stimulation of T47D cells, association of ErbB-2 with RAFTK was observed and found to be indirect and mediated by Src. Expression of wild-type RAFTK (WT) significantly increased MDA-MB-435 and MCF-7 breast cancer cell invasion, while expression of the kinase-mutated RAFTK-R457 (KM) or the Src binding site mutant RAFTK (402) did not affect this cell invasion. Furthermore, HRG leads to the activation of MAP kinase which is mediated by RAFTK. These findings indicate that RAFTK serves as a mediator and an integration point between the GAP proteins and HRG-mediated signaling in breast cancer cells, and implicate RAFTK involvement in the MAP kinase pathway and in breast cancer cell invasion.
Insights
RAFTK, a tyrosine kinase, mediates heregulin signaling in breast cancer cells. It influences cell invasion and MAP kinase activation, highlighting its role in tumor progression.
Area of Science:
- Cell Biology
- Molecular Oncology
- Signal Transduction
Background:
- Focal adhesions and actin cytoskeleton dynamics are crucial for cell behavior and tumor invasion.
- Mitogen stimulation alters actin cytoskeleton and tyrosine phosphorylation of focal adhesion proteins.
Purpose of the Study:
- Investigate the role of RAFTK (related to FAK) in heregulin-mediated signaling in breast cancer cells.
- Elucidate RAFTK's involvement in protein complex formation, phosphorylation events, and cell invasion.
Main Methods:
- Stimulation of T47D breast cancer cells with heregulin (HRG).
- Analysis of protein complex formation and tyrosine phosphorylation using immunoprecipitation and Western blotting.
- Site-directed mutagenesis of RAFTK to study specific binding and kinase activity.
- Assessment of breast cancer cell invasion (MDA-MB-435, MCF-7) upon RAFTK expression.
- Evaluation of MAP kinase activation.
Main Results:
- HRG stimulation induced RAFTK tyrosine phosphorylation and complex formation with p190 RhoGAP (p190), RasGAP, and ErbB-2.
- RAFTK mediated Src-dependent tyrosine phosphorylation of p190; mutation of the Src binding site abolished this.
- ErbB-2 association with RAFTK was indirect, mediated by Src.
- Wild-type RAFTK expression significantly increased breast cancer cell invasion, unlike kinase-dead or Src-binding site mutants.
- HRG-induced MAP kinase activation was RAFTK-dependent.
Conclusions:
- RAFTK acts as a mediator and integration point for heregulin signaling and GAP proteins in breast cancer.
- RAFTK is implicated in MAP kinase pathway activation and breast cancer cell invasion.
- RAFTK's role in mediating these pathways suggests its significance in tumor progression.