JNK Regulation of Bcl-2 Association with FAK and Paxillin in Differentiating Lens Fiber Cells

Abstract

Abstract: : Purpose: In differentiating lens fiber cells apoptotic-like processes lead to loss of nuclei and organelles, yet the cells survive. These differentiating cells express high levels of the cell survival protein Bcl-2. We have analyzed the mechanism by which Bcl-2 may promote lens fiber cell survival and the potential role of c-jun NH₂-terminal kinase (JNK) in regulating Bcl-2 function. Methods: For in vitro studies primary cultures were prepared from day 10 quail embryo lenses. Cultures were maintained until differentiated lentoid structures formed. The JNK specific inhibitor SP600125 was used to suppress JNK activity in the differentiated lens cultures. For in vivo studies embryonic day 10 chick lenses were separated into differentiation specific zones by microdissection. Protein localization was demonstrated by immunofluorescence. Molecular association in a signaling complex was determined by co-immunoprecipitation studies. Results: We have shown that Bcl-2, an anti-apoptotic protein, is highly expressed in the cortical fiber cell zone of the chick embryo lens, a region in which lens fiber cells undergo organelle loss. In this zone of lens fiber cell differentiation we found that Bcl-2 is localized to the plasma membrane. We explored the possibility that the membrane localization of Bcl-2 reflected its role in a complex with membrane associated signaling proteins that play a role in cell survival. Our results demonstrated that both focal adhesion kinase (FAK) and paxillin were highly associated with Bcl-2 in the lens cortical fiber cells and that this association was differentiation stage specific. Next, we investigated the signaling regulators of the association of Bcl-2 with the FAK/paxillin complex. Like Bcl-2, JNK, a MAP kinase implicated in cell survival, was found to be highly associated with FAK and paxillin in the cortical fiber zone. The functional role of JNK in Bcl-2 signaling of lens cell survival was analyzed by treatment of lens cultures with the JNK inhibitor followed by examination of the association of Bcl-2 with FAK and paxillin. We found that the linkage of Bcl-2 to both FAK and paxillin was dependent on JNK activity. Conclusions: JNK positively regulates the association of Bcl-2 with a FAK/paxillin signaling complex, which may be important for the function of Bcl-2 in fiber cell survival.

Keywords: signal transduction • apoptosis/cell death • cell adhesions/cell junctions