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A. S. Menko, G. F. Weber, L. Zhang, S. K. Joseph, M. Leonard, J. L. Walker; The Link Between c-Jun-N-Terminal Kinase (JNK), Bcl-2 and IP3R and the Regulation of Nuclear and Organelle Loss in the Lens. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1121.
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In addition to their well-known role in activating classical signaling pathways, integrin-linked adhesion complexes are emerging as sites for regulation of ER function, such as IP3 receptor (IP3R) release of Ca(2+) stores, a potential mechanism for signaling late stage lens differentiation events. The pro-survival molecule Bcl-2 can associate with both paxillin in integrin adhesion complexes and with the ER where it blocks activation of IP3R calcium channels, thereby protecting cells against apoptotic death signals. In this study we investigated the possibility that Bcl-2 and IP3R were associated with paxillin in differentiating lens fiber cells, and whether the MAP kinase JNK, which can phosphorylate both paxillin and Bcl-2, regulates the association between Bcl-2 with paxillin and the induction of the organelle-free zone (OFZ).
Differentiation-specific changes in linkage of ER proteins to paxillin were determined in chick embryo lenses microdissected into four regions of differentiation. Primary quail embryo lens cultures, which mimic lens differentiation as it occurs in vivo were used for functional studies with the JNK inhibitor SP600125 (25 µM). Protein-protein associations were examined by co-immunoprecipitation (IP) analysis, and protein localization by immunofluorescence analysis.
Co-IP analysis revealed that both Bcl-2 and IP3R were linked to paxillin in integrin focal adhesion complexes, most highly in differentiating lens fiber cells. This molecular complex has the potential to regulate the pathway that signals formation of the OFZ. Consistent with recruitment of Bcl-2 to this integrin-linked adhesion complex, we found that in differentiating cortical fiber cells Bcl-2 is highly localized at or near the plasma membrane. Inhibition of JNK activity in differentiating lens cell cultures caused the disassociation of Bcl-2 from paxillin adhesion complexes and induced formation of an OFZ. Dramatic loss of ER from the center of the lentoid was observed within 72 hr of exposure to the JNK inhibitor. Similar results were observed with a Golgi-specific marker (FTCD). The JNK inhibitor also induced nuclear loss, as evidenced by TUNEL staining, but the cells do not undergo apoptosis, mimicking formation of the OFZ as it occurs in vivo.
Inhibition of JNK signaling in differentiating lens fiber cells activates a pathway that can induce organelle loss, possibly through a mechanism involving loss of Bcl-2 from paxillin/Bcl-2/IP3R complexes.
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