Regulation of Signaling Pathways by A–Crystallin: A Possible Role for A–Crystallin Association With 6 Integrin Complexes

Abstract

Abstract: : Purpose: To examine the dependence of lens cell signaling pathways on αA–crystallin and determine whether this novel function for αA–crystallin may be related to its association with α6 integrin. Methods: Mouse lenses obtained from wild type or αA knockout lenses were microdissected into epithelial and cortical fiber fractions. Immunoblot analysis with antibodies to activated forms of specific cell signaling proteins (Src, p38, ERK and JNK) was performed. Analysis of these cell signaling pathways was also performed with control mouse lens cultures and those transfected with wild type αA–crystallin or the R116C αA–crystallin mutant. The association of αA–crystallin with α6 integrin in mouse epithelial and cortical fiber fractions was analyzed by immunoprecipitation with antibodies to α6 integrin followed by immunoblotting for αA–crystallin. Similar studies were performed with chicken embryo lenses microdissected into central and equatorial epithelia, cortical and nuclear fiber zones. Results: Src and p38 kinases were activated in the absence of functional αA–crystallin (knockout or R116C mutant). In contrast, signaling via the MAP kinase JNK was relatively unaffected. Signaling through ERK was enhanced only in the differentiating fiber cells of αA–/– lenses. In both adult mouse and embryonic chick lens we found that αA–crystallin was a component of α6 integrin complexes. In the mouse, this association was greater in the cortical fiber region than in the epithelial region. In the embryonic chicken lens, using an antibody to the lens differentiation–specific α6A integrin isoform, we found the highest association between αA–crystallin and α6A integrin in the differentiating fiber zones. Immunolocalization of αA–crystallin to cell–cell interfaces of cultured embryonic chicken lens cells, the site to which we have previously localized α6 integrin, supports the role of αA–crystallin in these important membrane–associated signaling complexes, and suggests that αA–crystallin may stabilize integrin–associated cell signaling pathways. Conclusions: Our work suggests that αA–crystallin is associated with α6 integrin complexes both during lens development and in the mature lens, where it is likely to function in regulating α6 integrin signaling pathways.

Keywords: crystallins • cell adhesions/cell junctions • signal transduction