Abstract
Abstract: :
Purpose: We have previously shown that inhibition of Src activity in lens epithelial cell cultures induces assembly of N–cadherin junctions, signaling cell compaction, cell cycle withdrawal and the initiation of differentiation. We now have investigated the role of Src kinases and the SHP2 tyrosine phosphatase in regulating N–cadherin junctional assembly in the differentiating lens. Methods: Microdissected embryonic day 10 chick lenses enabled us to use Western Blot analysis to determine in vivo expression of Src kinases and SHP2 and the activation state of the Src kinases at four distinct stages of lens cell differentiation. Differentiation–specific changes in N–cadherin complexes were examined in the microdissected fractions by immunoprecipitation of N–cadherin followed by Western Blotting for ß–catenin, α–catenin, Src and SHP2. Results: Assembly of cadherin cell–cell junctions involves the clustering of cadherin receptors and their linkage to the actin cytoskeleton, mediated through catenins. ß–catenin associates with the cadherin cytoplasmic domain and recruits α–catenin, to link the cadherin complex to the actin cytoskeleton. Tyrosine phosphorylation of ß–catenin is known to cause diminished linkage of the cadherin complex to the actin cytoskeleton; therefore, the action of kinases and phophatases associated with the cadherin complex are likely to be crucial to the regulation of cadherin function. Src kinases and the SHP2 phosphatase were expressed throughout lens cell differentiation. Src kinases were active at all stages of lens differentiation, but activity was highest in the central and equatorial epithelia. Active Src kinases were associated with N–cadherin complexes throughout differentiation. While ß–catenin association with N–cadherin remained similar throughout lens cell differentiation, ß–catenin phosphorylation was modulated with lens cell differentiation state. Consistent with the important role of α–catenin in cadherin junction stabilization, the induction of differentiation in the equatorial epithelium coincided with an increased association of α–catenin with the N–cadherin complex. This was paralleled by recruitment of SHP2 to N–cadherin complexes, suggesting that SHP2–mediated dephosphorylation of cadherin complex proteins is responsible for stabilizing cadherin junctions as lens cells differentiate. Conclusions: Our data demonstrates that differentiation–specific changes in N–cadherin/catenin complex formation are consistent with the regulation of cadherin junction formation by Src kinase/SHP2 phosphatase signaling.
Keywords: cell adhesions/cell junctions • signal transduction • phosphorylation