Biochemical and functional analysis of N-cadherin phosphorylation in embryonic chick tissues
The neural cell adhesion molecule N-cadherin can be phosphorylated and its function affected in oncogenic transformed cell lines. In several primary embryonic chick tissues, N-cadherin is known to be phosphorylated. However, the nature and function of these endogenous modifications remain unexplored. The objectives of this thesis are to first characterize the nature of this endogenous phosphorylation, followed by determining the consequences of modulating various protein kinases and phosphatases on aspects of N-cadherin expression and function. Phosphoamino acid and peptide mapping analyses indicate that N-cadherin from embryonic day 10 neural retina, brain, heart and lens are constitutively phosphorylated on the serine residues at multiple sites. Additional analysis in the retina reveals that N-cadherin can be phosphorylated by endogenous kinases on both serine and tyrosine, with the latter modification being normally kept at a minimal level by tyrosine phosphatases. These results indicate that N-cadherin expressed in primary embryonic tissues is the target of endogenous kinases and phosphatases of distinct functional classes. Several pharmacologic agents known to modulate kinase and phosphatase activities are used to determine the effects of altered N-cadherin phosphorylation state on its turnover, detergent solubility and adhesiveness. Retinal tissue cultures show that the turnover of N-cadherin into NCAD90 fragment is stimulated by enhanced tyrosine phosphorylation but attenuated by inhibitors of serine/threonine kinases. However, no significant alterations in N-cadherin detergent solubility and adhesiveness are observed. Since increased production of NCAD90 can be correlated with enhanced tyrosine phosphorylation of N-cadherin, the effects of insulin as well as insulin-like growth factor-I (IGF-I) on NCAD90 production and protein tyrosine phosphorylation are also examined. Insulin appears to stimulate NCAD90 production from treated retinal cultures although the increase is not statistically significant. Insulin also induces higher tyrosine phosphorylation of several retinal proteins despite having no specific effect on N-cadherin. The effects from insulin may have resulted partly through a cross activation of IGF-I receptors, since results of IGF-I treatment mimic those of insulin. In conclusion, these results suggest that the stability of N-cadherin on the membrane surface may depend on a critical balance of the activities from both serine/threonine and tyrosine kinases as well as phosphatases. The significance of this posttranslational modification, therefore, appears to be providing an epigenetic mechanism for modulating N-cadherin proteolysis to allow adhesive plasticity in the developing retina.
Lee, Mimi Meihui, "Biochemical and functional analysis of N-cadherin phosphorylation in embryonic chick tissues" (1996). ETD Collection for Thomas Jefferson University. AAI9625290.