Xinye Yin, Ph.D.

The University of Texas Graduate School of Biomedical Sciences
At San Antonio

Supervising Professor: Jean X. Jiang

DISSERTATION ABSTRACT

Gap junctions, composed of a family of proteins named connexins, are clusters of transmembrane channels that connect the cytoplasms of adjacent cells and allow passage of small molecules whose molecular weights are lower than 1000Da. The gap junction mediated cell-cell communications are important in maintaining cell homeostasis and function as indicated by a variety of human diseases caused by mutations of different connexin genes. Post-translational phosphorylation is a hallmark for most of connexins and has been previously indicated in regulation of gap junction communication. However, none of the phosphorylation sites in the animal system has been identified and the physiological significance and regulatory mechanisms of connexin phosphorylation are less certain. Thus, the first goal of this dissertation is to identify the in vivo phosphorylation site(s) of a connexin and study the function and regulation the specific phosphorylation. The chick lens is chosen as a model system since it contains a large network of gap junctions and connexin phosphorylation in chick lens primary culture has been shown to closely mimic the in vivo situation. Connexin (Cx) 45.6 is one of the two connexins that expressed in chick lens fibers and has been shown to play unique function in lens growth and homeostasis. Here, we identified two in vivo phosphorylation sites, Ser³⁶³ and Ser³⁷⁰ of Cx45.6 by mass spectrometry. Both serine residues are located in the COOH terminus of Cx45.6 and Ser³⁶³ is within casein kinase II phosphorylation consensus sequence. Data from in vitro and in vivo phosphorylation analysis confirmed that Ser³⁶³ was the targeting site by casein kinase II. To assess the function of Cx45.6 phosphorylation, exogenous wild type and phosphorylation site mutants of Cx45.6 were expressed in lens primary cultures by retroviral infection. The single site mutants, Cx45.6(S363A) and Cx45.6(S370A), were shown to have longer half-lives compared with the wild type Cx45.6. Moreover, the double site mutant Cx45.6(S363, 370A) had an even longer half-life than each single site mutant. These results suggest that phosphorylation on Ser³⁶³ and Ser³⁷⁰ are cooperatively involved in stimulation of Cx45.6 turnover.

In addition to phosphorylation, we observed another type of post-translational modification in Cx45.6 during lens development. Characterization of this type of modification is another major part of this dissertation. The majority of the truncated fragments were identified in the differentiated lens fibers. Interestingly, an apoptotic protease, caspase-3 was found to be responsible for cleavage of Cx45.6 and the cleavage site was identified at Glu³⁶⁷, close to the phosphorylation sites at Ser³⁶³ and Ser³⁷⁰. Furthermore, the cleavage of Cx45.6 was inhibited by phosphorylation at both serine residues, suggesting that phosphorylation of Cx45.6 alternatively regulates Cx45.6 turnover and its specific cleavage by caspase-3 during lens development.

This study, for the first time, identifies the in vivo phosphorylation sites of a connexin and reveals the functional roles of phosphorylation in a system that is close to the in vivo condition. In addition, this is the first report demonstrating that a connexin can be a direct target of an apoptotic protease and suggesting a novel function of a connexin in apoptosis and differentiation. The outcomes of these experimental findings will shed light on the regulation of gap junction channels by post-translational modifications in the other cells, organs and animal species as well as provide valuable insights into normal vertebrate lens biology.