POSTTRANSLATIONAL PHOSPHORYLATION OF LENS FIBER CONNEXIN46 - A SLOW OCCURRENCE

Purpose. To study in both in situ and primary cultures the posttransla tional phosphorylation of connexin46 (Cx46), one of two members of the connexin family of gap junction proteins expressed by lens fibers. Me thods. Phosphatase digestion, gel electrophoresis, cell culture, organ culture, immunoprecipitation, metabolic labeling, and phosphoamino ac id analysis were the methods used in this study. Results. Cx46 immunop recipitated from either rat or bovine lenses resulted in a shift to a more rapidly migrating species. During rat embryonic development, the more rapidly migrating, nonphosphorylated form of Cx46 was prevalent a t 15 days gestation; as development progressed, there was a loss of th e nonphosphorylated form with a concomitant increase in the phosphoryl ated form, such that by 28 days after birth only the phosphorylated fo rm was detectable. The rate of posttranslational phosphorylation was v ery slow compared to previously measured rates for connexin43. Primary cultures of rat embryonic lens epithelial cells, which contained diff erentiating lentoids, were labeled with S-35-methionine and chased for 8 days. Very low levels of Cx46 were detectable, and none of this lab eled material shifted to the slower mobility during the 8-day chase pe riod. Similarly, in organ culture of bovine lenses, Cx46 could be labe led with S-35-methionine, but the immunoprecipitated material remained in the rapidly migrating form for 1 week, the longest time measured. This immunoprecipitated material was shown to be serine-phosphorylated , which was insufficient to cause the electrophoretic mobility shift. Conclusions. There are low levels of Cx46 synthesis and phosphorylatio n in rat embryo lens primary cultures and a slow rate of phosphorylati on of Cx46 in bovine organ cultures.