Posttranslational phosphorylation of lens fiber connexin46: a slow occurrence.
OBJECTIVE
To study in both in situ and primary cultures the posttranslational phosphorylation of connexin46 (Cx46), one of two members of the connexin family of gap junction proteins expressed by lens fibers.


METHODS
Phosphatase digestion, gel electrophoresis, cell culture, organ culture, immunoprecipitation, metabolic labeling, and phosphoamino acid analysis were the methods used in this study.


RESULTS
Cx46 immunoprecipitated 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 at 15 days gestation; as development progressed, there was a loss of the nonphosphorylated form with a concomitant increase in the phosphorylated form, such that by 28 days after birth only the phosphorylated form was detectable. The rate of posttranslational phosphorylation was very slow compared to previously measured rates for connexin43. Primary cultures of rat embryonic lens epithelial cells, which contained differentiating lentoids, were labeled with 35S-methionine and chased for 8 days. Very low levels of Cx46 were detectable, and none of this labeled material shifted to the slower mobility during the 8-day chase period. Similarly, in organ culture of bovine lenses, Cx46 could be labeled with 35S-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 phosphorylation in rat embryo lens primary cultures and a slow rate of phosphorylation of Cx46 in bovine organ cultures.
Jiang
J X
JX
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115.
Paul
D L
DL
Goodenough
D A
DA
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D017630:Connexins / Q000096:biosynthesis / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian; D017629:Gap Junctions / Q000378:metabolism; D007908:Lens, Crystalline / Q000196:embryology / Q000378:metabolism*; D009924:Organ Culture Techniques; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D051381:Rats
D017630:Connexins; C082917:GJA3 protein, human