Phosphorylation of lens-fiber connexins in lens organ cultures.
Lens gap junction proteins, connexins [1], are known to be phosphorylated in vivo. Phosphorylated states of connexins were examined in lens cultures to define in vitro models for the study of the functions of lens connexin phosphorylation in lens biology. In organ and differentiated cell primary cultures, chick lens-fiber connexins, connexin45.6 and connexin56, were labeled with [32P]orthophosphate. Pulse-chase experiments of lens organ cultures with [35S]methionine demonstrated that connexin45.6 and connexin56 were properly processed into the phosphorylated forms observed in vivo. However, in lens cell primary cultures, both connexins had short half-lives, and connexin56 was degraded before it was phosphorylated into the form which showed the largest mobility shift. The data suggested that the phosphorylation patterns of connexins in lens organ cultures were similar to in vivo connexin phosphorylation, while primary cultures revealed abnormal rates of protein turnover and incomplete phosphorylation. Treatment of lens organ cultures with protein kinase inhibitors indicated that protein kinase C was involved in the phosphorylation of connexin45.6 and connexin56. Comparison of the phosphopeptide patterns by two-dimensional mapping suggested that protein kinase C was involved in the phosphorylation of connexin45.6 and that it phosphorylated the C-terminus of connexin45.6 in vitro.
Department of Biochemistry, University of Texas Health Science Center at San Antonio, 78284-7760, USA. jiangj@uthscsa.edu
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Jiang
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Goodenough
D000818:Animals; D002642:Chick Embryo; D017630:Connexins / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism*; D007908:Lens, Crystalline / Q000378:metabolism*; D009924:Organ Culture Techniques; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011499:Protein Processing, Post-Translational
D017630:Connexins; D005136:Eye Proteins; C082920:connexin 45; C078866:connexin 56; D011493:Protein Kinase C