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A. Sitaramayya, J.W. Crabb, A. Margulis, D.F. Matesic, V. Singh, S. Pulukuri; Analysis of Phosphorylation of Connexin 36 in Bovine Retina . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4583.
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Purpose: Gap junction-mediated intercellular communication between specific retinal neurons is known to be regulated by cyclic nucleotide-dependent protein kinases. Connexin 36 is a major gap junction protein in retina. We attempted here to demonstrate phosphorylation of connexin 36 by kinases responsive to cyclic nucleotides and calcium. Methods: Retinal homogenate was phosphorylated using 32P-ATP in the presence of cyclic AMP, cyclic GMP or calcium. Proteins of the membrane fraction and membranes enriched in gap junctions were resolved by electrophoresis, and phosphoproteins were detected by autoradiography. Phosphorylated membrane proteins were also dissolved in detergent and connexin 36 was immunoprecipitated with an anti-connexin 36 antibody. Immunoprecipitated proteins were separated by 1- or 2-D electrophoresis and phosphorylated proteins were detected by autoradiography. Proteins of interest were excised from 1- or 2-D gels, digested in situ with trypsin and identified by capillary LC MS/MS. Results: Specific retinal membrane proteins were phosphorylated in response to activation by cyclic AMP, cyclic GMP or calcium. Of them, a protein of about 36 kDa was phosphorylated only in incubations with calcium. However, immunoprecipitated connexin 36 was found not to be phosphorylated. The immunoprecipitate did contain other phosphorylated proteins, and the most prominent of them, a 58 kDa protein, was identified as beta tubulin. Alpha tubulin and trypsin inhibitor were also detected in the immunoprecipitate. Conclusions: These results suggest that connexin 36 may not be directly phosphorylated in response to changes in cyclic nucleotides or calcium. Proteins associated with connexin 36, though not necessarily the ones we identified so far, might be targets of phosphorylation and could be involved in the regulation of gap junction intercellular communication.
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