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Jean X. Jiang, Jialu Liu, Jose F. Ek Vitorin, Janis M. Burt, Sumin Gu, Qian Shi; In vivo Phosphorylation of Lens Connexin 50 by Protein Kinase A Enhances Gap Junction and Hemichannel Function. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3929.
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© ARVO (1962-2015); The Authors (2016-present)
Phosphorylation of connexins is an important mechanism in regulating the physiological function of gap junction channels in the lens. However, the in vivo role(s) of phosphorylation at certain residues of connexins is largely unknown.
Lens lysates were isolated from 3-month old chicken lenses and subjected to mass spectrometric analysis to identify in vivo phosphorylation sites. Tryptic peptide digestion and 2-dimensional thin layer chromatography were adopted to compare in vivo and in vitro phosphorylation by protein kinase A (PKA). Gap junction function was analyzed using both dye transfer and patch clamp electrophysiological approaches. Hemichannel activity was studied using dye uptake assay. Overall protein expression was analyzed using Western blots and the level of connexin (Cx) 50 on the plasma membrane was assessed by cell surface biotinylation.
We showed that Ser395 in the distal C-terminus of Cx50, a highly conserved residue across various species, is phosphorylated in the lens in vivo. Ser395 is located within a protein kinase A (PKA) consensus site. Comparison of two-dimensional thin-layer chromatography tryptic phosphopeptide profiles between in vitro PKA phosphorylation and in vivo phosphorylation using lens organ culture suggested that Ser395 was, indeed, phosphorylated in vivo by PKA. Moreover, the autoradiographic signal from the tryptic phosphopeptides containing phosphorylated Ser395 was enhanced after the treatment with a PKA activator, 8-Br-cAMP. Dye transfer and dye uptake studies showed that in the presence of either PKA activator forskolin or 8-Br-cAMP, both gap junction coupling and hemichannel activities of Cx50 were greatly increased, whereas this increase was significantly reduced in the cells expressing S395A mutant. This increase in gap junction and hemichannel activities induced by Ser395 phosphorylation is mediated neither through the enhancement of total expression level of Cx50 protein in the cell or on the cell surface nor the increased gap junctional plaques. Single channel recordings suggest that PKA-activation results in a shift toward higher single channel conductance values and, possibly, the preservation of high coupling at the macroscopic level.
These results suggest that Cx50 is in vivo phosphorylated by PKA at Ser395, and this phosphorylation plays an important role in promoting gap junction and hemichannel function in lens fibers.
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