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Jaya Aseervatham, Helen Wang, Alexsandra Espejo, Cheryl Mitchell, Mark Bedford, John O'Brien; Binding and regulation of Cx36 by 14-3-3 proteins. Invest. Ophthalmol. Vis. Sci. 2020;61(7):717.
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14-3-3 proteins are highly conserved and ubiquitously expressed phospho-serine/threonine binding proteins. They regulate a variety of neuronal processes such as neurite outgrowth, differentiation and synaptic transmission. Cx36 gap junctions in retina are subject to light-dependent plasticity and the coupling strength is regulated by phosphorylation of sites in the C terminus. This study was performed to examine whether 14-3-3 proteins are involved in regulation of Cx36 coupling.
Phosphorylation-specific protein binding was assessed by protein microarrays probed with synthetic peptides corresponding to the non-phosphorylated and S315 phosphorylated C terminus of Cx36. Pull-down assays employed the same peptides attached to streptavidin magnetic beads incubated with crude GST fusion proteins. Tracer coupling measurements were performed in HeLa cells transfected with Cx36 and Cx36 mutants with or without 14-3-3 siRNA. Immunohistochemistry was done on retina from C57BL6 mice that were light- or dark-adapted for 3 hours using antibodies for 14-3-3 protein isoforms β, γ, ε, η, ζ, σ, and τ/θ.
A “phospho-reader” protein microarray study revealed that 14-3-3 γ, η and σ bound to the S315 phosphorylated C-terminus of Cx36 and much less to non-phosphorylated CT. Binding was eliminated by truncation at S315. GST pull-down assays confirmed the peptide microarray results. Tracer coupling experiments indicated that Cx36 S315 truncation, non-phosphorylatable Cx36 S315A mutation or 14-3-3 η siRNA all produced the same disruption of Cx36 regulation, inverting regulation of coupling by protein kinase A such that PKA activation enhanced coupling. Confocal microscopy showed that all the 14-3-3 isoforms except ζ co-localized with Cx36 in the outer and inner plexiform layer. Zeta/delta was mostly present in the nucleus. The expression of 14-3-3‘s was significantly (p<0.05) higher in the light-adapted than in the dark-adapted state.
Several 14-3-3 proteins bind to the C terminus of Cx36 and this binding is involved in regulation of coupling by mediating a C-terminal functional switch. Light adaptation in retina enhances the association. Since Cx36 coupling is decreased in the light adapted retina, binding of 14-3-3 may directly or indirectly contribute to the closing of Cx36 channels and depends on the phosphorylation state of Cx36.
This is a 2020 ARVO Annual Meeting abstract.
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