Post-translational regulation of connexin 50 and the interaction with aquaporin 0 in lens fibers

The first goal of this dissertation is to elucidate the in vivo phosphorylation sites of Cx50, identify the responsible kinase(s), and characterize the potential functions. Previous studies in Dr. Jiang's laboratory have shown that chicken Cx50 is phosphorylated in vivo by casein kinase 2 (CK2) at Ser364, which not only facilitates Cx50 turnover but also prevents the caspase-3-mediated cleavage at the C-terminus during lens development. Here, I further identified Ser395 as a new in vivo phosphorylation site at the C-terminus of Cx50 by using mass spectrometry. This serine residue is highly conserved across various species and located within a protein kinase A (PKA) consensus site. Data from in vitro and in vivo phosphorylation analysis confirmed that Ser395 was the targeting site of PKA in the lens in vivo. Functional significance of this specific phosphorylation by PKA was further assessed in chicken embryo fibroblasts (CEF) with exogenous connexin expression through retroviral infection or in lens primary cultures. Dye transfer and dye uptake studies showed that in the presence of either PKA activator forskolin or 8-Br-cAMP, both Cx50 gap junction and hemichannel activities were significantly increased, whereas the corresponding activities for S395A mutant were reduced but not totally abolished. This increase is not likely through the enhancement of total Cx50 expression or Cx50 on the cell surface. Together, my findings suggest that Cx50 is phosphorylated by PKA at Ser395 in the lens in vivo and this phosphorylation plays an important role in promoting gap junction-mediated intercellular communication of lens fibers.;Aquaporin 0 (AQP0), also known as lens major intrinsic protein (MIP), is the most abundant membrane protein expressed in lens fibers. It not only serves as a water channel but also plays an important role in the cell-to-cell adhesion of lens fibers. Previous studies in Dr. Jiang's laboratory have shown that the C-terminus (CT) domain of AQP0 directly associates with the intracellular loop (IL) domain of Cx50 in the differentiating lens fibers. However, the physiological significance of this interaction remains unclear. Here, through the retroviral expression of wild-type lens connexins and the corresponding IL domain-swapped chimeras in embryonic chick lens, I demonstrated the importance of Cx50 IL domain in mediating the interaction with AQP0 in the lens in vivo. In the presence of AQP0, the gap junction conductance was significantly increased for homomeric Cx50 or heteromeric Cx50/Cx46, but not for chimeras whose Cx50 IL domain was replaced with those from other lens connexins (Cx50*46L or Cx50*43L). Moreover, the channel assembly rate and total electrical conductance of Cx50 were also increased in paired Xenopus oocytes in the presence of AQP0. In contrast to its effect on gap junctions, AQP0 had no effect on hemichannel activity and Cx50 expression level on the cell surface. Cell adhesion assay indicated that a fusion protein containing all three extracellular loop (EL) domains of AQP0 could efficiently block the adhesion function of AQP0 on the cell surface. Application of this fusion protein attenuated the stimulatory effect of AQP0 on Cx50 gap junctions. All these data suggest that the specific interaction between Cx50 and AQP0 enhances the ability of Cx50 to form functional gap junctions but not hemichannels, likely through the cell-to-cell adhesion function of AQP0.;In this study, I for the first time identified Ser395 as a new in vivo PKA phosphorylation site in lens Cx50 and further explored its functional role in promoting gap junction intercellular communication in CEF cells and lens primary cultures. The increase in gap junction and hemichannel activities induced by Ser395 phosphorylation is not likely mediated through the enhancement of total expression level of Cx50 in the cell or on the cell surface. In addition, I also revealed the physiological significance of the Cx50-AQP0 interaction in enhancing Cx50 to form functional gap junctions but not hemichannels through the cell-to-cell adhesion function of AQP0. The outcomes of these experimental findings will provide invaluable insights into the modulation of gap junction channels by post-translational modifications (PTM) as well as shed light on the regulation of gap junction function by the protein binding partners. (Abstract shortened by UMI.)...