(B) Immunofluorescence using pS365 and a monoclonal antibody for total Cx43 shows that pS365 resides predominantly in the plasma membrane (WT Cx43) and that the pS365 epitope is eliminated by conversion from serine to alanine or aspartate (S365A, S365D) (bar = 25 m). Phosphorylation on S365 creates the P1 isoform To study the role of S365 phosphorylation on the migratory isotypes of Cx43, we examined multiple clones of MDCK cell lines expressing Cx43 WT, S365A, and S365D. cells from ischemia and phorbol ester-induced down-regulation of channel conductance. Introduction Gap junctions are intercellular structures that facilitate direct communication among adjacent cells by allowing passage of ions and small metabolites (White and Paul, 1999; Saez et al., 2003; Sohl and Willecke, 2004; Laird, 2006). Vertebrate gap junctions, composed of integral membrane proteins encoded by the 21-member connexin gene family, are critically important in regulating embryonic development, coordinated contraction of excitable cells, tissue homeostasis, normal cell growth, and differentiation (Saez et al., 2003; Sohl and Willecke, 2004). Their functional importance has been shown by the linkage of connexin mutations to deafness and several diseases (Bergoffen et al., 1993; Gong et al., 1997; Kelsell et al., 1997; Laird, 2006) including oculodentodigital dysplasia, a disease linked to connexin43 (Cx43) mutations that can cause atrioseptal defects and arrhythmias (Paznekas et al., 2003). Cx43, the most ubiquitously expressed connexin, is differentially phosphorylated at a dozen or more serine residues throughout its life cycle (Lampe and Lau, 2004; Solan and Lampe, 2005). Regulation of Cx43 may occur at many different stages, including the oligomerization of six connexin proteins into a hemi-channel or connexon, connexon Revaprazan Hydrochloride transport to the plasma membrane, intercellular docking of connexons to form intercellular channels, condensation of hundreds to thousands of these channels into paracrystalline arrays (termed gap junctional plaques), gap junction channel gating, and turnover of plaques. There is correlative evidence that gap junctional communication and Cx43 levels may be controlled by phosphorylation of Cx43 at many of these steps; however, the mechanisms underlying these events Mmp9 have remained elusive. Cx43 from cultured cells commonly demonstrates multiple electrophoretic isoforms when analyzed by SDS-PAGE: a faster migrating form (sometimes referred to as P0 or NP) that includes the nonphosphorylated isoform, and multiple slower migrating forms (sometimes termed P1 and P2) (Musil and Goodenough, 1991). After alkaline phosphatase treatment in vitro, the phosphorylated species collapse to the fastest migrating form, suggesting that phosphorylation is the primary covalent modification resulting in the conformational change detected in SDS-PAGE analysis. Studies investigating phosphorylation in normal rat kidney (NRK) cells showed that Cx43 acquired resistance to Triton X-100 and was found in gap junction plaques when it had been phosphorylated to the P2 isoform (Musil and Goodenough, 1991). P2 formation requires phosphorylation at S325, S328, and/or S330 (Lampe et al., 2006). Myocardial ischemia leads to unspecified Cx43 dephosphorylation events and loss of localization to the intercalated disk, which likely contribute to contractile failure Revaprazan Hydrochloride and arrhythmias (Beardslee et al., 2000; Schulz et al., 2003). Thus, uncharacterized phosphorylation events have been correlated with changes in Cx43 localization, inclusion in gap junction structures, and acquisition of Triton X-100 insolubility. We have previously shown that Cx43 is phosphorylated at S368 in basal keratinocytes near the edge of a human wound, in heart tissue upon hypoxia, during S and G2/M phases of the cell cycle, and upon treatment with phorbol esters (Lampe et al., 1998; Solan et al., 2003; Richards et al., 2004; Ek-Vitorin et al., 2006). Phosphorylation at S368 reduces the conductivity of the gap junction channel, potentially causing the formation of communication compartments (Ek-Vitorin et al., 2006). In this paper, we show that Cx43 is phosphorylated on S365 in homeostatic cells and that this event is required for formation of the P1 isoform. Structural studies of the C-terminal region of Cx43 with a S365D mutation, intended to mimic phosphorylation, indicate that this change generates a different stable conformation than unphosphorylated, wild-type Cx43. Preparation and use of an antibody specific for Cx43 only when it is phosphorylated at S365 showed that this phosphorylation event occurs on Cx43 present specifically Revaprazan Hydrochloride in gap junction structures in cultured cells and in heart tissue. Phosphorylation at S365 was constitutive in heart tissue but absent during hypoxia when Cx43 loses its specific localization at the intercalated disk and phosphorylation at S368 increases (Ek-Vitorin et al., 2006). Western, immunoprecipitation, and in vitro kinase studies.