Key connexin 43 phosphorylation events regulate the gap junction life cycle

Abstract

Connexin 43 (Cx43), the most widely expressed and abundant vertebrate gap junction protein, is phosphorylated at multiple different serine residues during its life cycle. Cx43 is phosphorylated soon after synthesis and phosphorylation changes as it traffics through the endoplasmic reticulum and Golgi to the plasma membrane, ultimately forming a gap junction structure. The electrophoretic mobility of Cx43 changes as the protein proceeds through its life cycle, with prominent bands often labeled P0, P1 and P2. Many reports have indicated changes in "phosphorylation" based on these mobility shifts and others that occur in response to growth factors or other biological effectors. Here, we indicate how phosphospecific and epitope-specific antibodies can be utilized to show when and where certain phosphorylation events occur during the Cx43 life cycle. These reagents show that phosphorylation at S364 and/or S365 is involved in forming the P1 isoform, an event that apparently regulates trafficking to or within the plasma membrane. Phosphorylation at S325, S328 and/or S330 is necessary to form a P2 isoform; and this phosphorylation event is present only in gap junctions. Treatment with protein kinase C activators led to phosphorylation at S368, S279/S282 and S262 with a shift in mobility in CHO, but not MDCK, cells. The shift was dependent on mitogen-activated protein kinase activity but not phosphorylation at S279/S282. However, phosphorylation at S262 could explain the shift. By defining these phosphorylation events, we have begun to sort out the critical signaling pathways that regulate gap junction function.

Fig. 1

The P2 isoform of Cx43 is phosphorylated at S325/328 and/or 330. (A) Detection of Cx43 present in whole cell lysates via Western immunoblot with a mouse antibody to total Cx43 shows the characteristic 3 isoforms (first lane, note P0, P1 and P2). Triton X-100 insoluble extracts (Tx Ins, lanes 2 and 3) show predominately the P2 isoform while the rabbit antibody to Cx43 phosphorylated at S325/328/330 (pS325) shows exclusively the P2 isoform. Immunofluroescence detection of cells with both Total Cx43 and the p325 antibodies show extensive overlay in gap junctional regions (B-D).

Fig. 2

The CT antibody recognizes the P0 isoform and Cx43 present in cytoplasmic membranes. (A) The antibody to total Cx43 recognizes all 3 isoforms of Cx43 (First lane) while probing the same preparation with the CT antibody (second lane) shows predominately the P0 isoform. (B) Immunofluorescence detection of cells with both Total Cx43 and the CT antibodies show extensive overlay in cytoplasmic membrane regions (B-D).

Fig. 3

S262 phosphorylation appears to be involved in a shift to a P2 isoform position upon TPA treatment. MDCK cells expressing wild type Cx43 (MDCK), CHO cells, or HeLa cells expressing Cx43 with a serine to alanine mutation (HeLa-262A) were either treated (+) with PMA or not (-) and probed with the NT antibody to total Cx43 (Total), to Cx43 with S279/S282 phosphorylated (pS279), Cx43 with S368 phosphorylated (pS368) and Cx43 with S262 phosphorylated (pS262).

Fig. 4

Model of how Cx43 phosphorylation at S364/S365 and S325/S328/S330 could affect the gap junction life cycle.