Connexin43 (Cx43) one of the most widely expressed and abundant vertebrate space junction protein is phosphorylated at multiple different serine residues during its existence cycle. P1 isoform an event that apparently regulates trafficking to or within the plasma membrane. Phosphorylation at S325 328 and/or 330 is necessary to form a P2 isoform and this phosphorylation event is present only in space junctions. Treatment with protein kinase C activators led to phosphorylation at S368 S279/S282 and S262 having a shift in mobility in CHO cells but not MDCK cells. The shift was dependent on MAPK activity but not phosphorylation at S279/282. However phosphorylation at S262 could clarify the shift. By defining these phosphorylation events we have begun to be able to sort out the crucial signaling pathways that regulate space junction function. phosphorylated on S364 or S365 termed “CT” showed that these residues look like important for trafficking to the plasma membrane. Immunofluorescence staining showed that this antibody acknowledged Cx43 in the cytoplasm only and not in the plasma membrane (Fig. 2B-D) while immunoblots showed that in resting cells this antibody acknowledged primarily the P0 form of Cx43. Cell-surface biotinylation assays showed that essentially all isoforms including P0 could reach the plasma membrane while acquisition of Triton X-100 insolubility and inclusion in plaques was correlated with phosphorylation to the P2 form (Musil & Goodenough 1991 The practical relevance of the P1 form however has not been shown. Interestingly one MI 2 feature of the Rabbit Polyclonal to DQX1. “CT” antibody is definitely that it by no means recognizes the P1 form (Fig. 2 and Sosinsky et al. 2007 Since “CT” recognizes non-phosphorylated S364 and S365 and does not identify P1 it is likely that phosphorylation on one or both of these residues prospects to the P1 isoform. While the cell-surface biotinylation data shows the phosphorylation event leading the P1 isoform may occur in the plasma membrane (Musil & Goodenough 1991 the immunofluorescence data (Fig. 2) shows that this event is required for trafficking MI 2 from your cytoplasm to the plasma membrane (Fig. 4). Since hemichannels are made up of 6 connexins it may be that only a fraction of these need be phosphorylated to propel ahead trafficking. This would result in the cytoplasmic Cx43 becoming “CT” reactive i.e. not phosphorylated on S364 or S365. The P0 MI 2 or “CT” isoform in the plasma membrane could be diffuse and therefore undetectable by immunofluorescence until entering a space junction plaque where it would become more concentrated and eventually phosphorylated to the P2 isoform. Induced Phosphorylation Can Lead to a distinct P2 Treatment of cells with numerous stimuli can result in a shift of Cx43 to slower migrating forms and is often associated with downregulation of space junctional communication. Several studies have focused on using growth factors and PMA in combination with MAPK and PKC inhibitors to correlate changes in Cx43 isoform migration with shutdown of space junctional communication. In MI 2 one study IAR6.1 cells which endogenously express Cx43 and help to make P2 in resting cells exhibited a decrease in space junctional communication and a migration shift in response to both PMA and EGF (Rivedal & Opsahl 2001 In these cells inhibition of ERK1/2 but not PKC inhibition could inhibit the migration shift in response to PMA and EGF although it did not reverse PMA induced inhibition of space junctional communication. This led the authors to conclude the migration shift was due to phosphorylation on Cx43 via ERK1/2. However which sites might be responsible was not identified. The sites where ERK1/2 phosphorylates Cx43 have been determined to be S255 S279 and S282 and when crazy type Cx43 or S279/S282/S255A mutant Cx43 were indicated in HeLa cells EGF treatment led to a migration shift in both crazy type and mutant expressing cells although inhibition of communication was only observed in crazy type Cx43 expressing cells (Warn-Cramer et al. 1998 Warn-Cramer et al. 1996 Inhibition of ERK1/2 reversed both of these effects. Both of these MI 2 studies are consistent with the idea that ERK1/2 activation can lead to P2 formation although not through phosphorylation on S279.