The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the ras oncogene modulate expression and phosphorylation of gap junction proteins.

Abstract

Gap junctional intercellular communication is inhibited in response to tumor promoters and oncogene transformation, suggesting that loss of this function is an important step in tumor formation. To elucidate the molecular mechanisms responsible for this inhibition, we examined the expression of gap junction proteins and mRNA in mouse primary keratinocytes after treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or ras transformation. During normal cell growth, keratinocytes express the a, (connexin 43) and P (connexin 26) proteins. Within 5 min of TPA treatment, the al protein became rapidly phosphorylated on serine residues and its expression was dramatically reduced by 24 h. The 12 protein, after an initial increase in expression, was also significantly reduced 24 h after treatment with TPA. ras transformation caused changes similar to those induced by TPA. The al protein underwent an increase in serine phosphorylation, although its expression declined only slightly, while 012 expression was greatly reduced.The effects of TPA and ras on a, expression were additive; treatment of ras-transformed cells with TPA resulted in increased a, phosphorylation, with greatly decreased protein levels, much lower than those generated by either agent alone. These data provide a likely explanation for the similar and synergistic inhibition of gap junctional intercellular communication by phorbol esters and ras.

Cyclin-dependent kinase antagonizes promyelocytic leukemia zinc-finger through phosphorylation

“…Primary murine keratinocytes were maintained in culture as previously described (Brissette et al, 1991). Labeling proceeded for 4 h, starting 48 h after transfection (COS cells).…”

A Self-restricted CD38-connexin 43 Cross-talk Affects NAD+ and Cyclic ADP-ribose Metabolism and Regulates Intracellular Calcium in 3T3 Fibroblasts

“…ϩ Transport Mediated by Cx43 Hemichannels-Phosphorylated versus nonphosphorylated Cx43 channels and hemichannels are known to feature remarkably different activities of solute exchange, which are also differentially regulated by the multiple sites of phosphorylation recognized by the several protein kinases acting on Cx43 (35,36,38,39). Moreover, although this effect was observed in a quite restricted range of expression of nonphosphorylated Cx43 (see "Discussion"), also the specific NAD ϩ transporting activity is much higher in the native CD38 Ϫ cells than in the same cADPR-loaded cells or in the CD38 ϩ fibroblasts.…”

Regulation of gap junctions by protein phosphorylation

“…Frequently, in cells where the unphosphorylated form of Cx43 (Cx43-NP) predominates under basal conditions, stimulation of PKC with a tumor promoter phorbol ester (e.g., TPA) leads to rapid cell uncoupling and shifts the electrophoretic mobility of Cx43 forms (30,45,52,53,55). By contrast, in cells where the phosphorylated forms of Cx43 predominate (e.g., rat cardiocytes) TPA promotes intercellular communication with no detectable changes in the state of phosphorylation, as evaluated by Western blots (42).…”

“…Various phospho-forms of Cx43 can be resolved by immunoblotting, making it a useful technique to evaluate its state of phosphorylation (13)(14)(15)20,25,29,30,33,35,37,41,42,(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56). Nonetheless, not all changes in the state of phosphorylation of Cx43 alter the electrophoretic mobility of the protein (42).…”