Phosphorylation of the 27-kDa Gap Junction Protein by Protein Kinase C In Vitro and in Rat Hepatocytes

Abstract

We previously demonstrated that the 27-kDa major component protein in rat liver gap junctions was phosphorylated by protein kinase C in vitro (Takeda, A. et al. (1987) FEBS Lett. 210, 169-172). In this study, we examined this further and examined the phosphorylation of the 27-kDa gap junction protein in rat hepatocytes by metabolically labeling cells with [32P]orthophosphate and using a monoclonal antibody to immunoprecipitate the protein. The in vitro phosphorylation was inhibited by monoclonal antibodies recognizing the carboxyl- (C-)terminal domain of the 27-kDa protein. Protease digestion analysis revealed that phosphorylation occurred at the C-terminal domain. In rat hepatocytes, the phorbol esters, 12-O-tetradecanoylphorbol-13-acetate and phorbol-12,13-dibutyrate, stimulated the 27-kDa protein phosphorylation, whereas 4 alpha-phorbol-12,13-didecanoate did not. 1-Oleoyl-2-acetyl-sn-glycerol also stimulated the 27-kDa protein phosphorylation. In addition, norepinephrine stimulated the phosphorylation and pretreatment of hepatocytes with staurosporine, a potent inhibitor of protein kinase C, inhibited this stimulatory effect of norepinephrine. Both in vitro and in hepatocytes, analysis of chemical cleavage of the 27-kDa phosphoprotein revealed that phosphorylation occurred mainly at a 10-kDa fragment which the antibodies recognized. These results indicate that protein kinase C phosphorylates the 27-kDa gap junction protein, not only in vitro but also in hepatocytes, at the C-terminal domain of the protein.

Regulation of gap junctions by protein phosphorylation

“…Activation of either cAMP-dPK or PKC increases the state of phosphorylation of Cx32 (34,63,70,72) and at least the effect of cAMP-dPK is temporally correlated with an increased junctional conductance (g j ). Moreover, Chanson et al (71) reported that an increase in intracellular [cAMP] in a human colonic T84 cell line induces a rapid (<20 min) increase in intercellular gap junctional communication mediated by Cx32 gap junctions, that is directly related to an increase in fluid secretion.…”

“…cAMP increases the amount of metabolically labelled Cx32 in primary cell cultures of fetal hepatocytes (70), implying either an increased rate of synthesis or a reduced rate of degradation. The stoichiometry of phosphorylation of Cx32 in vitro is low; by PKC it approaches 1 mol/mol and by cAMP-dPK it is about 0.1 mol of P i /mol of protein (34,63,72). Nevertheless, the stoichiometry of phosphorylation in vivo and the cell compartment in which Cx32 phosphorylation occurs have not been determined.…”

“…Moreover, in isolated liver gap junctions previously phosphorylated by PKC, treatment with cAMP-dPK does not increase the incorporation of 32 P into Cx32. On the other hand, if gap junctions are first phosphorylated by cAMP-dPK, treatment with PKC increases the incorporation of 32 P into Cx32, suggesting that PKC can phosphorylate other amino acyl residues besides those phosphorylated by cAMP-dPK (72). Using synthetic peptides corresponding to regions of the C-terminus of Cx32, it has been demonstrated that Ser 233 is phosphorylated by both cAMP-dPK and PKC (34).…”

Connexins as Substrates for Protein Kinases and Phosphoprotein Phosphatases

“…The most frequently used experimental approaches to demonstrate that a particular Cx is a phosphoprotein include metabolic labeling of cultured cells with 32 P followed by immunoprecipitation and alkaline phosphatase treatment, phosphoamino acid analysis (Sáez et al 1986;Takeda et al, 1989;Musil et al, 1990;Crow et al, 1990;Sáez et al, 1990;Lau et al, 1992;Goldberg and Lau, 1993;Kurata and Lau, 1994;Doble et al, 1996;Warn-Cramer et al, 1996;Mikalsen et al, 1997;Cheng and Louis, 1999) or two-dimensional phosphopeptide mapping (Sáez et al, 1990;Kurata and Lau, 1994;Díez et al, 1995;Loo et al, 1995;WarnCramer et al, 1996;Berthoud et al, 1997;Díez et al, 1998;Kanemitsu et al, 1998) in vitro phosphorylation assays using fusion proteins or synthetic peptides containing the putative phosphorylation site(s) and purified protein kinases (Sáez et al, 1990;Loo et al, 1995;WarnCramer et al, 1996;Berthoud et al, 1997;Kanemitsu et al, 1998;Shah et al, 2002;O'Brien et al, 2004;Ouyang et al, 2005;Yogo et al, 2006;Alev et al, 2008;Morel et al, 2010); treatment of cultured cells with specific protein kinase or phosphoprotein phosphatase activators or inhibitors to alter 32 P incorporation or the immunoblot pattern of connexins (Lau et al, 1992;Husøy et al, 1993;Guan et al, 1996;Berthoud et al, 1997;…”

“…The first reports that demonstrated a particular Cx to be a phosphoprotein using metabolic labeling with 32 P showed phosphorylation of Cx32 in hepatocytes (treated with phorbol esters, OAG, forskolin or cAMP analogs)( (Sáez et a., 1986;Takeda et al, 1989;Sáez et al, 1990) and phosphorylation of Cx43 in uninfected and Rous sarcoma virus (RSV)-transformed fibroblasts (Crow et al, 1990). Phosphoamino acid analysis indicated that hepatocyte Cx32 and Cx43 in uninfected fibroblasts were phosphorylated on seryl residues (Takeda et al, 1989;Crow et al, 1990;Sáez et al, 1990), but Cx43 was also phosphorylated in tyrosyl residues in RSV-transformed fibroblasts (Crow et al, 1990).…”