Carboxylmethylation of the catalytic subunit of protein phosphatase 2A in insulin-secreting cells: evidence for functional consequences on enzyme activity and insulin secretion.

Abstract

We report the carboxylmethylation of a 36-kDa protein in intact normal rat islets and clonal beta (INS-1) cells. This protein was predominantly cytosolic. Its carboxylmethylation, as assessed by vapor phase equilibration assay, was resistant to inhibition by N-acetyl-S-trans, trans-farnesyl-L-cysteine, a competitive substrate for cysteine methyl transferases. These data suggest that the methylated C-terminal amino acid is not cysteine. The methylated protein was identified as the catalytic subunit of protein phosphatase 2A (PP2Ac) by immunoblotting. The carboxylmethylation of the PP2Ac increased its catalytic activity, suggesting a key role in the functional regulation of PP2A. Therefore, we studied okadaic acid, a selective inhibitor of PP2A that acts by an unknown mechanism. Okadaic acid (but not 1-nor-okadaone, its inactive analog) inhibited (Ki = 10 nM) the carboxylmethylation of PP2Ac and phosphatase activity in the cytosolic fraction (from normal rat islets and clonal beta-cells) as well as in intact rat islets. Furthermore, methylated PP2Ac underwent rapid demethylation (t 1/2 = 40 min) catalyzed by a methyl esterase localized in islet homogenates. Ebelactone, a purported inhibitor of methyl esterases, significantly delayed (> 200 min) the demethylation of PP2Ac. Furthermore, ebelactone reversibly inhibited glucose- and ketoisocaproate-induced insulin secretion from normal rat islets. These data identify, for the first time, a methylation-demethylation cycle for PP2Ac in the beta-cell and suggest a key functional relationship between PP2A activity and the carboxylmethylation of its catalytic subunit. These findings thus suggest a negative modulatory role for PP2A in nutrient-induced insulin exocytosis.

Methylation of the Protein Phosphatase 2A Catalytic Subunit Is Essential for Association of Bα Regulatory Subunit But Not SG2NA, Striatin, or Polyomavirus Middle Tumor Antigen

“…Then, l-[methyl-3 H]methionine (Amersham Pharmacia Biotech, Piscataway, NJ) was added to a final concentration of 100 Ci/ml. Preincubation of the cells with 5 M cycloheximide and 30 g/ml puromycin prevents translational incorporation of subsequently added radiolabeled methionine into newly synthesized proteins (Favre et al, 1994;Kowluru et al, 1996); the fact that no radioactivity was detected in certain PP2A mutants indicates that protein synthesis was indeed completely inhibited. After labeling for 5 h, the cells were washed, scraped into 1 ml of ice-cold wash buffer, and centrifuged at 3000 ϫ g for 1 min.…”

“…The effect of methylation on PP2A activity is controversial, with some studies indicating up to a twofold increase in specific activity (Favre et al, 1994;Kowluru et al, 1996) and another finding no effect (De Baere et al, 1999). Although C subunit methylation appears to occur in vivo in a cell cycle-regulated manner (Floer and Stock, 1994;Turowski et al, 1995), the molecular mechanisms controlling such changes and the PP2A function(s) affected are not yet understood.…”

Role of Janus kinase-2 in insulin-mediated phosphorylation and inactivation of protein phosphatase-2A and its impact on upstream insulin signalling components

“…So far, over 15 regulatory subunits have been identified for PP-2A, which, in addition to regulating enzymic activity, are predicted to be involved in the subcellular targeting of the holoenzyme. In addition to regulatory subunits, PP-2A activity is also influenced by catalytic subunit phosphorylation [5][6][7][8] and carboxymethylation [9,10].…”

Activation of Acetyl-CoA Carboxylase by a Glutamate- and Magnesium-Sensitive Protein Phosphatase in the Islet β-Cell

“…The C subunit of PP2A (PP2Ac) undergoes posttranslational modifications, including phosphorylation (at Tyr-307) and carboxyl methylation (at Leu-309). Such modification steps appear to influence the catalytic activity of PP2A (5,7,8). Recently, we immunologically identified PP2A in clonal ␤-cells, normal rat islets, and human islets (7).…”

“…Such modification steps appear to influence the catalytic activity of PP2A (5,7,8). Recently, we immunologically identified PP2A in clonal ␤-cells, normal rat islets, and human islets (7). We demonstrated further that PP2Ac undergoes methylation at its COOHterminal leucine and that the carboxyl methylation of PP2Ac is associated with a modest but significant increase in its catalytic activity (7); this finding is compatible with published evidence by Favre et al (8).…”