Regulation by glucose and calcium of the carboxylmethylation of the catalytic subunit of protein phosphatase 2A in insulin-secreting INS-1 cells

Abstract

Palanivel, Rengasamy, Rajakrishnan Veluthakal, and Anjaneyulu Kowluru. Regulation by glucose and calcium of the carboxylmethylation of the catalytic subunit of protein phosphatase 2A in insulin-secreting INS-1 cells. Am J Physiol Endocrinol Metab 286: E1032-E1041, 2004. First published February 17, 2004 10.1152/ajpendo.00587.2003.-Previously, we reported that the catalytic subunit of protein phosphatase 2A (PP2Ac) undergoes carboxylmethylation (CML) at its COOH-terminal leucine, and that inhibitors of such a posttranslational modification markedly attenuate nutrientinduced insulin secretion from isolated ␤-cells. More recent studies have suggested direct inhibitory effects of glucose metabolites on PP2A activity in isolated ␤-cells, implying that inhibition of PP2A leads to stimulation of insulin secretion. Because the CML of PP2Ac has been shown to facilitate the holoenzyme assembly and subsequent functional activation of PP2A, we investigated putative regulation by glucose of the CML of PP2Ac in insulin-secreting (INS)-1 cells. Our data indicated a marked inhibition by specific intermediates of glucose metabolism (e.g., citrate and phosphoenolpyruvate) of the CML of PP2Ac in INS-1 cell lysates. Such inhibitory effects were also demonstrable in intact cells by glucose. Mannoheptulose, an inhibitor of glucose metabolism, completely prevented inhibitory effects of glucose on the CML of PP2Ac. Moreover, glucose-mediated inhibition of the CML of PP2Ac was resistant to diazoxide, suggesting that glucose metabolism and the generation of glucose metabolites might control inhibition of the CML of PP2Ac. A membrane-depolarizing concentration of KCl also induced inhibition of the CML of PP2Ac in intact INS cells. On the basis of these data, we propose that glucose metabolism and increase in intracellular calcium facilitate inhibition of the CML of PP2Ac, resulting in functional inactivation of PP2A. This, in turn, might retain the key signaling proteins of the insulin exocytotic cascade in their phosphorylated state, leading to stimulated insulin secretion. glucose metabolism; insulin-secreting cells THE PHOSPHORYLATION STATUS of proteins is regulated by the balance of activities of protein kinases and phosphatases, which induce the addition and removal of phosphoprotein phosphate, respectively (8, 42). Although several previous studies were focused on the identification and characterization of protein kinases in islets (4, 11, 16), little information is available on the localization and regulation of phosphoprotein phosphatases in the pancreatic ␤-cell. With use of relatively specific inhibitors of protein phosphatase function, recent studies (1,2,23,31,36,40) have suggested important roles for various phosphoprotein phosphatases, such as protein phosphatase 2A (PP2A), in the pancreatic ␤-cell function. For example, okadaic acid (OKA), a selective inhibitor of protein phosphatases, stimulates basal and cAMP-stimulated insulin secretion from electrically permeabilized islets (31). There is also evidence to indicate inhi...

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