Protein Phosphate in the Regulation of Protein Phosphorylation and Insulin Secretion




Parameswara, Vinay K.


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Parameswara, Vinay K., Protein Phosphatase 2A in the Regulation of Ca2+- Sensitive Protein Phosphorylation and Insulin Secretion. Doctor of Philosophy (Biomedical Sciences), May 2003; 191 pp., 28 illustrations; 5 tables; 250 references. Type 2 diabetes is characterized by insufficient insulin secretion in the midst of increased demand from concomitant insulin resistance of peripheral tissues. More specifically, the diabetic β-cell is characterized by impaired responsiveness to D-glucose, the primary physiological regulator of insulin secretion, necessitating that the mechanism of glucose-induced insulin secretion from the β-cell of the pancreas is critically dependent on an elevation of cytosolic calcium as a trigger signal but is also dependent on reversible protein phosphorylation. Accordingly, a number of protein kinases are activated by glucose, or by incretin hormones that enhance glucose-induced insulin secretion. This dissertation however stems from a general hypothesis that protein phosphorylation and insulin secretion may also be controlled via the regulation of protein phosphatases (PP). Initially, a panel of specific antibodies was used to profile the expression of known PP species in the β-cell. By immunoblotting cultured clonal β-cells, INS-1, were shown to express various protein phosphatases namely PP 1, 2A, 2B, 2C, 4 and 6, but with distinct subcellular localization suggesting that these phosphatases regulated distinct functions within the β-cells. Of particular interest, PP-2A holoenzyme was localized to purified fractions of insulin secretory granules suggesting an involvement in insulin regulation. Selective inhibition of PP-2A in the presence of endothall or low concentrations of okadaic acid, increased insulin secretion in the presence of glucose in INS-1 cells. In order to discern potential substrates of PP-2A and thus-mechanisms of action, microcystin immobilized to sepharose was employed to affinity purify phosphatase species from β-cell lysates and proteins complexed with them. Fractions containing PP-2A also contained synapsin I and a specific interaction of these proteins was confirmed by co-immunoprecipitation from INS-1 cell lysates. In contrast, PP-1 was not associated with synapsin I. That synapsin I is indeed a substrate for PP-2A in INS-1 cells was confirmed via the demonstration that synapsin I phosphorylation was increased by okadaic acid under conditions that increased insulin release. Okadaic acid also induced the autophosphrylation and activation of CaMKII, a Ca2+-dependent kinase that phosphorylates synapsin I; suggesting CaMKII may mediate PP-2A effects on insulin secretion. The elimination of syanpsin I, markedly modulates glucose homeostasis of mice and subtly modulates insulin release. In summary these studies document that the modulation of PP-2A in β-cells dramatically influences insulin secretion reinforcing a concept that the control of protein phosphatase may have a critical role in the regulation of insulin secretion. These data suggest that a role of PP-2A on insulin secretion is mediated in part through the regulation of CaMKII activity and synapsin I-phosphorylation.