Protein phosphatase and kinase activities possibly involved in exocytosis regulation in Paramecium tetraurelia

摘要: In Paramecium tetraurelia cells synchronous exocytosis induced by aminoethyldextran (AED) is accompanied an equally rapid dephosphorylation of a 63 kDa phosphoprotein (PP63) within 80 ms. vivo, rephosphorylation occurs few seconds after AED triggering. homogenates (P)P63 can be solubilized in all three phosphorylation states (phosphorylated, dephosphorylated and rephosphorylated) thus tested vitro. By using chelators different divalent cations, de- PP63 P63 respectively achieved endogenous protein phosphatase/kinase system. Dephosphorylation the presence EDTA, whereas EGTA this was concealed kinase(s), indicating that calcium-independent. Results obtained with phosphatase inhibitors (okadaic acid, calyculin A) allowed us to exclude serine/threonine type I (with selective sensitivity Paramecium). Protein 2C also less likely candidate because its requirement for high Mg2+ concentrations. According previous evidence 2B (calcineurin; CaN) possibly involved. We have now found bovine brain CaN dephosphorylates Taking into account specific requirements vitro, p-nitrophenyl phosphate as substrate, we isolated cytosolic similar characteristics combined preparative gel electrophoresis affinity-column chromatography. vitro (after 32P labelling vivo). Using various combinations ion exchange, affinity hydrophobic interaction chromatography kinases from soluble fraction, i.e. cAMP-dependent kinase (PKA), cGMP-dependent (PKG) casein kinase. Among tested, PKA cannot phosphorylate P63, either PKG or On basis these findings propose system involved regulation P. cells.