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

Abstract

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

Comparison of in Vivo and in Vitro Phosphorylation of the Exocytosis-Sensitive Protein PP63/Parafusin by Differential MALDI Mass Spectrometric Peptide Mapping

“…As reported earlier, we have enriched two protein kinases, casein kinase type II (CK) and cGMP-dependent kinase (PKG), from 100000g supernatants of Paramecium cell homogenates which are able to phosphorylate PP63/pf in vitro (12). The scope of this study was to determine whether one of these kinases is involved in the reversible phosphorylation cycle of PP63/pf during regulated exo-and endocytosis in vivo.…”

“…All solvents were of the highest quality available. Materials for isolation of protein kinases and for in vivo and in vitro phosphorylation studies were as reported previously (12).…”

“…The isolation of a cGMP-dependent protein kinase (PKG) and a casein kinase (CK) was carried out as reported previously (12,13).…”

“…Regulation of PP63/pf phosphorylation involves several protein kinases and phosphatases (12). PP63-1/pf-1 contains respectively.…”

“…For further identification of protein kinases putatively involved in the phosphorylation of PP63/pf in vivo, picomolar quantities of two recombinant His-tagged isoforms His-PP63-1/pf-1 and His-PP63-2/pf-2 were assayed in vitro with Ser/Thr-specific protein kinases isolated from P. tetraurelia (12,13) and subsequently analyzed analogously. By using casein kinase type II protein kinase (CK) (13) and cyclic GMP-dependent protein kinase (PKG) (12), we identified multiple serine and threonine phosphorylation sites in vitro, only some of which correspond to those in vivo. This indicates that in vitro protein phosphorylation by selected kinases does not necessarily reflect the in vivo regulation of the protein substrate.…”