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 1 (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 PP63in 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.
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July 1996
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Research Article|
July 01 1996
Protein phosphatase and kinase activities possibly involved in exocytosis regulation in Paramecium tetraurelia
Roland KISSMEHL;
Roland KISSMEHL
1Faculty of Biology, University of Konstanz, P.O. Box 5560, D-78434 Konstanz, Federal Republic of Germany
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Tilman TREPTAU;
Tilman TREPTAU
1Faculty of Biology, University of Konstanz, P.O. Box 5560, D-78434 Konstanz, Federal Republic of Germany
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Hans Werner HOFER;
Hans Werner HOFER
1Faculty of Biology, University of Konstanz, P.O. Box 5560, D-78434 Konstanz, Federal Republic of Germany
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Helmut PLATTNER
Helmut PLATTNER
*
*To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
January 02 1996
Revision Received:
February 23 1996
Accepted:
February 27 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 317 (1): 65–76.
Article history
Received:
January 02 1996
Revision Received:
February 23 1996
Accepted:
February 27 1996
Citation
Roland KISSMEHL, Tilman TREPTAU, Hans Werner HOFER, Helmut PLATTNER; Protein phosphatase and kinase activities possibly involved in exocytosis regulation in Paramecium tetraurelia. Biochem J 1 July 1996; 317 (1): 65–76. doi: https://doi.org/10.1042/bj3170065
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