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Comparison of in vivo and in vitro phosphorylation of the exocytosis-sensitive protein PP63/Parafusin by differential MALDI mass spectrometric peptide mapping

Comparison of in vivo and in vitro phosphorylation of the exocytosis-sensitive protein PP63/Parafusin by differential MALDI mass spectrometric peptide mapping

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KUSSMANN, Martin, Karin HAUSER, Roland KISSMEHL, Jason BREED, Helmut PLATTNER, Peter ROEPSTORFF, 1999. Comparison of in vivo and in vitro phosphorylation of the exocytosis-sensitive protein PP63/Parafusin by differential MALDI mass spectrometric peptide mapping. In: Biochemistry. 38(24), pp. 7780-7790. ISSN 0006-2960. eISSN 1520-4995. Available under: doi: 10.1021/bi982888y

@article{Kussmann1999Compa-8220, title={Comparison of in vivo and in vitro phosphorylation of the exocytosis-sensitive protein PP63/Parafusin by differential MALDI mass spectrometric peptide mapping}, year={1999}, doi={10.1021/bi982888y}, number={24}, volume={38}, issn={0006-2960}, journal={Biochemistry}, pages={7780--7790}, author={Kussmann, Martin and Hauser, Karin and Kissmehl, Roland and Breed, Jason and Plattner, Helmut and Roepstorff, Peter} }

<rdf:RDF xmlns:dcterms="http://purl.org/dc/terms/" xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:bibo="http://purl.org/ontology/bibo/" xmlns:dspace="http://digital-repositories.org/ontologies/dspace/0.1.0#" xmlns:foaf="http://xmlns.com/foaf/0.1/" xmlns:void="http://rdfs.org/ns/void#" xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > <rdf:Description rdf:about="https://kops.uni-konstanz.de/rdf/resource/123456789/8220"> <dcterms:abstract xml:lang="deu">PP63 (parafusin) is a 63 kDa phosphoprotein, which exists in at least two different isoforms. It is very rapidly (80 ms) dephosphorylated during triggered trichocyst exocytosis. This occurs selectively in exocytosis-competent Paramecium tetraurelia strains. At least two protein kinases isolated from Paramecium, casein kinase type II kinase and cGMP-dependent kinase, are able to phosphorylate the two recombinant PP63/parafusin isoforms, both with phosphoglucomutase activity, in vitro. By performing mass spectrometric peptide mapping, we have investigated in vitro phosphorylation of recombinant PP63/ parafusin by these kinases in comparison to in vivo phosphorylation of native PP63/parafusin isolated from Paramecium homogenates. Low picomolar quantities of proteolytic digests of recombinant and native PP63/parafusin, prior to and following alkaline phosphatase treatment, were directly analyzed by MALDI mass spectrometry. In native PP63-1/parafusin-1, six of 64 serine and threonine residues (S-196, T-205, T-280, T-371, T-373, and T-469) were found definitely, 27 were found possibly phosphorylated, 28 were identified as nonphosphorylated, and three were not covered by mapping. Three of the six certainly phosphorylated amino acids represent consensus phosphorylation sites for casein kinase II or cGMPdependent protein kinase. In vitro phosphorylation studies of recombinant PP63/parafusin confirm that some of the sites found were used in vivo; however, also significant differences with respect to in vivo phosphorylation of native PP63/parafusin were observed. The two Paramecium protein kinases that were used do not preferably phosphorylate expected consensus sites in vitro. Homology structure modeling of PP63/parafusin with rabbit phosphoglucomutase revealed that the majority of residues found phosphorylated is located on the surface of the molecule.</dcterms:abstract> <dc:language>eng</dc:language> <dcterms:issued>1999</dcterms:issued> <dc:contributor>Hauser, Karin</dc:contributor> <dspace:hasBitstream rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/8220/1/Comparison_of_in_vivo_and_in_vitro_phosphorylation_of_the_exocytosis_sensitive_protein_PP63.pdf"/> <dcterms:title>Comparison of in vivo and in vitro phosphorylation of the exocytosis-sensitive protein PP63/Parafusin by differential MALDI mass spectrometric peptide mapping</dcterms:title> <dcterms:bibliographicCitation>First publ. in: Biochemistry ; 38 (1999). - S. 7780-7790</dcterms:bibliographicCitation> <dc:creator>Hauser, Karin</dc:creator> <dcterms:hasPart rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/8220/1/Comparison_of_in_vivo_and_in_vitro_phosphorylation_of_the_exocytosis_sensitive_protein_PP63.pdf"/> <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T17:41:32Z</dc:date> <dc:creator>Kussmann, Martin</dc:creator> <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/> <foaf:homepage rdf:resource="http://localhost:8080/jspui"/> <dc:rights>deposit-license</dc:rights> <dc:creator>Breed, Jason</dc:creator> <dc:creator>Roepstorff, Peter</dc:creator> <dc:contributor>Kussmann, Martin</dc:contributor> <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/rdf/resource/123456789/28"/> <dc:contributor>Breed, Jason</dc:contributor> <dc:contributor>Plattner, Helmut</dc:contributor> <dc:creator>Plattner, Helmut</dc:creator> <dc:contributor>Roepstorff, Peter</dc:contributor> <bibo:uri rdf:resource="http://kops.uni-konstanz.de/handle/123456789/8220"/> <dcterms:rights rdf:resource="https://creativecommons.org/licenses/by-nc-nd/2.0/legalcode"/> <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/rdf/resource/123456789/28"/> <dc:creator>Kissmehl, Roland</dc:creator> <dc:contributor>Kissmehl, Roland</dc:contributor> <dc:format>application/pdf</dc:format> </rdf:Description> </rdf:RDF>

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