Regulation of Synaptotagmin I Phosphorylation by Multiple Protein Kinases
: Synaptotagmin I has been suggested to function as a low‐affinity calcium sensor for calcium‐triggered exocytosis from neurons and neuroendocrine cells. We have studied the phosphorylation of synaptotagmin I by a variety of protein kinases in vitro and in intact preparations. Syntagl, the purified,...
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| Veröffentlicht in: | Journal of neurochemistry 1999-09, Vol.73 (3), p.921-932 |
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| creator | Hilfiker, Sabine Pieribone, Vincent A. Nordstedt, Christer Greengard, Paul Czernik, Andrew J. |
| description | : Synaptotagmin I has been suggested to function as a low‐affinity calcium sensor for calcium‐triggered exocytosis from neurons and neuroendocrine cells. We have studied the phosphorylation of synaptotagmin I by a variety of protein kinases in vitro and in intact preparations. Syntagl, the purified, recombinant, cytoplasmic domain of rat synaptotagmin I, was an effective substrate in vitro for Ca2+/calmodulin‐dependent protein kinase II (CaMKII), protein kinase C (PKC), and casein kinase II (caskII). Sequencing of tryptic phosphopeptides from syntagl revealed that CaMKII and PKC phosphorylated the same residue, corresponding to Thr112, whereas CaskII phosphorylated two residues, corresponding to Thr125 and Thr128. Endogenous synaptotagmin I was phosphorylated on purified synaptic vesicles by all three kinases. In contrast, no phosphorylation was observed on clathrin‐coated vesicles, suggesting that phosphorylation of synaptotagmin I in vivo occurs only at specific stage(s) of the synaptic vesicle life cycle. In rat brain synaptosomes and PC12 cells, K+‐evoked depolarization or treatment with phorbol ester caused an increase in the phosphorylation state of synaptotagmin I at Thr112. The results suggest the possibility that the phosphorylation of synaptotagmin I by CaMKII and PKC contributes to the mechanism(s) by which these two kinases regulate neurotransmitter release. |
| doi_str_mv | 10.1046/j.1471-4159.1999.0730921.x |
| format | Article |
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We have studied the phosphorylation of synaptotagmin I by a variety of protein kinases in vitro and in intact preparations. Syntagl, the purified, recombinant, cytoplasmic domain of rat synaptotagmin I, was an effective substrate in vitro for Ca2+/calmodulin‐dependent protein kinase II (CaMKII), protein kinase C (PKC), and casein kinase II (caskII). Sequencing of tryptic phosphopeptides from syntagl revealed that CaMKII and PKC phosphorylated the same residue, corresponding to Thr112, whereas CaskII phosphorylated two residues, corresponding to Thr125 and Thr128. Endogenous synaptotagmin I was phosphorylated on purified synaptic vesicles by all three kinases. In contrast, no phosphorylation was observed on clathrin‐coated vesicles, suggesting that phosphorylation of synaptotagmin I in vivo occurs only at specific stage(s) of the synaptic vesicle life cycle. In rat brain synaptosomes and PC12 cells, K+‐evoked depolarization or treatment with phorbol ester caused an increase in the phosphorylation state of synaptotagmin I at Thr112. The results suggest the possibility that the phosphorylation of synaptotagmin I by CaMKII and PKC contributes to the mechanism(s) by which these two kinases regulate neurotransmitter release.</description><identifier>ISSN: 0022-3042</identifier><identifier>EISSN: 1471-4159</identifier><identifier>DOI: 10.1046/j.1471-4159.1999.0730921.x</identifier><identifier>PMID: 10461881</identifier><identifier>CODEN: JONRA9</identifier><language>eng</language><publisher>Oxford UK: Blackwell Science Ltd</publisher><subject>Amino Acid Sequence ; Animals ; Biological and medical sciences ; Calcium-Binding Proteins ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Calcium-Calmodulin-Dependent Protein Kinases - metabolism ; Casein Kinase II ; Cell Differentiation ; Cell physiology ; Clathrin - pharmacology ; Conserved Sequence ; Fundamental and applied biological sciences. Psychology ; Humans ; Isoenzymes - metabolism ; Membrane Glycoproteins - metabolism ; Molecular and cellular biology ; Molecular Sequence Data ; Nerve Tissue Proteins - metabolism ; Neurotransmitter release ; PC12 Cells ; Peptide Mapping ; Phosphoamino Acids - metabolism ; Phosphorylation ; Protein Kinase C - metabolism ; Protein Kinases - metabolism ; Protein-Serine-Threonine Kinases - metabolism ; Rats ; Secretion. Exocytosis ; Synaptic vesicles ; Synaptosomes ; Synaptosomes - metabolism ; Synaptotagmin ; Synaptotagmin I ; Synaptotagmins</subject><ispartof>Journal of neurochemistry, 1999-09, Vol.73 (3), p.921-932</ispartof><rights>1999 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5001-c4bc40c04787ac451ec2e18911b485bb3d033eb2e1610436b022b968f7af5e833</citedby><cites>FETCH-LOGICAL-c5001-c4bc40c04787ac451ec2e18911b485bb3d033eb2e1610436b022b968f7af5e833</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1046%2Fj.1471-4159.1999.0730921.x$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1046%2Fj.1471-4159.1999.0730921.x$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,776,780,1411,1427,27902,27903,45552,45553,46386,46810</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=1953233$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10461881$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hilfiker, Sabine</creatorcontrib><creatorcontrib>Pieribone, Vincent A.</creatorcontrib><creatorcontrib>Nordstedt, Christer</creatorcontrib><creatorcontrib>Greengard, Paul</creatorcontrib><creatorcontrib>Czernik, Andrew J.</creatorcontrib><title>Regulation of Synaptotagmin I Phosphorylation by Multiple Protein Kinases</title><title>Journal of neurochemistry</title><addtitle>J Neurochem</addtitle><description>: Synaptotagmin I has been suggested to function as a low‐affinity calcium sensor for calcium‐triggered exocytosis from neurons and neuroendocrine cells. We have studied the phosphorylation of synaptotagmin I by a variety of protein kinases in vitro and in intact preparations. Syntagl, the purified, recombinant, cytoplasmic domain of rat synaptotagmin I, was an effective substrate in vitro for Ca2+/calmodulin‐dependent protein kinase II (CaMKII), protein kinase C (PKC), and casein kinase II (caskII). Sequencing of tryptic phosphopeptides from syntagl revealed that CaMKII and PKC phosphorylated the same residue, corresponding to Thr112, whereas CaskII phosphorylated two residues, corresponding to Thr125 and Thr128. Endogenous synaptotagmin I was phosphorylated on purified synaptic vesicles by all three kinases. In contrast, no phosphorylation was observed on clathrin‐coated vesicles, suggesting that phosphorylation of synaptotagmin I in vivo occurs only at specific stage(s) of the synaptic vesicle life cycle. In rat brain synaptosomes and PC12 cells, K+‐evoked depolarization or treatment with phorbol ester caused an increase in the phosphorylation state of synaptotagmin I at Thr112. The results suggest the possibility that the phosphorylation of synaptotagmin I by CaMKII and PKC contributes to the mechanism(s) by which these two kinases regulate neurotransmitter release.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>Calcium-Binding Proteins</subject><subject>Calcium-Calmodulin-Dependent Protein Kinase Type 2</subject><subject>Calcium-Calmodulin-Dependent Protein Kinases - metabolism</subject><subject>Casein Kinase II</subject><subject>Cell Differentiation</subject><subject>Cell physiology</subject><subject>Clathrin - pharmacology</subject><subject>Conserved Sequence</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Humans</subject><subject>Isoenzymes - metabolism</subject><subject>Membrane Glycoproteins - metabolism</subject><subject>Molecular and cellular biology</subject><subject>Molecular Sequence Data</subject><subject>Nerve Tissue Proteins - metabolism</subject><subject>Neurotransmitter release</subject><subject>PC12 Cells</subject><subject>Peptide Mapping</subject><subject>Phosphoamino Acids - metabolism</subject><subject>Phosphorylation</subject><subject>Protein Kinase C - metabolism</subject><subject>Protein Kinases - metabolism</subject><subject>Protein-Serine-Threonine Kinases - metabolism</subject><subject>Rats</subject><subject>Secretion. Exocytosis</subject><subject>Synaptic vesicles</subject><subject>Synaptosomes</subject><subject>Synaptosomes - metabolism</subject><subject>Synaptotagmin</subject><subject>Synaptotagmin I</subject><subject>Synaptotagmins</subject><issn>0022-3042</issn><issn>1471-4159</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1999</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqVkNtOxCAQhonR6Hp4BdMY410rU2gL3unGw3qOh2sCSLWbbqmljfbtpdlGvTNeEeCbmX8-hPYAR4BpejiPgGYQUkh4BJzzCGcE8xiizxU0-f5aRROM4zgkmMYbaNO5OcaQ0hTW0cbQBhiDCZo9mNeulG1hq8DmwWNfybq1rXxdFFUwC-7frKvfbNOPiOqDm65si7o0wX1jW-Opq6KSzrhttJbL0pmd8dxCz2enT9OL8PrufDY9vg514gOEmipNscY0Y5nUNAGjYwOMAyjKEqXICybEKP-W-pQkVX4HxVOWZzJPDCNkCx0s-9aNfe-Ma8WicNqUpayM7ZxIOWeUx-xPEDI_iWbcg0dLUDfWucbkom6KhWx6AVgMqsRcDFrFoFUMxsVoXHz64t1xSqcW5uVX6VKxB_ZHQDoty7yRlS7cD8cTEpNhrZMl9lGUpv9HAnF5Ox0v5AvpSJxn</recordid><startdate>199909</startdate><enddate>199909</enddate><creator>Hilfiker, Sabine</creator><creator>Pieribone, Vincent A.</creator><creator>Nordstedt, Christer</creator><creator>Greengard, Paul</creator><creator>Czernik, Andrew J.</creator><general>Blackwell Science Ltd</general><general>Blackwell</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7TK</scope><scope>7X8</scope></search><sort><creationdate>199909</creationdate><title>Regulation of Synaptotagmin I Phosphorylation by Multiple Protein Kinases</title><author>Hilfiker, Sabine ; Pieribone, Vincent A. ; Nordstedt, Christer ; Greengard, Paul ; Czernik, Andrew J.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5001-c4bc40c04787ac451ec2e18911b485bb3d033eb2e1610436b022b968f7af5e833</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1999</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>Calcium-Binding Proteins</topic><topic>Calcium-Calmodulin-Dependent Protein Kinase Type 2</topic><topic>Calcium-Calmodulin-Dependent Protein Kinases - metabolism</topic><topic>Casein Kinase II</topic><topic>Cell Differentiation</topic><topic>Cell physiology</topic><topic>Clathrin - pharmacology</topic><topic>Conserved Sequence</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Humans</topic><topic>Isoenzymes - metabolism</topic><topic>Membrane Glycoproteins - metabolism</topic><topic>Molecular and cellular biology</topic><topic>Molecular Sequence Data</topic><topic>Nerve Tissue Proteins - metabolism</topic><topic>Neurotransmitter release</topic><topic>PC12 Cells</topic><topic>Peptide Mapping</topic><topic>Phosphoamino Acids - metabolism</topic><topic>Phosphorylation</topic><topic>Protein Kinase C - metabolism</topic><topic>Protein Kinases - metabolism</topic><topic>Protein-Serine-Threonine Kinases - metabolism</topic><topic>Rats</topic><topic>Secretion. Exocytosis</topic><topic>Synaptic vesicles</topic><topic>Synaptosomes</topic><topic>Synaptosomes - metabolism</topic><topic>Synaptotagmin</topic><topic>Synaptotagmin I</topic><topic>Synaptotagmins</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hilfiker, Sabine</creatorcontrib><creatorcontrib>Pieribone, Vincent A.</creatorcontrib><creatorcontrib>Nordstedt, Christer</creatorcontrib><creatorcontrib>Greengard, Paul</creatorcontrib><creatorcontrib>Czernik, Andrew J.</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Neurosciences Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of neurochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hilfiker, Sabine</au><au>Pieribone, Vincent A.</au><au>Nordstedt, Christer</au><au>Greengard, Paul</au><au>Czernik, Andrew J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Regulation of Synaptotagmin I Phosphorylation by Multiple Protein Kinases</atitle><jtitle>Journal of neurochemistry</jtitle><addtitle>J Neurochem</addtitle><date>1999-09</date><risdate>1999</risdate><volume>73</volume><issue>3</issue><spage>921</spage><epage>932</epage><pages>921-932</pages><issn>0022-3042</issn><eissn>1471-4159</eissn><coden>JONRA9</coden><abstract>: Synaptotagmin I has been suggested to function as a low‐affinity calcium sensor for calcium‐triggered exocytosis from neurons and neuroendocrine cells. We have studied the phosphorylation of synaptotagmin I by a variety of protein kinases in vitro and in intact preparations. Syntagl, the purified, recombinant, cytoplasmic domain of rat synaptotagmin I, was an effective substrate in vitro for Ca2+/calmodulin‐dependent protein kinase II (CaMKII), protein kinase C (PKC), and casein kinase II (caskII). Sequencing of tryptic phosphopeptides from syntagl revealed that CaMKII and PKC phosphorylated the same residue, corresponding to Thr112, whereas CaskII phosphorylated two residues, corresponding to Thr125 and Thr128. Endogenous synaptotagmin I was phosphorylated on purified synaptic vesicles by all three kinases. In contrast, no phosphorylation was observed on clathrin‐coated vesicles, suggesting that phosphorylation of synaptotagmin I in vivo occurs only at specific stage(s) of the synaptic vesicle life cycle. In rat brain synaptosomes and PC12 cells, K+‐evoked depolarization or treatment with phorbol ester caused an increase in the phosphorylation state of synaptotagmin I at Thr112. The results suggest the possibility that the phosphorylation of synaptotagmin I by CaMKII and PKC contributes to the mechanism(s) by which these two kinases regulate neurotransmitter release.</abstract><cop>Oxford UK</cop><pub>Blackwell Science Ltd</pub><pmid>10461881</pmid><doi>10.1046/j.1471-4159.1999.0730921.x</doi><tpages>12</tpages></addata></record> |
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| subjects | Amino Acid Sequence Animals Biological and medical sciences Calcium-Binding Proteins Calcium-Calmodulin-Dependent Protein Kinase Type 2 Calcium-Calmodulin-Dependent Protein Kinases - metabolism Casein Kinase II Cell Differentiation Cell physiology Clathrin - pharmacology Conserved Sequence Fundamental and applied biological sciences. Psychology Humans Isoenzymes - metabolism Membrane Glycoproteins - metabolism Molecular and cellular biology Molecular Sequence Data Nerve Tissue Proteins - metabolism Neurotransmitter release PC12 Cells Peptide Mapping Phosphoamino Acids - metabolism Phosphorylation Protein Kinase C - metabolism Protein Kinases - metabolism Protein-Serine-Threonine Kinases - metabolism Rats Secretion. Exocytosis Synaptic vesicles Synaptosomes Synaptosomes - metabolism Synaptotagmin Synaptotagmin I Synaptotagmins |
| title | Regulation of Synaptotagmin I Phosphorylation by Multiple Protein Kinases |
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