Searching for novel Cdk5 substrates in brain by comparative phosphoproteomics of wild type and Cdk5-/- mice
Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as...
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| Veröffentlicht in: | PloS one 2014-03, Vol.9 (3), p.e90363 |
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| creator | Contreras-Vallejos, Erick Utreras, Elías Bórquez, Daniel A Prochazkova, Michaela Terse, Anita Jaffe, Howard Toledo, Andrea Arruti, Cristina Pant, Harish C Kulkarni, Ashok B González-Billault, Christian |
| description | Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5-/- embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5-/- brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate. |
| doi_str_mv | 10.1371/journal.pone.0090363 |
| format | Article |
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Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5-/- embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5-/- brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0090363</identifier><identifier>PMID: 24658276</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Affinity chromatography ; Alanine ; Alzheimer's disease ; Animals ; Apoptosis ; Axon guidance ; Axonal plasticity ; Axonogenesis ; Biological activity ; Biology ; Biology and life sciences ; Brain ; Brain - metabolism ; Brain research ; Cell adhesion & migration ; Cell cycle ; Cell Line ; Cell migration ; Chromatography ; Cyclin-dependent kinase 5 ; Cyclin-Dependent Kinase 5 - genetics ; Cyclin-Dependent Kinase 5 - metabolism ; Cyclin-Dependent Kinase 5 - physiology ; Cyclin-dependent kinases ; Cytoskeleton ; Embryos ; Gene Deletion ; Gene expression ; Genomics ; Hippocampus ; Intracellular Signaling Peptides and Proteins - metabolism ; Kinases ; Laboratories ; MARCKS protein ; Mass Spectrometry ; Mass spectroscopy ; Membrane Proteins - metabolism ; Mice ; Myristoylated Alanine-Rich C Kinase Substrate ; Nerve Tissue Proteins - metabolism ; Nervous system ; Neurological disorders ; Neurons ; Phosphoproteins ; Phosphoproteins - chemistry ; Phosphoproteins - metabolism ; Phosphorylation ; Post-translation ; Proline ; Protein kinase C ; Protein-serine/threonine kinase ; Proteins ; Proteomics ; Purines - pharmacology ; Quantitation ; Receptors, N-Methyl-D-Aspartate - metabolism ; Research and Analysis Methods ; Roscovitine ; Scientific imaging ; Substrates ; Synaptic plasticity ; Threonine</subject><ispartof>PloS one, 2014-03, Vol.9 (3), p.e90363</ispartof><rights>2014 Contreras-Vallejos et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2014 Contreras-Vallejos et al 2014 Contreras-Vallejos et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c592t-8b33c6988f5518755fc09f155980692217b63d1bb78d43bbb8654d99319da7ef3</citedby><cites>FETCH-LOGICAL-c592t-8b33c6988f5518755fc09f155980692217b63d1bb78d43bbb8654d99319da7ef3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962345/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962345/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2095,2914,23846,27903,27904,53770,53772,79347,79348</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24658276$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Paudel, Hemant K.</contributor><creatorcontrib>Contreras-Vallejos, Erick</creatorcontrib><creatorcontrib>Utreras, Elías</creatorcontrib><creatorcontrib>Bórquez, Daniel A</creatorcontrib><creatorcontrib>Prochazkova, Michaela</creatorcontrib><creatorcontrib>Terse, Anita</creatorcontrib><creatorcontrib>Jaffe, Howard</creatorcontrib><creatorcontrib>Toledo, Andrea</creatorcontrib><creatorcontrib>Arruti, Cristina</creatorcontrib><creatorcontrib>Pant, Harish C</creatorcontrib><creatorcontrib>Kulkarni, Ashok B</creatorcontrib><creatorcontrib>González-Billault, Christian</creatorcontrib><title>Searching for novel Cdk5 substrates in brain by comparative phosphoproteomics of wild type and Cdk5-/- mice</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5-/- embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5-/- brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate.</description><subject>Affinity chromatography</subject><subject>Alanine</subject><subject>Alzheimer's disease</subject><subject>Animals</subject><subject>Apoptosis</subject><subject>Axon guidance</subject><subject>Axonal plasticity</subject><subject>Axonogenesis</subject><subject>Biological activity</subject><subject>Biology</subject><subject>Biology and life sciences</subject><subject>Brain</subject><subject>Brain - metabolism</subject><subject>Brain research</subject><subject>Cell adhesion & migration</subject><subject>Cell cycle</subject><subject>Cell Line</subject><subject>Cell migration</subject><subject>Chromatography</subject><subject>Cyclin-dependent kinase 5</subject><subject>Cyclin-Dependent Kinase 5 - genetics</subject><subject>Cyclin-Dependent Kinase 5 - metabolism</subject><subject>Cyclin-Dependent Kinase 5 - physiology</subject><subject>Cyclin-dependent kinases</subject><subject>Cytoskeleton</subject><subject>Embryos</subject><subject>Gene Deletion</subject><subject>Gene expression</subject><subject>Genomics</subject><subject>Hippocampus</subject><subject>Intracellular Signaling Peptides and Proteins - metabolism</subject><subject>Kinases</subject><subject>Laboratories</subject><subject>MARCKS protein</subject><subject>Mass Spectrometry</subject><subject>Mass spectroscopy</subject><subject>Membrane Proteins - metabolism</subject><subject>Mice</subject><subject>Myristoylated Alanine-Rich C Kinase Substrate</subject><subject>Nerve Tissue Proteins - metabolism</subject><subject>Nervous system</subject><subject>Neurological disorders</subject><subject>Neurons</subject><subject>Phosphoproteins</subject><subject>Phosphoproteins - chemistry</subject><subject>Phosphoproteins - metabolism</subject><subject>Phosphorylation</subject><subject>Post-translation</subject><subject>Proline</subject><subject>Protein kinase C</subject><subject>Protein-serine/threonine kinase</subject><subject>Proteins</subject><subject>Proteomics</subject><subject>Purines - pharmacology</subject><subject>Quantitation</subject><subject>Receptors, N-Methyl-D-Aspartate - metabolism</subject><subject>Research and Analysis Methods</subject><subject>Roscovitine</subject><subject>Scientific imaging</subject><subject>Substrates</subject><subject>Synaptic 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Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Contreras-Vallejos, Erick</au><au>Utreras, Elías</au><au>Bórquez, Daniel A</au><au>Prochazkova, Michaela</au><au>Terse, Anita</au><au>Jaffe, Howard</au><au>Toledo, Andrea</au><au>Arruti, Cristina</au><au>Pant, Harish C</au><au>Kulkarni, Ashok B</au><au>González-Billault, Christian</au><au>Paudel, Hemant K.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Searching for novel Cdk5 substrates in brain by comparative phosphoproteomics of wild type and Cdk5-/- mice</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2014-03-21</date><risdate>2014</risdate><volume>9</volume><issue>3</issue><spage>e90363</spage><pages>e90363-</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5-/- embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5-/- brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>24658276</pmid><doi>10.1371/journal.pone.0090363</doi><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2014-03, Vol.9 (3), p.e90363 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1509205532 |
| source | PubMed (Medline); MEDLINE; Public Library of Science; DOAJ Directory of Open Access Journals; Free Full-Text Journals in Chemistry; EZB Electronic Journals Library |
| subjects | Affinity chromatography Alanine Alzheimer's disease Animals Apoptosis Axon guidance Axonal plasticity Axonogenesis Biological activity Biology Biology and life sciences Brain Brain - metabolism Brain research Cell adhesion & migration Cell cycle Cell Line Cell migration Chromatography Cyclin-dependent kinase 5 Cyclin-Dependent Kinase 5 - genetics Cyclin-Dependent Kinase 5 - metabolism Cyclin-Dependent Kinase 5 - physiology Cyclin-dependent kinases Cytoskeleton Embryos Gene Deletion Gene expression Genomics Hippocampus Intracellular Signaling Peptides and Proteins - metabolism Kinases Laboratories MARCKS protein Mass Spectrometry Mass spectroscopy Membrane Proteins - metabolism Mice Myristoylated Alanine-Rich C Kinase Substrate Nerve Tissue Proteins - metabolism Nervous system Neurological disorders Neurons Phosphoproteins Phosphoproteins - chemistry Phosphoproteins - metabolism Phosphorylation Post-translation Proline Protein kinase C Protein-serine/threonine kinase Proteins Proteomics Purines - pharmacology Quantitation Receptors, N-Methyl-D-Aspartate - metabolism Research and Analysis Methods Roscovitine Scientific imaging Substrates Synaptic plasticity Threonine |
| title | Searching for novel Cdk5 substrates in brain by comparative phosphoproteomics of wild type and Cdk5-/- mice |
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