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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...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962345/ https://www.ncbi.nlm.nih.gov/pubmed/24658276 http://dx.doi.org/10.1371/journal.pone.0090363 |
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author | 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 |
author_facet | 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 |
author_sort | Contreras-Vallejos, Erick |
collection | PubMed |
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. |
format | Online Article Text |
id | pubmed-3962345 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-39623452014-03-24 Searching for Novel Cdk5 Substrates in Brain by Comparative Phosphoproteomics of Wild Type and Cdk5(−/−) Mice 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 PLoS One Research Article 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. Public Library of Science 2014-03-21 /pmc/articles/PMC3962345/ /pubmed/24658276 http://dx.doi.org/10.1371/journal.pone.0090363 Text en © 2014 Contreras-Vallejos et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article 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 Searching for Novel Cdk5 Substrates in Brain by Comparative Phosphoproteomics of Wild Type and Cdk5(−/−) Mice |
title | Searching for Novel Cdk5 Substrates in Brain by Comparative Phosphoproteomics of Wild Type and Cdk5(−/−) Mice |
title_full | Searching for Novel Cdk5 Substrates in Brain by Comparative Phosphoproteomics of Wild Type and Cdk5(−/−) Mice |
title_fullStr | Searching for Novel Cdk5 Substrates in Brain by Comparative Phosphoproteomics of Wild Type and Cdk5(−/−) Mice |
title_full_unstemmed | Searching for Novel Cdk5 Substrates in Brain by Comparative Phosphoproteomics of Wild Type and Cdk5(−/−) Mice |
title_short | Searching for Novel Cdk5 Substrates in Brain by Comparative Phosphoproteomics of Wild Type and Cdk5(−/−) Mice |
title_sort | searching for novel cdk5 substrates in brain by comparative phosphoproteomics of wild type and cdk5(−/−) mice |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962345/ https://www.ncbi.nlm.nih.gov/pubmed/24658276 http://dx.doi.org/10.1371/journal.pone.0090363 |
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