title: Searching for novel Cdk5 substrates in brain by comparative phosphoproteomics of wild type and Cdk5-/- mice.
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.
authors.affiliation: Laboratory of Cellular and Neuronal Dynamics, Department of Biology, Faculty of Sciences, Universidad de Chile, Santiago, Chile.
authors.forename: Erick
authors.initials: E
authors.lastname: Contreras-Vallejos
authors.affiliation: Laboratory of Cellular and Neuronal Dynamics, Department of Biology, Faculty of Sciences, Universidad de Chile, Santiago, Chile.
authors.forename: Elías
authors.initials: E
authors.lastname: Utreras
authors.affiliation: Laboratory of Cellular and Neuronal Dynamics, Department of Biology, Faculty of Sciences, Universidad de Chile, Santiago, Chile.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Bórquez
authors.affiliation: Functional Genomics Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda MD, USA.
authors.forename: Michaela
authors.initials: M
authors.lastname: Prochazkova
authors.affiliation: Functional Genomics Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda MD, USA.
authors.forename: Anita
authors.initials: A
authors.lastname: Terse
authors.affiliation: Protein and Peptide Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda MD, USA.
authors.forename: Howard
authors.initials: H
authors.lastname: Jaffe
authors.affiliation: Laboratorio de Cultivo de Tejidos, Sección Biología Celular, Departamento de Biología Celular y Molecular, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay.
authors.forename: Andrea
authors.initials: A
authors.lastname: Toledo
authors.affiliation: Laboratorio de Cultivo de Tejidos, Sección Biología Celular, Departamento de Biología Celular y Molecular, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay.
authors.forename: Cristina
authors.initials: C
authors.lastname: Arruti
authors.affiliation: Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda MD, USA.
authors.forename: Harish C
authors.initials: HC
authors.lastname: Pant
authors.affiliation: Functional Genomics Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda MD, USA.
authors.forename: Ashok B
authors.initials: AB
authors.lastname: Kulkarni
authors.affiliation: Laboratory of Cellular and Neuronal Dynamics, Department of Biology, Faculty of Sciences, Universidad de Chile, Santiago, Chile.
authors.forename: Christian
authors.initials: C
authors.lastname: González-Billault
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D002460:Cell Line; D051360:Cyclin-Dependent Kinase 5 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017353:Gene Deletion; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics; D011687:Purines / Q000494:pharmacology; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D000077546:Roscovitine
chemical_list: C121673:Gprin1 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011687:Purines; D016194:Receptors, N-Methyl-D-Aspartate; D000077546:Roscovitine; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D051360:Cyclin-Dependent Kinase 5; C495906:Cdk5 protein, mouse