Searching for novel Cdk5 substrates in brain by comparative phosphoproteomics of wild type and Cdk5-/- mice

Erick Contreras-Vallejos, Elías Utreras, Daniel A. Bórquez, Michaela Prochazkova, Anita Terse, Howard Jaffe, Andrea Toledo, Cristina Arruti, Harish C. Pant, Ashok B. Kulkarni, Christian González-Billault
PLoS ONE. 2014-03-21; 9(3): e90363
DOI: 10.1371/journal.pone.0090363

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1. PLoS One. 2014 Mar 21;9(3):e90363. doi: 10.1371/journal.pone.0090363. eCollection
2014.

Searching for novel Cdk5 substrates in brain by comparative phosphoproteomics of
wild type and Cdk5-/- mice.

Contreras-Vallejos E(1), Utreras E(1), Bórquez DA(1), Prochazkova M(2), Terse
A(2), Jaffe H(3), Toledo A(4), Arruti C(4), Pant HC(5), Kulkarni AB(2),
González-Billault C(1).

Author information:
(1)Laboratory of Cellular and Neuronal Dynamics, Department of Biology, Faculty
of Sciences, Universidad de Chile, Santiago, Chile.
(2)Functional Genomics Section, National Institute of Dental and Craniofacial
Research, National Institutes of Health, Bethesda MD, USA.
(3)Protein and Peptide Facility, National Institute of Neurological Disorders and
Stroke, National Institutes of Health, Bethesda MD, USA.
(4)Laboratorio de Cultivo de Tejidos, Sección Biología Celular, Departamento de
Biología Celular y Molecular, Facultad de Ciencias, Universidad de la República,
Montevideo, Uruguay.
(5)Laboratory of Neurochemistry, National Institute of Neurological Disorders and
Stroke, National Institutes of Health, Bethesda MD, USA.

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: 10.1371/journal.pone.0090363
PMCID: PMC3962345
PMID: 24658276 [Indexed for MEDLINE]


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