Searching for Novel Cdk5 Substrates in Brain by Comparative Phosphoproteomics of Wild Type and Cdk5−/− Mice

Erick Contreras-Vallejos,Daniel A Bórquez,Harish C Pant,Christian González-Billault,Anita Terse,Howard Jaffe,Andrea Toledo,Cristina Arruti,Elías Utreras,Michaela Prochazkova,Ashok B Kulkarni,Hemant K Paudel

doi:10.1371/journal.pone.0090363

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.

Highlights

The complexity of cell functions derives from the particular content of individual proteins at a given time, and from their post-translational modifications
A decreased phosphorylation at these sites may reflect the indirect effects of other proline-directed kinases regulated by Cyclin-dependent kinase 5 (Cdk5)
In order to get an overview of the altered phosphoprotein profile in Cdk52/2 brains, we classified all of the phosphorylated proteins with the canonical Cdk5 motif which we identified in our experimental analysis in three different groups, namely signaling pathways (Figure 1A), molecular functions (Figure 1B) and biological processes (Figure 1C) according to their annotation in PANTHER

Summary

Introduction

The complexity of cell functions derives from the particular content of individual proteins at a given time, and from their post-translational modifications. The most common post-translational modification of proteins is the phosphorylation of serine, threonine, and tyrosine residues. This is important to cellular mechanisms that integrate extracellular and intracellular signals through selective phosphorylation of proteins leading to specific functional outcomes. Protein phosphorylation plays a key role in many cellular functions such as cell signaling, apoptosis, cell migration, cytoskeletal dynamics and brain development. Cdk is known to phosphorylate cytoskeletal proteins, signaling molecules, ion channels and regulatory proteins that participate in the normal function of the brain and during neurodegenerative disorders [1,2,4,6]. A detailed analysis of Cdk52/2 mice, which display perinatal lethality and extensive neuronal migration defects, revealed that Cdk serves as a key regulator of neuronal migration, neurite outgrowth, and axonal path finding and dendrite development [7,8]

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