1,3-PBIT dihydrobromide Introduction Cyclin dependent kinase

Introduction Cyclin dependent kinases (CDKs) are a family of proline-directed serine/threonine kinases with roles in regulation of crucial cellular processes such as 1,3-PBIT dihydrobromide and transcription [1]. One of the unifying features of this family is its dependence on association with cyclins/cyclin-like regulatory proteins for activation [1,2]. In addition, some CDKs also require phosphorylation of a key threonine residue in the activation loop to stabilize the CDK-Cyclin binding leading to complete activation of the kinase [[1], [2], [3]]. CDK5 is one of the unique members of this family with a primary role in cell migration rather than cell cycle [4,5]. It was initially identified for its essential role in neuronal development including neuronal migration and differentiation; and normal synaptic function post-natally [4,5] but has since been implicated in non-neuronal functions as well [6,7]. Its primary cognate neuronal activator is p35 which has no sequence similarity with classical cyclin proteins, however, structural analysis has revealed that it adopts a cyclin-like fold that mediates interaction with CDK5 [8,9]. Unlike other CDKs, the activation loop phosphorylation is dispensable for activation of CDK5 and it solely depends on binding to its activator to adopt a catalytically active conformation [10]. While CDK5 is a relatively stable protein, p35 has a short half-life owing to its proteasomal degradation which is triggered upon its phosphorylation by CDK5, a feedback mechanism likely to control the duration and level of CDK5 activation [11]. Given its role in important cellular processes, it is not surprising that deregulation of CDK5 activity has been implicated in many pathophysiological conditions including neurodegenerative diseases, cancer and type II diabetes [[12], [13], [14]]. One of the main mechanisms that results in overactivation of CDK5 is calpain mediated cleavage of p35 to generate its 10 kDa shorter counterpart, p25 [15]. This shorter version maintains the CDK5 binding region and has a ∼5–10 times longer half-life than p35 resulting in prolonged activation and mislocalization of CDK5 leading to sustained phosphorylation of its substrates [5]. Therefore, it is important to investigate mechanisms that regulate CDK5 activity in a manner independent of the degradative clearance of its activator. Here we identify a novel phosphosite and provide the biochemical basis of how phosphorylation of this residue may negatively regulate CDK5 by abolishing its interaction with p35. Together, our study uncovers a phosphoregulatory mechanism that renders CDK5 inactive leading to inhibition of cell migration and promotion of cell proliferation.
Materials and methods Reagents, Antibodies and Plasmid Constructs: All reagents and chemicals were of analytical grade. The purified recombinant CDK5:p35 complex (Catalog#14–477) was purchased from Millipore-Sigma. Recombinant shrimp alkaline phosphatase (rSAP) was from New England Biolabs (NEB). Protease and phosphatase inihibitors were from Fisher Scientific and Sigma, respectively. ProLong Gold Antifade Mountant with DAPI was from ThermoFisher Scientific. Antibodies against CDK5 (Cat#sc-6247) and p35 (Cat#sc-820) were from Santa Cruz Biotechnology. Antibodies against Tubulin (Cat#2128), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cat#5174) and phospho-Histone H3 Serine 10 (Cat#53348) were from Cell Signaling. IRDye 680RD and IRDye 800CW secondary antibodies for western blotting were from Li-COR Biosciences and goat anti-rabbit Alexafluor-647 conjugated antibody for immunofluorescence was from Invitrogen. CDK5 (WT and D144N) and p35-HA constructs were gifts from David S. Park (University of Ottawa, Ottawa) and Edward Giniger (NIH/NINDS), respectively. Point mutants were generated using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). The mutations and the integrity of the rest of the insert were confirmed by sequencing. Cell Culture and Transfection: Cos7 cells (American Type Culture Collection) were cultured in Dulbecco's Modified Eagle Medium (DMEM; HyClone) supplemented with 10% Fetal Bovine Serum (FBS; HyClone) and Penicillin, Streptomycin, and Glutamine (Corning). Cells were maintained in a humidified atmosphere containing 5% CO2 at 37 °C. Transfections were performed using TransIT®-LT1 (MirusBio) according to the manufacturer's protocol.