Cyclin-dependent kinase 5 (Cdk5)-p35 is a proline-directed Ser/Thr kinase which plays a key role in neuronal migration, neurite outgrowth, and spine formation during brain development. has yet to be investigated. Phosphorylation of drebrin is indicated in non-neural cells [23], [24]. Although neuronal phosphorylation is also suggested [25], the exact phosphorylation site(s), its kinase, and role remain unknown. In this study, we found that drebrin is phosphorylated by Cdk5-p35. We additionally identified Ser142 and Ser342 in drebrin at Cdk5-phosphorylation sites and identified the role of neuronal migration in the embryonic cortex. Materials and Methods Ethics Statement All animal experiments were performed according to the guidelines for animal experimentation of Tokyo Metropolitan University. The study was approved by the Research Ethics Committee of Tokyo Metropolitan University (approval number, 24C45). All efforts were made to reduce the suffering of animals used. Antibodies and Chemicals Monoclonal anti-drebrin (M2F6) and monoclonal anti-GFP were purchased from MBL (Nagoya, Japan). Monoclonal anti-neuron-specific class III -tubulin (Tuj1) was obtained from Genzyme-Techne (Minneapolis, MN), anti-actin was obtained from Sigma-Aldrich (St Louis, MO), and monoclonal anti-myc (4A6) was obtained from Millipore (Billerica, Fisetin inhibition MA). Fisetin inhibition Anti-Homer2a was used as described previously [26]. Horseradish Peroxidase (HRP)-conjugated anti-mouse IgG and HRP-conjugated anti-rabbit IgG were purchased from DAKO (Glostrup, Denmark). Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit, Alexa Fluor 546 goat anti-rabbit IgG, Alexa Fluor 647 goat anti-mouse, and Alexa Fluor 647 goat anti-rabbit were purchased from HDAC5 Invitrogen (Carlsbad, CA). Tetramethylrhodamine isothiocyanate-phallidin (TRITC-phalloidin) was purchased from Sigma-Aldrich. And roscovitine and Phos-tag acrylamide were obtained from Wako (Osaka, Japan). Production and Purification of Anti-phospho-Ser142 of Drebrin Phospho-Ser142 peptide C-LARLS(pS)PVHR and non-phospho-Ser142 peptide C-LARLSSPVHR were obtained from BEX (Tokyo, Japan). The keyhole limpet hemocyanin (KLH)-conjugated phospho-Ser142 peptide was immunized into 15-weeks-old female rabbits (New Zealand White, Sankyo Labo, Tokyo, Japan) using TiterMax Gold (Funakoshi, Tokyo, Japan) as adjuvant. The anti-phospho-Ser142 antibody (pS142) was purified from antiserum using a phospho-Ser142-bound column after removing non-phospho-antibody with a Ser142 peptide-bound column. Plasmids Construction HA-Cdk5, kinase-negative Fisetin inhibition HA-Cdk5 (Cdk5-D144N), and p35-myc have been described previously [27], [28]. pEGFP-drebrin A, pEGFP-drebrin E, and pEGFP-ins2 have also been described previously [25]. The N-terminal fragment (pEGFP-drebrin A-NT) and C-terminal fragment (pEGFP-drebrin-CT) of Drebrin A were constructed by PCR-based deletion mutagenesis using pEGFP-drebrin A as a template. The primers used were: and as forward and reverse primers, respectively, for the N-terminal fragment, and for the C-terminal fragment. Nonphosphorylation Ala and phosphorylation-mimic Asp mutants of drebrins were constructed by site-directed mutagenesis using pEGFP-drebrin A, pEGFP-drebrin E, pEGFP-drebrin A-NT, pEGFP-drebrin-CT, pEGFP-ins2 and pET19b-drebrin A and pET19b-drebrin E as templates. The primers used were as follows: and for S142A, and for S342A, and for T346A, and for T356A, and for T377A, and for S383A, and for T392A, and for S142D, and for S342D. pEGFP-drebrin A-WT, S142A/S342A, and S142D/S342D were subcloned into site of pCAG-GFP-MCS2 [29], and DsRed-monomer derived from pDsRed-monomer-N1 was subcloned into site of pCAGGS. Mutations were confirmed by DNA sequences. Expression and Purification of Recombinant Drebrin pET19b-drebrin A, pET19b-drebrin E, and their Ala mutants were expressed in BL21-CodonPlus (DE3)-RP cells and obtained as a heat-stable supernatant of the cell extracts [30]. The amount of Fisetin inhibition drebrin was estimated by Coomassie Brilliant Blue staining of gels using bovine serum albumin as the standard. Preparation of Actin Gels from Mouse Brain Actin gels were prepared from mouse brains according to the method described previously by Taguchi phosphorylation. Mass Spectrometric Analysis Protein bands were stained using the ProteoSilver Plus Silver Stain Kit (Sigma, St. Louis, MO) according to the manufacturers protocol and excised from the polyacrylamide gel. After washing, the gels were digested by incubation in buffer with trypsin [32]. The tryptic digests were analyzed by an LC-MS/MS system as described previously [33]. Database search was performed using MASCOT software (version 2.2.1., Matrix Science Ltd., London) and the NCBI Refseq sequence Fisetin inhibition database under the parameters as described previously [34]. Phosphorylation of Drebrin Cdk5-p35 was expressed and purified from Sf9 cells (Clontech, Palo Alto, CA) infected by Baculovirus encoding Cdk5 and p35, as described previously [35]. Drebrin at 50 g/mL was phosphorylated by Cdk5-p35 at 37C for 1 h in the presence of 0.1 mM [-32P]ATP. Phosphorylation was detected by autoradiography after 10% polyacrylamide gel SDS-PAGE, and the extent of phosphorylation was quantified using a FLA7000 bioimage analyzer (GE Healthcare, Tokyo, Japan). Cell Culture and Transfection COS-7 and Neuro2A cells were obtained from Japanese Collection of Research Bioresources (Osaka, Japan) and maintained in Dulbeccos modified Eagles medium (DMEM) containing 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Cells were plated at a density of 3104 cells/cm2 for immunoblotting or at a density of 1 1.5104 cells/cm2 on.