QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 27 November 2007
doi: 10.1242/jcs.018218

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miyamoto, Y. Articles by Tanoue, A. Search for Related Content PubMed PubMed Citation Articles by Miyamoto, Y. Articles by Tanoue, A.

Cdk5 regulates differentiation of oligodendrocyte precursor cells through the direct phosphorylation of paxillin

¹ Department of Pharmacology, National Research Institute for Child Health and Development, Okura, Setagaya, Tokyo 157-8535, Japan
² Department of Cell and Neurobiology, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
³ Department of Biological Science and Technology, and Tissue Research Center, Tokyo University of Science, Yamazaki, Noda City, Chiba 278-8510, Japan
⁴ Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Minami-Osawa, Hachiohji City, Tokyo 194-8511, Japan

View larger version (54K): [in this window] [in a new window]   Fig. 1. Cdk5 is necessary for the differentiation of FBD-102b cells. (A) After induction of differentiation, FBD-102b-cell morphology was observed using phase-contrast microscopy. Representative images of cells are shown. (B) FBD-102b cells were randomly selected and assigned to one of three categories of complexity; low (two or less primary processes longer than a cell body, minimal development of secondary and tertiary processes), medium (three or more primary processes, moderate development of secondary and tertiary processes), and high (five or more primary processes, extensive development of secondary and tertiary. The percentagees of the cells in each category and the representative images of cells are shown. (C) FBD-102b cells were stained with antibodies against NG2 (green) or MBP (red) before or 4 days after induction of differentiation. (D) After induction of differentiation, FBD-102b cells treated with or without 10 µM roscovitine were lysed and immunoblotted with antibodies against MBP, PLP1 and β-actin. (E) RT-PCR analyses for MBP, PLP1, and β-actin were performed on differentiating cells treated with or without 10 µM roscovitine. (F) Representative images of cells treated with or without 10 µM roscovitine 4 days after induction of differentiation. The percentages of the cells in each category are shown in the bar graph. (G) The number of processes of the cells transfected with siRNAs for Cdk5 and luciferase is shown (left). Lysates from the transfected cells were immunoblotted with antibodies against Cdk5, paxillin and β-actin (right). Data were evaluated using Student's t-test (*P<0.01).

View larger version (72K): [in this window] [in a new window]   Fig. 2. Cdk5 is necessary for the differentiation of primary OLs. (A) After induction of differentiation, primary OL morphology was observed using phase-contrast microscopy. Representative images of cells in each category are shown. (B) Percentages of the cells treated with or without 10 µM roscovitine in each category after induction of differentiation. (C) Primary OLs treated with or without 10 µM roscovitine immunostained with anti-MBP antibody (red) before or 3 days after induction of differentiation. Cells can only be identified by DAPI staining of the nuclei (blue). (D) Percentages of MBP-positive cells (red) from the total number of cells. Data were evaluated using Student's t-test (*P<0.01). (E) After induction of differentiation, primary OLs treated with or without 10 µM roscovitine were lysed and immunoblotted with antibodies against MBP, PLP1 and β-actin. (F) Primary OPCs were infected with Cdk5, p35 or luciferase shRNA retroviral vectors. At 3 days after induction of differentiation, lysates from the infected cells were immunoblotted with antibodies against Cdk5, p35 or β-actin. (G) Representative fluorescence images of MBP- and GFP-positive cells are shown. Arrows indicate GFP-expressing cells. (H) Percentages of MBP-positive cells (red) against the total number of infected cells. Data were evaluated using Student's t-test (*P<0.01).

View larger version (19K): [in this window] [in a new window]   Fig. 3. Expression and the activity of Cdk5 are increased following differentiation of OLs. (A) After induction of differentiation, FBD-102b cells were lysed and immunoblotted with an anti-Cdk5 antibody (upper panel). RT-PCR analysis for Cdk5 mRNA was performed in FBD-102b cells after induction of differentiation (lower panel). The internal control β-actin is also shown. (B) After induction of differentiation, endogenous Cdk5 proteins were immunoprecipitated from the lysates of FBD-102b cells with anti-Cdk5 antibody, incubated with buffer containing histone H1 as the substrate, and immunoblotted with an antibody specific for a phosphorylated consensus-Cdk5 motif. The band intensity of phosphorylated histone H1 was quantified. (C) After induction of differentiation, primary OLs were lysed and immunoblotted with anti-Cdk5 antibody. (D) Activity of endogenous Cdk5 in primary OLs was measured after induction of differentiation using histone H1 as the substrate. The band intensity of phosphorylated histone H1 was quantified.

View larger version (70K): [in this window] [in a new window]   Fig. 4. Paxillin is necessary for OL differentiation. (A) Primary OPCs were infected with paxillin103, paxillin1206 or luciferase shRNA retroviral vectors. At 3 days after induction of differentiation, lysates from infected cells were immunoblotted with antibodies against paxillin, Cdk5 and β-actin. (B) Representative fluorescence images of MBP and GFP-positive cells. Arrows indicate GFP-expressing cells. (C) Percentages of MBP-positive cells (red) against the total number of infected cells. Data were evaluated using Student's t-test (*P<0.01).

View larger version (52K): [in this window] [in a new window]   Fig. 5. Cdk5 phosphorylation of paxillin mediates differentiation of OLs. (A) Amino acid sequences of human, mouse and rat paxillin. The consensus Cdk5 phosphorylation sequences are underlined. (B) FBD-102b cells were transfected with plasmid encoding GFP or the GFP-paxillin mutants, and the morphologies of the cells were observed using fluorescence microscopy. Representative fluorescence images of cells are shown. Arrows indicate cell adhesion. (C) Number of processes of cells transfected with the paxillin mutants. Data were evaluated using Student's t-test (*P<0.01). (D) FBD-102b cells were transfected with plasmid encoding GFP or the GFP-paxillin mutants. At 4 days after induction of differentiation, lysates from transfected cells were immunoblotted with antibodies against GFP and β-actin. The asterisk indicates a non-specific band. (E) Primary OPCs were infected with retrovirus expressing GFP, GFP-wt-paxillin, GFP-paxillin-S83A or GFP-paxillin-S244A. Representative fluorescence images of MBP- and GFP-positive cells are shown 3 days after induction of differentiation. Arrows indicate GFP-expressing cells. (F) Percentages of MBP-positive cells (red) against the total number of infected cells. Data were evaluated using Student's t-test (*P<0.01).

View larger version (48K): [in this window] [in a new window]   Fig. 6. Cdk5 forms a complex with paxillin. (A) In 293T cells, the immunoprecipitates of FLAG-Cdk5 were immunoblotted with an anti-GFP antibody to detect GFP-tagged paxillin. The total protein levels of Cdk5 and paxillin are also shown. (B) 293T cells were co-transfected with plasmids encoding LD1, LD2, LD3, LD4/5, LIM1, LIM2, LIM3 or LIM4 domains of paxillin and FLAG-Cdk5. Lysates of the cells were immunoprecipitated with anti-FLAG antibody and then immunoblotted with anti-HA antibody. Expression of paxillin domains and Cdk5 is also shown. Boxed bands indicate specific binding. (C) In 293T cells, the immunoprecipitates of GFP-wt-paxillin and GFP-paxillin-S244A were immunoblotted with anti-FAK antibody. The total protein levels of FAK and paxillin are also shown.

View larger version (21K): [in this window] [in a new window]   Fig. 7. Cdk5 phosphorylates paxillin at Ser244 in vitro. (A) Immobilized paxillin constructs were incubated with recombinant Cdk5 and p25, and immunoblotted with antibody recognizing phosphorylated Cdk substrate. Total levels of GFP-paxillin are also shown. (B) Quantification of the intensity of the bands shown in the immunoblots.

View larger version (40K): [in this window] [in a new window]   Fig. 8. Cdk5 phosphorylates paxillin and forms a complex with paxillin after OL differentiation. (A) Primary OLs were immunostained with antibodies against Cdk5 (red) and paxillin (green) before or 3 days after induction of differentiation. Panels a-f are magnifications of the boxed areas as indicated. (B) Three days after induction of differentiation, immunoprecipitates of endogenous Cdk5 and paxillin in the lysates of OLs were immunoblotted with their respective antibodies. The total proteins levels of Cdk5 and paxillin are also shown. (C) Immunoprecipitates of endogenous paxillin from the lysates of OLs treated with or without 10 µM roscovitine immunoblotted with antibody recognizing phosphorylated Cdk substrate before or 3 days after induction of differentiation. Total protein levels of paxillin and β-actin are also shown. (D) Quantification of the intensity of the bands shown in the immunoblots. Data were evaluated using Student's t-test (*P<0.01). (E) Before or 3 days after induction of differentiation, immunoprecipitates of endogenous FAK in lysates of OLs treated with or without 10 µM roscovitine were immunoblotted with antibody against paxillin. Total protein levels of paxillin and FAK are also shown.