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

First published online 19 August 2008
doi: 10.1242/jcs.026757

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend 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 Google Scholar Google Scholar Articles by Kang, T.-H. Articles by Kim, K.-T. Search for Related Content PubMed PubMed Citation Articles by Kang, T.-H. Articles by Kim, K.-T. Social Bookmarking                         What's this?

VRK1 phosphorylates CREB and mediates CCND1 expression

Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology (POSTECH), San-31, Hyoja-Dong, Pohang 790-784, Republic of Korea

View larger version (50K): [in this window] [in a new window]   Fig. 1. Requirement of VRK1 for DNA replication. (A) Immunofluorescence of VRK1, VRK2 and VRK3 (red), BrdU (green) and DNA (Hoechst 33342, blue) from cells expressing pFlag-VRK. (B) The expression levels of Flag-labeled VRK1, VRK2 and VRK3 were determined by immunoblotting with the indicated antibodies. (C) HeLa cells transfected with control siRNA, siVRK1(CAAGGAACCTGGTGTTGAA; sequence effective only on human VRK1 not rat VRK1), or siVRK1 plus Flag-ratVRK1 (rVRK1) were immunostained for BrdU (green) and DNA (red, propidium iodide, PI). (D) The level of endogenous VRKs was measured by immunoblotting with the indicated antibodies. The error bars represent the mean ± s.d. from n>300 transfected cells. Scale bars: 20 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 2. Induction of CCND1 by VRK1. (A) Cytoplasmic (Cyt.) and nuclear (Nuc.) lysates from HeLa cells transfected with control vector (vector, pDsRed1-C1) or pDsRed1-C1-VRK1 were immunoblotted with the indicated antibodies. (B) HeLa cells transfected with mock vector (pFlag-CMV2), control siRNA (siCont), siVRK1 or siVRK3 were fractionated into cytoplasmic and nuclear lysates and then immunoblotted with the indicated antibodies. (C) Total protein lysates from HeLa cells transfected with control siRNA (siCont) or siVRK1 for the indicated periods were immunoblotted with the indicated antibodies. (D) HeLa cells transfected with control vector (vector, pFlag) or pFlag-VRK1 were treated with DMSO (vehicle), MG132 or actinomycin D (ActD) for 5 hours, followed by immunoblotting (IB) with the indicated antibodies and northern blotting (NB) with an antisense CCND1 probe. Ethidium bromide (EtBr) stained 18S and 28S ribosomal RNA (rRNA) was used as a loading control.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Kinase activity of VRK1 is required for the nuclear accumulation of CCND1. (A) Immunofluorescence of CCND1 (green) and VRK1 (red) from cells expressing wild-type (WT) or the kinase-dead (KD) mutant of VRK1. (B) Cytoplasmic (Cyt.) and nuclear (Nuc.) lysates of HeLa cells transfected with mock vector (pFlag-CMV2), kinase-dead (VRK1-KD) or wild-type (VRK1-WT) VRK1 were immunoblotted with the indicated antibodies. Protein disulfide isomerase (PDI) and Lamin B were used as markers for the cytoplasm and nucleus, respectively. Scale bars: 20 µm.

View larger version (27K): [in this window] [in a new window]   Fig. 4. Activation of cAMP-response element (CRE) in the CCND1 promoter by VRK1. (A) The luciferase activity of cells transfected with kinase-dead (KD) or wild-type (WT) VRK1 was measured. The results represent mean ± s.d. from experiments performed triplicate. (B,C) ChIP assay measuring the CREB-P (B) and ATF2 (C) binding to CRE in the CCND1 promoter of HeLa cells transfected with either KD-VRK1 or WT-VRK1.

View larger version (43K): [in this window] [in a new window]   Fig. 5. Physical interaction of VRK1 with CREB in a cell-cycle-regulated manner. (A) Protein extracts from HeLa cells transfected with pFlag-VRK1 were analyzed by immunoprecipitation with an anti-Flag antibody. Coprecipitated CREB proteins were detected by immunoblotting using the anti-CREB antibody. Mouse immunoglobulin-G1 (mIg-G1) was used as an immunoprecipitation control. (B) Total cell lysates from HeLa cells were incubated with the GST-CREB protein and pulled down with glutathione-conjugated beads. Bound proteins were evaluated by immunoblotting using anti-VRK isoform-specific antibodies. (C) Recombinant full-length or fragments of CREB (F1-F4) were used for binding analyses with VRK1. Bound VRK1 protein was detected by immunoblotting using the anti-VRK1 antibody. Recombinant proteins were detected using the anti-GST antibody. bZIP, basic leucine zipper; CAD, constitutive activation domain; KID, kinase-inducible domain; Q, glutamine-rich domain. (D,E) The cell cycle was synchronized in S phase using a double thymidine block (D) or in M phase by treatment with nocodazole (E), and then released at the indicated time by removing the cell cycle inhibitors. Expression of VRK1 and CREB was assessed by immunoblotting and interactions were investigated by immunoprecipitation. P-H3 S10 (phosphorylated histone 3) was used as a mitotic marker.

View larger version (42K): [in this window] [in a new window]   Fig. 6. VRK1 phosphorylates CREB at Ser133. (A) Phosphorylation of CREB by wild-type or kinase-dead VRK1 was assessed by in vitro kinase assays. GST-CREB-fragments (F1-F4), wild-type CREB or site-directed mutated CREB (S133A and S142A) proteins were used as substrates. (B,C) Cytoplasmic and nuclear lysates from HeLa cells transfected with kinase-dead (KD) or wild-type (WT) VRK1 (B), or with control siRNA (siCont) or siVRK1 (C) were immunoblotted with the indicated antibodies. Protein disulfide isomerase (PDI) and Lamin B were used as fraction markers for the cytoplasm and nucleus, respectively. (D,E) HeLa cells transfected with WT-VRK1 or KD-VRK1 were immunostained for CREB-P S133 (green) (D) or CREB (E). Scale bars: 20 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 7. VRK1 mediates Myc-induced CCND1 expression. (A) HeLa cells were cotransfected with pHA empty vector (Vector) or pHA-Myc and control siRNA (siCont) or siVRK1. Cells were immunoblotted with the indicated antibodies. (B) Immunofluorescence of Myc-EBFP (blue), VRK1 (green) and CCND1 (red) from cells transfected with siCont or siVRK1. Arrowheads and arrow denote untransfected cells and cells that do not show knockdown, respectively. Scale bars: 20 µm. (C) ChIP assay measuring Myc binding to conserved Myc-binding sites in the VRK1 promoter of HeLa cells transfected with pHA empty vector or pHA-Myc. (D) Cells were maintained without serum for 24 hours then fetal bovine serum was added to 5% and cells incubated for a further 3 hours. Cells were then immunostained for VRK1 (red), Myc (green) and DNA (Hoechst 33342, blue). DIC, digital interference contrast image. Scale bar: 30 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter