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First published online 13 December 2005
doi: 10.1242/jcs.02699

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Nuclear protein NP60 regulates p38 MAPK activity

Jing Fu^1,*, Ziqiang Yang^1,*, Jinxue Wei¹, Jiahuai Han² and Jun Gu^1,

¹ National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, People's Republic of China
² Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

View larger version (50K): [in a new window]   Fig. 1. Sequence and structure analysis of NP60. (A) ORF sequence of NP60. (B) Chromosome localization and genome structure of NP60. (C) The structure of NP60 at the level of the protein illustrated schematically. The motifs of PWWP, AT hook and the NAD-binding region are indicated (analyzed at http://smart.embl-heidelberg.de).

View larger version (40K): [in a new window]   Fig. 2. Expression and cellular localization of NP60. (A) Total cell extracts from RAW264.7 cells were resolved by SDS-PAGE, and western blotting was performed using anti-NP60 antibody. (B) Total RNA was extracted from different cell lines, and used for PCR amplification with the primers as described in the Materials and Methods. NP60 is a 483 bp fragment. Cytoplasmic and nuclear proteins were prepared from RAW264.7 cells (C) and NP60-transfected 293T cells (D), resolved by SDS-PAGE and western blotted with antibodies against NP60 and actin. (E) 293T cells transfected with NP60-myc/his were fixed after transfection and used for immunohistochemistry assay. Signal was detected using his antibody; DNA was stained using DAPI. Bar, 10 µm.

View larger version (37K): [in a new window]   Fig. 3. Interaction of NP60 with p38. (A) Bacterially expressed NP60, its truncated mutants, PWWP, AT-hook and NAD, ATF2 were coated on 96-well plates and incubated with p38. The interaction was determined by ELISA. Values are means ± s.e.m. (B) Bacterially expressed GST-NP60 and His-p38 were incubated and the reaction complex was pulled down with glutathione Sepharose 4B, and detected on a blot using p38 antibody. (C) Bacterially expressed GST-NP60 and His-p38 were incubated and the reaction complex was pulled down with Ni-NTA agarose and detected by NP60 antibody on a blot. (D) Bacterially expressed GST-NP60 and its different truncated mutant proteins were incubated with His-p38 and the reaction complex pulled down with glutathione Sepharose 4B, and detected on a blot using p38 antibody. (E) Vectors that express myc-NP60 and Flag-p38 were co-transfected into 293T cells. Total protein was extracted after transfection and precipitated with myc antibody. The precipitate was examined by immunoblotting with either p38 antibody or myc antibody as indicated. (F) myc-NP60 was co-transfected with Flag-JNK or Flag-ERK into 293T cells. Total protein was extracted after transfection and precipitated with myc antibody. The precipitation was examined by immunoblotting with flag or myc antibodies as indicated.

View larger version (37K): [in a new window]   Fig. 4. Induction of p38 phosphorylation by NP60. (A) Flag-p38 was co-transfected with myc-NP60 in 293T cells. Cell extracts were prepared 24 hours after transfection. Total proteins were analyzed by immunoblotting with anti-phospho-p38 (*P-p38) antibody. Equal loading was confirmed by immunoblotting with anti-p38 antibody as indicated. (B) Cell extracts were prepared from NP60-transfected 293T cells. Total proteins were analyzed by immunoblotting with anti-phospho-p38 antibody. Equal loading was confirmed by immunoblotting with anti-p38 antibody as indicated. (C) Cell extracts of truncated NP60 mutants co-transfected with p38. Total proteins were analyzed by immunoblotting with anti-phospho-p38 antibody. (D) 293T cells were co-transfected with myc-NP60 and Flag-JNK1. Cell extracts were prepared after transfection. Total proteins were analyzed by immunoblotting with anti-phospho-JNK antibody. (E) 293T cells were co-transfected with myc-NP60 and Flag-ERK2. Total proteins were analyzed by immunoblotting with anti-phospho-ERK antibody. (F) 293T cells were transfected with interference plasmid mu6, or the plasmid containing interference sequence of NP60 at two concentrations. Total RNA was extracted after transfection for 48 hours and used for RT-RCR analysis with NP60 primers. (G) 293T cells were transfected with interference plasmid mu6 and the plasmid containing interference sequence of NP60. Cells were treated with sorbitol for 30 minutes after transfection for 48 hours. Total proteins were extracted and subjected to western blot assay using anti-phospho-p38, or anti-phospho-JNK or anti-phospho-ERK as indicated. Normalization of loading was assessed using p38 antibody.

View larger version (47K): [in a new window]   Fig. 5. Phosphorylation assay for p38 in vitro. (A) NP60 expressed in 293T cells was incubated with p38 in the presence of [-32P]ATP with or without SB203580. The reaction complex was resolved by SDS-PAGE, and visualized by autoradiography. (B) NP60 incubated with p38 and ATF2 in the presence of [-32P]ATP.

View larger version (37K): [in a new window]   Fig. 6 . Effect of MAPKKs on the phosphorylation of p38 induced by NP60. (A) Flag-tagged wild-type p38 (wt) and myc-NP60 were co-transfected into 293T cells. SB203580 (2 µM) was added to the medium 4 hours after transfection as indicated. Phosphorylation of p38 was analyzed by western blotting with anti-phospho-p38 (left panel). Flag-tagged mutant p38 [p38(M)] and myc-NP60 were co-transfected into 293T cells. Phosphorylation of p38 was analyzed as above (right panel). (B) Flag-p38 and myc-NP60 were co-transfected into 293T cells together with MKK6b(A) or MKK4(A). Phosphorylation of p38 was analyzed as in A. (C) Flag-p38 and myc-NP60 were co-transfected into 293T cells together with MKK6b or MKK4. Phosphorylation of p38 was analyzed as in A.

View larger version (21K): [in a new window]   Fig. 7 . Effect of NP60 on the activation of ATF2. (A) 293T cells were grown on 35 mm dishes. Luciferase reporter plasmid (5xGAL, 2 µg), pRL-SV40 (100 ng), GAL-ATF2 (200 ng), NP60 (200 ng) or MKK6b (200 ng) and FLAG-p38 (200 ng) were co-transfected into 293T cells in different combinations as indicated. Luciferase activity was measured 24 hours after transfection. Values are the means ± s.e.m. (B) Flag-p38 was co-expressed with myc-NP60 in 293T cells. Flag-p38 was isolated by anti-Flag antibody. It was then incubated with ATF2 fusion protein in the presence of ATP. The reaction complex was resolved by SDS-PAGE and western blotting was performed using anti-phospho-ATF2 antibody.