spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 30 May 2006
doi: 10.1242/jcs.02995

Journal of Cell Science 119, 2563-2571 (2006)
Published by The Company of Biologists 2006
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Maertens, G. N.
Right arrow Articles by Engelman, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maertens, G. N.
Right arrow Articles by Engelman, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Transcriptional co-activator p75 binds and tethers the Myc-interacting protein JPO2 to chromatin

G. N. Maertens*, P. Cherepanov{ddagger} and A. Engelman§

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Department of Pathology, Harvard Medical School, Boston, MA 02115, USA


Figure 1
View larger version (37K):

[in a new window]
  		Fig. 1. Transcriptional co-activator p75 interacts with JPO2. (A) Reciprocal co-immunoprecipitations of Flag-JPO2 and HA-p75. 293T cells co-transfected with HA-p75 and Flag-JPO2 or Flag-Mob2 expression constructs were lysed 24 hours post-transfection. Proteins immunoprecipitated (IP) with anti-Flag (left panel) or anti-HA (right panel) antibodies were detected by western blotting (WB). The migration positions of molecular mass standards are indicated to the left of each panel. (B) Flag-JPO2 complexes with endogenous p75. Lysates of 293T cells transfected with Flag-JPO2 were immunoprecipitated with anti-Flag or anti-p75/p52 antibodies. (C) Interaction between endogenous JPO2 and p75 proteins. Extracts of non-transfected 293T cells were immunoprecipitated with monoclonal anti-p75/p52 antibody, and JPO2 was detected using rabbit anti-JPO2 antibodies. The asterisk indicates a nonspecific protein detected during western blotting. (D) Overexpression of p75 post-transcriptionally augments the steady-state level of Flag-JPO2 protein. 293T cells were transfected with variable amounts of pBHA-p75 (lanes 2 and 6, 1 µg; lanes 3 and 7, 2 µg; lanes 4 and 8, 3 µg) with Flag-JPO2 (lanes 1-4) or Flag-Mob2 (lanes 5-8). Lanes 1 and 5, pBHA-p75 was omitted from transfection. The total amount of DNA in each transfection was adjusted to 4 µg using pcDNA6.V5HisB (Invitrogen). Cells were harvested 24 hours post-transfection, and 15 µg of total cellular protein was analyzed by SDS-PAGE and western blotting.

 

Figure 2
View larger version (52K):

[in a new window]
  		Fig. 2. Co-localization of JPO2 with wild-type and NLS-deficient p75. (A) EGFP-JPO2 accumulates in nuclei with a heterogeneous distribution pattern. (B) EGFP-JPO2 and HcRed1-p75 co-localize in cell nuclei. (C) EGFP-GST-p75K150A (K150A) sequesters HcRed1-JPO2 to the cell cytoplasm. (D) Co-localization of EGFP-p75326-530 (p75/Ct) and HcRed1-JPO2. Images were acquired 24 hours post-transfection. Bars, 10 µm.

 

Figure 3
View larger version (29K):

[in a new window]
  		Fig. 3. HIV-1 IN and JPO2 compete for binding to the p75 IBD. (A) The p75 IBD is necessary and sufficient for the interaction with JPO2. Lanes 1 and 2; input levels of BSA and 35S-labeled JPO2, respectively. Lanes 3-9: GST, GST-p75 or the indicated p75 deletion mutant pre-bound to GS beads was incubated with 35S-labeled JPO2. BSA (10 µg) was included in each reaction as an internal specificity control. Bound proteins separated by SDS-PAGE were stained with Coomassie Blue (top panel); JPO2 was detected by autoradiography (bottom panel). (B) HIV-1 IN competes with JPO2 for binding to p75 IBD. Lanes 1-4: input BSA, IN-His6, p75 and 35S-labeled JPO2, respectively. Lanes 5-10: His6-tagged HIV-1 IN was incubated with p75 and/or JPO2 in the presence of Ni-NTA beads and BSA, and bound proteins fractionated by SDS-PAGE were detected by staining with Coomassie Blue (top panel) or autoradiography (bottom panel). Migration positions of molecular mass markers, IN-His6, p75, BSA and JPO2 are indicated.

 

Figure 4
View larger version (39K):

[in a new window]
  		Fig. 4. Identification of the p75-binding domain in JPO2. (A) JPO2 (wt) or its deletion mutants (indicated) produced by in vitro transcription and translation were incubated with GST or GST-p75347-471 pre-bound to GS beads in the presence of BSA. Eluted proteins fractionated by SDS-PAGE were visualized by autoradiography (top panels) or staining with Coomassie Blue (bottom panels). Migration positions of molecular mass markers, GST and GST-p75347-471are indicated. None of the JPO2 deletions mutants bound to GST alone (data not shown). (B) Schematic representation of wild-type JPO2 and deletion mutants used in panel A. Predicted {alpha} helices and ß strands are indicated below the full-length sequence as filled and hatched boxes, respectively. Binding affinities of deletion mutant proteins relative to wild-type protein are indicated (+, +/- or -).

 

Figure 5
View larger version (24K):

[in a new window]
  		Fig. 5. Conservation of the p75-binding domain in JPO2 among mammalian and avian species. Sequence alignment of the p75-binding domain (human residues 58-128) among known JPO2 orthologs. Amino acids identical in all species are highlighted in red, and sequence identity in at least 70% of the species as well as conservative amino acid substitutions are in yellow. Predicted secondary structural elements ({alpha}-helices, wavy lines; ß-strands, arrows) are shown at the bottom; the minimal binding region identified in Fig. 4 is indicated. Numbering on the top of the alignment corresponds to the human sequence. Accession numbers for JPO2 orthologs are: chimpanzee (Pan troglodytes), XP 527681; mouse (Mus musculus), NP 666152; rat (Rattus norvegicus), NP 001030125; cow (Bos taurus), XP 592513; dog (Canis familiaris), XP 539464; chicken (Gallus gallus), NP 001026153. The alignment was created using ESPript (Gouet et al., 1999Go).

 

Figure 6
View larger version (76K):

[in a new window]
  		Fig. 6. p75 tethers JPO2 to chromosomes. 293T-si1340/1428 cells depleted for endogenous p75 or control 293T-siScram cells were transfected with the EGFP-JPO2 expression construct. At 48 hours post-transfection, fixed cells were immunostained for p75 (red). EGFP-JPO2 was detected by epifluorescence (green) and DNA by staining with DRAQ5 (blue). Bars, 10 µm.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2006