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First published online December 22, 2004
doi: 10.1242/10.1242/jcs.01600

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Activity and subcellular compartmentalization of peroxisome proliferator-activated receptor are altered by the centrosome-associated protein CAP350

¹ Department of Biochemistry, McMaster University, Hamilton, ON L8N 3Z5, Canada
² Department of Cell Biology, University of Alberta, Edmonton, AB T6G 2H7, Canada

View larger version (5K): [in a new window]   Fig. 1. Schematic of human CAP350. The CAP350 cDNA is predicted to encode a protein of 3117 amino acids. The locations of the conserved CAP-Gly domain and of the two putative LxxLL nuclear hormone receptor-interacting signature motifs in CAP350 are indicated. KM3 indicates the extent of two independently isolated, partial HeLa cell cDNA clones obtained from yeast two-hybrid screens for PPAR-interacting proteins (Miyata et al., 1996) and corresponds to amino acids 572-1176 of CAP350.

View larger version (58K): [in a new window]   Fig. 2. Expression pattern of CAP350 mRNA. A northern blot of RNAs isolated from different human tissues was probed with a 32P-labeled partial cDNA for CAP350. The same blot was probed with 32P-labeled -actin to serve as an internal standard for RNA loading. The positions of size markers, in kilobases (kb), are indicated.

View larger version (36K): [in a new window]   Fig. 3. CAP350 interacts with PPARs and LXR, but not RXR, in vitro. (A) Human PPAR and firefly luciferase (Luc) as a control for nonspecific protein binding, and (B) PPAR2, PPAR, LXR and RXR synthesized in vitro and labeled with L-[35S]methionine were incubated with immobilized GST-KM3 or GST alone in the presence or absence of cognate ligand, as indicated. Bound radiolabeled proteins were analyzed by SDS-PAGE. The ligands used were Panagonist B for PPARs, 22-[R]-hydroxycholesterol for LXR, and 9-cis retinoic acid for RXR. Lanes designated Load (1/10) had 10% of the L-[35S]methionine-labeled protein added to each of the respective binding assays.

View larger version (69K): [in a new window]   Fig. 4. CAP350 redirects PPAR subcellular localization. (A) NIH3T3 cells were transfected separately with expression plasmids for PPAR (panels A-C) or CAP350 (panels D-F) and subjected to immunostaining with anti-PPAR antibody (green) or anti-c-Myc antibody (red) to detect tagged CAP350, as indicated. Nuclei were stained with Hoechst. Panels C and F show the respective merged images. Representative images are shown. PPAR is found distributed throughout the nucleus and is absent from the cytoplasm. CAP350 is present in the nucleus and concentrates in discrete subnuclear foci, as well as in the perinuclear region and on cytoskeletal microfilaments. (See also movie 1 in supplementary material.) (B) NIH3T3 cells were cotransfected with expression vectors for PPAR and CAP350, which were visualized in the same cell. Panel D is the merged image in which colocalization of PPAR and CAP350 is shown in yellow. (See also Movie 2 in supplementary material.) Bars, 10 µm.

View larger version (60K): [in a new window]   Fig. 5. CAP350 does not affect RXR or TR subcellular localization. Representative images of NIH3T3 cells transfected with an expression vector for RXR alone (A-C) or TR alone (J-L) or cotransfected with expression vectors for RXR and CAP350 (D-F) or TR and CAP350 (M-O), and analyzed by indirect immunofluoresence microscopy with anti-RXR antibody or anti-TR antibody (green) and anti-c-Myc antibody (red) to detect tagged CAP350. The merged images indicate that CAP350 does not alter the subcellular localization of RXR or TR (F and O, respectively). Nuclei were detected by staining with Hoechst (B and K). (G-I) PPAR ligand does not affect the subcellular relocalization of PPAR in the presence of CAP350. NIH3T3 cells were transfected with expression vectors for PPAR and CAP350 in the presence of the PPAR ligand Panagonist B and subjected to immunostaining with anti-PPAR antibody (green) and anti-c-Myc antibody (red) to detect tagged CAP350. The merged image (I) shows that ligand does not alter the colocalization of PPAR and CAP350. Bars, 10 µm.

View larger version (84K): [in a new window]   Fig. 6. CAP350 colocalizes with the centrosome. (A) NIH3T3 cells were cotransfected with expression plasmids for CAP350 and ECFP-Golgi in the absence (panels A-C) or presence (panels D-F) of Brefeldin A. CAP350 was detected by immunostaining with anti-c-Myc antibody (red). The merged images show that CAP350 does not colocalize with the Golgi complex. (B) CAP350 is present in the centrosome. NIH3T3 cells were transfected with the expression plasmid for CAP350 and subjected to immunostaining with anti-pericentrin antibody (green) (panel A) and anti-c-Myc antibody (red) to detect tagged CAP350 (panel B). The merged image indicates colocalization of CAP350 with pericentrin in the centrosome, as shown by the yellow color. (See also Movie 3 in supplementary material.) (C) CAP350 colocalizes with -tubulin. NIH3T3 cells were cotransfected with plasmids expressing EYFP--tubulin (panel A) and CAP350 (panel B). CAP350 was detected by immunostaining with anti-c-Myc antibody (red). The colocalization of CAP350 with EYFP--tubulin is seen as yellow in the merged image (panel C). Bars, 10 µm.

View larger version (111K): [in a new window]   Fig. 7. CAP350 colocalizes with intermediate filaments (IF). (A) NIH3T3 cells were transfected with expression plasmid for CAP350 and subjected to immunostaining with anti-cytokeratin antibody (green; panel A) and anti-c-Myc antibody (red) to detect tagged CAP350 (panel B). The merged image (panel C) shows colocalization of CAP350 and cytokeratin, a marker of IFs, as indicated by the yellow color. (B) CAP350 does not colocalize with mitochondria. NIH3T3 cells were cotransfected with expression plasmids for ECFP-Mito (panel A) and CAP350 (panel B). CAP350 was detected by immunostaining with anti-c-Myc antibody (red). The merged image (panel C) shows that CAP350 does not colocalize with mitochondria. (C) CAP350 does not colocalize with actin filaments. NIH3T3 cells transfected with expression plasmid for CAP350 were analyzed by staining with phalloidin-FITC to detect actin (green) (panel A) and anti-c-Myc antibody (red) to detect CAP350 (panel E). The merged image (panel C) shows that CAP350 does not colocalize with actin filaments. Bars, 10 µm.

View larger version (98K): [in a new window]   Fig. 8. CAP350 and PPAR colocalize to novel subnuclear foci. NIH3T3 cells were cotransfected with expression plasmids for CAP350 and GFP-SF2/ASF (A-D), GFP-NUP98 (E-H), or GFP-PML (I-K), as indicated. CAP350 was detected by immunostaining with anti-c-Myc antibody (red). Nuclei were detected by staining with Hoechst. The respective merged images indicate that the CAP350-containing subnuclear foci do not represent speckles, Nup98 bodies or PML bodies. Formation of CAP350-PPAR subnuclear foci does not require active transcription. NIH3T3 cells were cotransfected with plasmids expressing CAP350 and PPAR in the presence of the transcriptional inhibitor, actinomycin D (M-P). CAP350 and PPAR were detected by immunostaining with anti-c-Myc antibody (red) and anti-PPAR antibody (green), respectively. Nuclei were detected by staining with Hoechst. The merged image (P) indicates that the colocalization of CAP350 and PPAR in subnuclear bodies does not require ongoing RNA synthesis. Bars, 10 µm.

View larger version (43K): [in a new window]   Fig. 9. An amino-terminal fragment of CAP350 localizes to subnuclear foci and recruits PPAR to these foci. NIH3T3 cells were cotransfected with expression plasmids for EYFP-CAP3501-890 (green) and mRFP-PPAR (red) (A-C) or for EYFP-CAP3501-890(LSHAA) and mRFP-PPAR (D-F) and subjected to live cell imaging. Panels C and F show the merged images of panels A and B and panels D and E, respectively. Representative images are shown. EYFP-CAP3501-890 and EYFP-CAP3501-890(LSHAA) concentrate in subnuclear foci but are not present in the centrosome or in cytoskeletal microfilaments. mRFP-PPAR colocalizes in subnuclear foci with EYFP-CAP3501-890 but not with EYFP-CAP3501-890(LSHAA) (panel F). Bar, 10 µm. (G) Mutation of the LXXLL motif in the amino-terminal fragment of CAP350 does not abrogate its binding to PPAR in vitro. In vitro synthesized, L-[35S]methionine-labeled PPAR or firefly luciferase (Luc) (a control for nonspecific protein binding) synthesized in vitro was incubated with immobilized GST-CAP3501-890 or GST-CAP3501-890(LSHAA), as indicated, and bound radiolabeled proteins were analyzed by SDS-PAGE. The left lanes show parallel binding reactions carried out with labeled luciferase as a negative control. Lanes designated Load (1/10) had 10% of the L-[35S]methionine-labeled protein added to each of the respective binding assays.

View larger version (15K): [in a new window]   Fig. 10. CAP350 antagonizes transcriptional transactivation by endogenous PPAR in vivo. (A) H4IIEC3 cells were transfected with the reporter plasmid pTK-PPRE(x3)-Luc in the presence or absence of the PPAR ligand Wy-14,643 (100 µM) and increasing amounts of the CAP350 expression vector pMT-hCAP350 (0-0.5 µg/plate), as indicated. Luciferase activity was measured 48 hours posttransfection. The values are the average (±s.d.) fold-induction relative to untreated cells (taken as 1) from three independent transfections carried out in triplicate and normalized for protein and the levels of expression of ß-galactosidase from the plasmid pCMVLacZ used to control for transfection efficiency.(B) An amino-terminal fragment of CAP350 antagonizes PPAR-mediated transcriptional activity in an LXXLL-dependent manner. H4IIEC3 cells were transfected with the reporter plasmid pTK-PPRE(x3)-Luc in the presence or absence of the PPAR ligand, Wy-14,643 (100 µM) and expression vectors for EYFP-CAP350, EYFP-CAP3501-890, EYFP-CAP3501-890(LSHAA) or EYFP alone (designated by the (–) symbol), as indicated. Luciferase activity was measured as above.

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