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PKCζ mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation.

Heo KS, Lee H, Nigro P, Thomas T, Le NT, Chang E, McClain C, Reinhart-King CA, King MR, Berk BC, Fujiwara K, Woo CH, Abe J - J. Cell Biol. (2011)

Bottom Line: Atherosclerosis is readily observed in regions of blood vessels where disturbed blood flow (d-flow) is known to occur.En face confocal microscopy revealed increases in nonnuclear p53 expression, nitrotyrosine staining, and apoptosis in aortic EC located in d-flow areas in wild-type mice, but these effects were significantly decreased in p53(-/-) mice.We propose a novel mechanism for p53 SUMOylation mediated by the PKCζ-PIASy interaction during d-flow-mediated EC apoptosis, which has potential relevance to early events of atherosclerosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Aab Cardiovascular Research Institute, University of Rochester, Rochester, NY 14642, USA.

ABSTRACT
Atherosclerosis is readily observed in regions of blood vessels where disturbed blood flow (d-flow) is known to occur. A positive correlation between protein kinase C ζ (PKCζ) activation and d-flow has been reported, but the exact role of d-flow-mediated PKCζ activation in atherosclerosis remains unclear. We tested the hypothesis that PKCζ activation by d-flow induces endothelial cell (EC) apoptosis by regulating p53. We found that d-flow-mediated peroxynitrite (ONOO(-)) increased PKCζ activation, which subsequently induced p53 SUMOylation, p53-Bcl-2 binding, and EC apoptosis. Both d-flow and ONOO(-) increased the association of PKCζ with protein inhibitor of activated STATy (PIASy) via the Siz/PIAS-RING domain (amino acids 301-410) of PIASy, and overexpression of this domain of PIASy disrupted the PKCζ-PIASy interaction and PKCζ-mediated p53 SUMOylation. En face confocal microscopy revealed increases in nonnuclear p53 expression, nitrotyrosine staining, and apoptosis in aortic EC located in d-flow areas in wild-type mice, but these effects were significantly decreased in p53(-/-) mice. We propose a novel mechanism for p53 SUMOylation mediated by the PKCζ-PIASy interaction during d-flow-mediated EC apoptosis, which has potential relevance to early events of atherosclerosis.

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PKCζ–PIASy association is critical for p53 SUMOylation and p53–Bcl-2 binding. (A) HUVECs were stimulated with 100 µM ONOO– for the indicated times and subjected to immunoprecipitation with anti-PIASy followed by Western blotting with anti-PKCζ (top). (B and C) Association between PKCζ and PIASy was tested by a mammalian two-hybrid assay. HeLa cells were transfected with plasmids containing Gal4-PKCζ wild type and VP16-PIASy (B) or truncated mutants of VP16-PIASy (C) as well as the Gal4-responsive luciferase reporter pG5-luc. After 24 h of transfection, cells were stimulated with 100 µM ONOO− or vehicle for 16 h, and luciferase activity was quantified. Luciferase activity was normalized with the Renilla luciferase (Luc.) activity (Woo et al., 2008). Data are representative of three experiments using two or more different preparations of ECs (means ± SD; **, P < 0.01). (D) PIASy binding to PKCζ occurs via a domain consisting of aa 301–410 of PIASy. HeLa cells were transfected with each of the Flag-tagged PIASy fragments, and then pull-down assays were preformed using anti-Flag and IgG Sepharose beads in the presence of GST-fused recombinant PKCζ. Association of PIASy fragments with GST-PKCζ was assayed by Western blotting with anti-PKCζ. (bottom) PIASy fragment expression was detected by Western blotting with anti-Flag. (E) PIASy Fr3, but not Fr4, inhibited PKCζ–PIASy association. HUVECs were cotransfected with HA-tagged PKCζ wild type, Myc-tagged PIASy wild type, and Flag-tagged PIASy Fr3 or Fr4 for 24 h. Myc-PIASy wild type was immunoprecipitated with anti-Myc followed by immunoblotting with anti-HA (top). The expression of PKCζ, PIASy, and PIASy fragments was detected by Western blotting with specific antibodies. Data are representative of three independent experiments. (F) HUVECs were transfected with Flag-tagged PIASy Fr3 or Fr4 or control vectors for 24 h and then stimulated by d-flow for 3 h. p53 was immunoprecipitated using anti-p53, and d-flow–induced p53 SUMOylation was analyzed by immunoblotting with anti-SUMO2/3 (top). The expression of p53, SUMO, and PIASy fragments was detected by Western blotting with specific antibodies. Data are representative of three independent experiments. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation. WT, wild type.
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fig8: PKCζ–PIASy association is critical for p53 SUMOylation and p53–Bcl-2 binding. (A) HUVECs were stimulated with 100 µM ONOO– for the indicated times and subjected to immunoprecipitation with anti-PIASy followed by Western blotting with anti-PKCζ (top). (B and C) Association between PKCζ and PIASy was tested by a mammalian two-hybrid assay. HeLa cells were transfected with plasmids containing Gal4-PKCζ wild type and VP16-PIASy (B) or truncated mutants of VP16-PIASy (C) as well as the Gal4-responsive luciferase reporter pG5-luc. After 24 h of transfection, cells were stimulated with 100 µM ONOO− or vehicle for 16 h, and luciferase activity was quantified. Luciferase activity was normalized with the Renilla luciferase (Luc.) activity (Woo et al., 2008). Data are representative of three experiments using two or more different preparations of ECs (means ± SD; **, P < 0.01). (D) PIASy binding to PKCζ occurs via a domain consisting of aa 301–410 of PIASy. HeLa cells were transfected with each of the Flag-tagged PIASy fragments, and then pull-down assays were preformed using anti-Flag and IgG Sepharose beads in the presence of GST-fused recombinant PKCζ. Association of PIASy fragments with GST-PKCζ was assayed by Western blotting with anti-PKCζ. (bottom) PIASy fragment expression was detected by Western blotting with anti-Flag. (E) PIASy Fr3, but not Fr4, inhibited PKCζ–PIASy association. HUVECs were cotransfected with HA-tagged PKCζ wild type, Myc-tagged PIASy wild type, and Flag-tagged PIASy Fr3 or Fr4 for 24 h. Myc-PIASy wild type was immunoprecipitated with anti-Myc followed by immunoblotting with anti-HA (top). The expression of PKCζ, PIASy, and PIASy fragments was detected by Western blotting with specific antibodies. Data are representative of three independent experiments. (F) HUVECs were transfected with Flag-tagged PIASy Fr3 or Fr4 or control vectors for 24 h and then stimulated by d-flow for 3 h. p53 was immunoprecipitated using anti-p53, and d-flow–induced p53 SUMOylation was analyzed by immunoblotting with anti-SUMO2/3 (top). The expression of p53, SUMO, and PIASy fragments was detected by Western blotting with specific antibodies. Data are representative of three independent experiments. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation. WT, wild type.

Mentions: As PIASy played a critical role in CATζ-mediated p53 SUMOylation (Fig. 4 B), PKCζ might directly phosphorylate PIASy and activate its E3 SUMO ligase activity. To test this possibility, we incubated recombinant PKCζ with GST-tagged PIASy fragments and found that none of the fragments was phosphorylated by PKCζ, whereas autophosphorylation of PKCζ was detected (Fig. S3). Although it does not phosphorylate PIASy, PKCζ may still interact with PIASy. We cotransfected HeLa cells with HA-tagged PKCζ and myc-tagged PIASy and performed a coimmunoprecipitation assay in which PKCζ and PIASy were coimmunoprecipitated (unpublished data). To confirm this interaction between endogenous PKCζ and PIASy, we stimulated HUVECs with ONOO− for the indicated times and performed coimmunoprecipitation using anti-PKCζ. PIASy was indeed coimmunoprecipitated by anti-PKCζ, and ONOO− stimulation increased the PKCζ–PIASy interaction (Fig. 8 A). Next, to determine the PIASy-binding regions of PKCζ, we generated four PKCζ-truncated mutants and evaluated their association with PIASy using a mammalian two-hybrid assay (Fig. 8, B and C). Plasmids encoding the GAL4–DNA-binding domain and PKCζ (full length or one of the truncated forms) were constructed using the pBIND vector. A plasmid containing VP16-PIASy was constructed using the pACT vector. As expected, wild-type PKCζ bound to PIASy, and ONOO− increased this association (Fig. 8 B). More importantly, however, we found that the C-terminal kinase domain (aa 401–587) was required for the PKCζ–PIASy association (Fig. 8 C). Next, to determine the PKCζ binding site of PIASy, we coexpressed GST-fused PKCζ and Flag-tagged PIASy fragments in HeLa cells and performed coimmunoprecipitation using anti-Flag. We found that PIASy fragment 3 (Fr 3; aa 301–410), which contains the Siz/PIAS RING domain, interacted with PKCζ (Fig. 8 D). In a separate experiment, we examined this fragment could interfere with the PKCζ–PIASy association. HeLa cells were cotransfected with wild-type PKCζ and PIASy together with PIASy Fr 3. Consistent with the coimmunoprecipitation results, PIASy Fr 3 significantly inhibited the PKCζ–PIASy interaction (Fig. 8 E). To demonstrate the importance of the PKCζ–PIASy interaction for p53 SUMOylation, ECs expressing PIASy fragments were stimulated by d-flow. As shown in Fig. 8 F, only PIASy Fr 3 inhibited d-flow–mediated p53 SUMOylation, suggesting that p53 SUMOylation depends on PKCζ–PIASy binding.


PKCζ mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation.

Heo KS, Lee H, Nigro P, Thomas T, Le NT, Chang E, McClain C, Reinhart-King CA, King MR, Berk BC, Fujiwara K, Woo CH, Abe J - J. Cell Biol. (2011)

PKCζ–PIASy association is critical for p53 SUMOylation and p53–Bcl-2 binding. (A) HUVECs were stimulated with 100 µM ONOO– for the indicated times and subjected to immunoprecipitation with anti-PIASy followed by Western blotting with anti-PKCζ (top). (B and C) Association between PKCζ and PIASy was tested by a mammalian two-hybrid assay. HeLa cells were transfected with plasmids containing Gal4-PKCζ wild type and VP16-PIASy (B) or truncated mutants of VP16-PIASy (C) as well as the Gal4-responsive luciferase reporter pG5-luc. After 24 h of transfection, cells were stimulated with 100 µM ONOO− or vehicle for 16 h, and luciferase activity was quantified. Luciferase activity was normalized with the Renilla luciferase (Luc.) activity (Woo et al., 2008). Data are representative of three experiments using two or more different preparations of ECs (means ± SD; **, P < 0.01). (D) PIASy binding to PKCζ occurs via a domain consisting of aa 301–410 of PIASy. HeLa cells were transfected with each of the Flag-tagged PIASy fragments, and then pull-down assays were preformed using anti-Flag and IgG Sepharose beads in the presence of GST-fused recombinant PKCζ. Association of PIASy fragments with GST-PKCζ was assayed by Western blotting with anti-PKCζ. (bottom) PIASy fragment expression was detected by Western blotting with anti-Flag. (E) PIASy Fr3, but not Fr4, inhibited PKCζ–PIASy association. HUVECs were cotransfected with HA-tagged PKCζ wild type, Myc-tagged PIASy wild type, and Flag-tagged PIASy Fr3 or Fr4 for 24 h. Myc-PIASy wild type was immunoprecipitated with anti-Myc followed by immunoblotting with anti-HA (top). The expression of PKCζ, PIASy, and PIASy fragments was detected by Western blotting with specific antibodies. Data are representative of three independent experiments. (F) HUVECs were transfected with Flag-tagged PIASy Fr3 or Fr4 or control vectors for 24 h and then stimulated by d-flow for 3 h. p53 was immunoprecipitated using anti-p53, and d-flow–induced p53 SUMOylation was analyzed by immunoblotting with anti-SUMO2/3 (top). The expression of p53, SUMO, and PIASy fragments was detected by Western blotting with specific antibodies. Data are representative of three independent experiments. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation. WT, wild type.
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fig8: PKCζ–PIASy association is critical for p53 SUMOylation and p53–Bcl-2 binding. (A) HUVECs were stimulated with 100 µM ONOO– for the indicated times and subjected to immunoprecipitation with anti-PIASy followed by Western blotting with anti-PKCζ (top). (B and C) Association between PKCζ and PIASy was tested by a mammalian two-hybrid assay. HeLa cells were transfected with plasmids containing Gal4-PKCζ wild type and VP16-PIASy (B) or truncated mutants of VP16-PIASy (C) as well as the Gal4-responsive luciferase reporter pG5-luc. After 24 h of transfection, cells were stimulated with 100 µM ONOO− or vehicle for 16 h, and luciferase activity was quantified. Luciferase activity was normalized with the Renilla luciferase (Luc.) activity (Woo et al., 2008). Data are representative of three experiments using two or more different preparations of ECs (means ± SD; **, P < 0.01). (D) PIASy binding to PKCζ occurs via a domain consisting of aa 301–410 of PIASy. HeLa cells were transfected with each of the Flag-tagged PIASy fragments, and then pull-down assays were preformed using anti-Flag and IgG Sepharose beads in the presence of GST-fused recombinant PKCζ. Association of PIASy fragments with GST-PKCζ was assayed by Western blotting with anti-PKCζ. (bottom) PIASy fragment expression was detected by Western blotting with anti-Flag. (E) PIASy Fr3, but not Fr4, inhibited PKCζ–PIASy association. HUVECs were cotransfected with HA-tagged PKCζ wild type, Myc-tagged PIASy wild type, and Flag-tagged PIASy Fr3 or Fr4 for 24 h. Myc-PIASy wild type was immunoprecipitated with anti-Myc followed by immunoblotting with anti-HA (top). The expression of PKCζ, PIASy, and PIASy fragments was detected by Western blotting with specific antibodies. Data are representative of three independent experiments. (F) HUVECs were transfected with Flag-tagged PIASy Fr3 or Fr4 or control vectors for 24 h and then stimulated by d-flow for 3 h. p53 was immunoprecipitated using anti-p53, and d-flow–induced p53 SUMOylation was analyzed by immunoblotting with anti-SUMO2/3 (top). The expression of p53, SUMO, and PIASy fragments was detected by Western blotting with specific antibodies. Data are representative of three independent experiments. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation. WT, wild type.
Mentions: As PIASy played a critical role in CATζ-mediated p53 SUMOylation (Fig. 4 B), PKCζ might directly phosphorylate PIASy and activate its E3 SUMO ligase activity. To test this possibility, we incubated recombinant PKCζ with GST-tagged PIASy fragments and found that none of the fragments was phosphorylated by PKCζ, whereas autophosphorylation of PKCζ was detected (Fig. S3). Although it does not phosphorylate PIASy, PKCζ may still interact with PIASy. We cotransfected HeLa cells with HA-tagged PKCζ and myc-tagged PIASy and performed a coimmunoprecipitation assay in which PKCζ and PIASy were coimmunoprecipitated (unpublished data). To confirm this interaction between endogenous PKCζ and PIASy, we stimulated HUVECs with ONOO− for the indicated times and performed coimmunoprecipitation using anti-PKCζ. PIASy was indeed coimmunoprecipitated by anti-PKCζ, and ONOO− stimulation increased the PKCζ–PIASy interaction (Fig. 8 A). Next, to determine the PIASy-binding regions of PKCζ, we generated four PKCζ-truncated mutants and evaluated their association with PIASy using a mammalian two-hybrid assay (Fig. 8, B and C). Plasmids encoding the GAL4–DNA-binding domain and PKCζ (full length or one of the truncated forms) were constructed using the pBIND vector. A plasmid containing VP16-PIASy was constructed using the pACT vector. As expected, wild-type PKCζ bound to PIASy, and ONOO− increased this association (Fig. 8 B). More importantly, however, we found that the C-terminal kinase domain (aa 401–587) was required for the PKCζ–PIASy association (Fig. 8 C). Next, to determine the PKCζ binding site of PIASy, we coexpressed GST-fused PKCζ and Flag-tagged PIASy fragments in HeLa cells and performed coimmunoprecipitation using anti-Flag. We found that PIASy fragment 3 (Fr 3; aa 301–410), which contains the Siz/PIAS RING domain, interacted with PKCζ (Fig. 8 D). In a separate experiment, we examined this fragment could interfere with the PKCζ–PIASy association. HeLa cells were cotransfected with wild-type PKCζ and PIASy together with PIASy Fr 3. Consistent with the coimmunoprecipitation results, PIASy Fr 3 significantly inhibited the PKCζ–PIASy interaction (Fig. 8 E). To demonstrate the importance of the PKCζ–PIASy interaction for p53 SUMOylation, ECs expressing PIASy fragments were stimulated by d-flow. As shown in Fig. 8 F, only PIASy Fr 3 inhibited d-flow–mediated p53 SUMOylation, suggesting that p53 SUMOylation depends on PKCζ–PIASy binding.

Bottom Line: Atherosclerosis is readily observed in regions of blood vessels where disturbed blood flow (d-flow) is known to occur.En face confocal microscopy revealed increases in nonnuclear p53 expression, nitrotyrosine staining, and apoptosis in aortic EC located in d-flow areas in wild-type mice, but these effects were significantly decreased in p53(-/-) mice.We propose a novel mechanism for p53 SUMOylation mediated by the PKCζ-PIASy interaction during d-flow-mediated EC apoptosis, which has potential relevance to early events of atherosclerosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Aab Cardiovascular Research Institute, University of Rochester, Rochester, NY 14642, USA.

ABSTRACT
Atherosclerosis is readily observed in regions of blood vessels where disturbed blood flow (d-flow) is known to occur. A positive correlation between protein kinase C ζ (PKCζ) activation and d-flow has been reported, but the exact role of d-flow-mediated PKCζ activation in atherosclerosis remains unclear. We tested the hypothesis that PKCζ activation by d-flow induces endothelial cell (EC) apoptosis by regulating p53. We found that d-flow-mediated peroxynitrite (ONOO(-)) increased PKCζ activation, which subsequently induced p53 SUMOylation, p53-Bcl-2 binding, and EC apoptosis. Both d-flow and ONOO(-) increased the association of PKCζ with protein inhibitor of activated STATy (PIASy) via the Siz/PIAS-RING domain (amino acids 301-410) of PIASy, and overexpression of this domain of PIASy disrupted the PKCζ-PIASy interaction and PKCζ-mediated p53 SUMOylation. En face confocal microscopy revealed increases in nonnuclear p53 expression, nitrotyrosine staining, and apoptosis in aortic EC located in d-flow areas in wild-type mice, but these effects were significantly decreased in p53(-/-) mice. We propose a novel mechanism for p53 SUMOylation mediated by the PKCζ-PIASy interaction during d-flow-mediated EC apoptosis, which has potential relevance to early events of atherosclerosis.

Show MeSH
Related in: MedlinePlus