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Stress-induced alternative splice forms of MDM2 and MDMX modulate the p53-pathway in distinct ways.

Jacob AG, Singh RK, Comiskey DF, Rouhier MF, Mohammad F, Bebee TW, Chandler DS - PLoS ONE (2014)

Bottom Line: We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2.Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21.In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response.

View Article: PubMed Central - PubMed

Affiliation: From the Center for Childhood Cancer at the Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States of America; The Department of Pediatrics, and Molecular, Cellular and Developmental Biology (MCDB) program, The Ohio State University, Columbus, Ohio, United States of America; Center for RNA Biology, Wexner Medical Center, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
MDM2 and MDMX are the chief negative regulators of the tumor-suppressor protein p53 and are essential for maintaining homeostasis within the cell. In response to genotoxic stress and also in several cancer types, MDM2 and MDMX are alternatively spliced. The splice variants MDM2-ALT1 and MDMX-ALT2 lack the p53-binding domain and are incapable of negatively regulating p53. However, they retain the RING domain that facilitates dimerization of the full-length MDM proteins. Concordantly, MDM2-ALT1 has been shown to lead to the stabilization of p53 through its interaction with and inactivation of full-length MDM2. The impact of MDM2-ALT1 expression on the p53 pathway and the nature of its interaction with MDMX remain unclear. Also, the role of the architecturally similar MDMX-ALT2 and its influence of the MDM2-MDMX-p53 axis are yet to be elucidated. We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2. Additionally, we demonstrate that MDMX-ALT2 is able to dimerize with both full-length MDMX and MDM2 and that the expression of MDM2-ALT1 and MDMX-ALT2 leads to the upregulation of p53 protein, and also of its downstream target p21. Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21. Finally we present evidence that MDM2-ALT1 and MDMX-ALT2 expression can activate subtly distinct subsets of p53-transcriptional targets implying that these splice variants can modulate the p53 tumor suppressor pathway in unique ways. In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response.

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MDM2-ALT1 and MDMX-ALT2 interact with full-length MDM2 and MDMX.A. Full-length MDM2 is encoded by exons 3 to 12 of the MDM2 gene and consists of the N-terminal p53-binding domain, the nuclear localization (NLS) and export signals (NES), the central ARF binding and Zinc finger domains and the C-terminal RING domain. MDM2-ALT1 comprises only exons 3 and 12 spliced together and the protein lacks the p53-binding domain. However, it retains the RING domain. B. Full-length MDMX, a close family member of MDM2, also comprises an N-terminal p53-binding domain, a central Zinc finger domain and a C-terminal RING domain and is encoded by exons 2 to 11 of the MDMX gene. MDMX-ALT2 consists of exons 2,3,10 and 11 and the protein is architecturally similar to MDM2-ALT1 in that it lacks the p53-binding domain but retains the RING domain. C. Myc-tagged constructs of LacZ, MDM2-ALT1 or MDMX-ALT2 were transfected into MCF7 cells. Immunoprecipitation of the myc-tagged proteins revealed the specific binding of full-length MDM2 to MDM2-ALT1 and MDMX-ALT2 and not to negative control protein myc-LacZ (compare lanes 2 and 3 to lane 1). Experiments were repeated a minimum of three times and consistent results were observed. Representative gel images are presented in the figure. D. Myc-tagged MDM2-ALT1 and MDMX-ALT2 co-immunoprecipitate with full-length MDMX while the negative control protein myc-LacZ does not interact with MDMX (compare lanes 2 and 3 to lane 1). These results were observed in two independent trials and representative images are shown.
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pone-0104444-g001: MDM2-ALT1 and MDMX-ALT2 interact with full-length MDM2 and MDMX.A. Full-length MDM2 is encoded by exons 3 to 12 of the MDM2 gene and consists of the N-terminal p53-binding domain, the nuclear localization (NLS) and export signals (NES), the central ARF binding and Zinc finger domains and the C-terminal RING domain. MDM2-ALT1 comprises only exons 3 and 12 spliced together and the protein lacks the p53-binding domain. However, it retains the RING domain. B. Full-length MDMX, a close family member of MDM2, also comprises an N-terminal p53-binding domain, a central Zinc finger domain and a C-terminal RING domain and is encoded by exons 2 to 11 of the MDMX gene. MDMX-ALT2 consists of exons 2,3,10 and 11 and the protein is architecturally similar to MDM2-ALT1 in that it lacks the p53-binding domain but retains the RING domain. C. Myc-tagged constructs of LacZ, MDM2-ALT1 or MDMX-ALT2 were transfected into MCF7 cells. Immunoprecipitation of the myc-tagged proteins revealed the specific binding of full-length MDM2 to MDM2-ALT1 and MDMX-ALT2 and not to negative control protein myc-LacZ (compare lanes 2 and 3 to lane 1). Experiments were repeated a minimum of three times and consistent results were observed. Representative gel images are presented in the figure. D. Myc-tagged MDM2-ALT1 and MDMX-ALT2 co-immunoprecipitate with full-length MDMX while the negative control protein myc-LacZ does not interact with MDMX (compare lanes 2 and 3 to lane 1). These results were observed in two independent trials and representative images are shown.

Mentions: MDM2-ALT1, a major stress- inducible splice variant of MDM2 lacks a p53-binding domain but contains an intact RING domain ([36] and fig 1A). The RING domain facilitates the homodimerization of MDM2 and also hetero-dimerization with MDMX [14], [52]. Previous reports have demonstrated the interaction of MDM2-ALT1 with full-length MDM2, which potentially affects the functions of MDM2 [30], [32]. We wanted to test the possibility that MDM2-ALT1 could also directly form heterodimers with MDMX. To this end, we transfected MCF7 cells with plasmids expressing myc-MDM2-ALT1 or a negative control myc-GFP and performed an immunoprecipitation of the myc-tagged proteins. The immunoprecipitated samples were then probed with antibodies specific for MDM2 or MDMX to identify interaction of MDM2-ALT1 and MDMX-ALT2 with the endogenous isoforms of MDM2 and MDMX (Fig 1 and S1). Consistent with previous reports, we observed the direct interaction of MDM2-ALT1 with full-length MDM2 (Fig 1C and S1A). We additionally performed reciprocal immunoprecipitation of MDM2 to validate interaction of endogenous MDM2 with MDM2-ALT1 in cells expressing control (myc-LacZ) or myc-MDM2-ALT1. Results indicate that myc-MDM2-ALT1 interacted with endogenous MDM2 (Figure S2). We also observed that MDM2-ALT1 interacts directly with full-length MDMX (Fig 1D and S1B). The specificity of these interactions is demonstrated by the fact that myc-tagged LacZ or GFP did not co-immunoprecipitate with either endogenous MDM2 or MDMX (Fig 1C and 1D).


Stress-induced alternative splice forms of MDM2 and MDMX modulate the p53-pathway in distinct ways.

Jacob AG, Singh RK, Comiskey DF, Rouhier MF, Mohammad F, Bebee TW, Chandler DS - PLoS ONE (2014)

MDM2-ALT1 and MDMX-ALT2 interact with full-length MDM2 and MDMX.A. Full-length MDM2 is encoded by exons 3 to 12 of the MDM2 gene and consists of the N-terminal p53-binding domain, the nuclear localization (NLS) and export signals (NES), the central ARF binding and Zinc finger domains and the C-terminal RING domain. MDM2-ALT1 comprises only exons 3 and 12 spliced together and the protein lacks the p53-binding domain. However, it retains the RING domain. B. Full-length MDMX, a close family member of MDM2, also comprises an N-terminal p53-binding domain, a central Zinc finger domain and a C-terminal RING domain and is encoded by exons 2 to 11 of the MDMX gene. MDMX-ALT2 consists of exons 2,3,10 and 11 and the protein is architecturally similar to MDM2-ALT1 in that it lacks the p53-binding domain but retains the RING domain. C. Myc-tagged constructs of LacZ, MDM2-ALT1 or MDMX-ALT2 were transfected into MCF7 cells. Immunoprecipitation of the myc-tagged proteins revealed the specific binding of full-length MDM2 to MDM2-ALT1 and MDMX-ALT2 and not to negative control protein myc-LacZ (compare lanes 2 and 3 to lane 1). Experiments were repeated a minimum of three times and consistent results were observed. Representative gel images are presented in the figure. D. Myc-tagged MDM2-ALT1 and MDMX-ALT2 co-immunoprecipitate with full-length MDMX while the negative control protein myc-LacZ does not interact with MDMX (compare lanes 2 and 3 to lane 1). These results were observed in two independent trials and representative images are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4126728&req=5

pone-0104444-g001: MDM2-ALT1 and MDMX-ALT2 interact with full-length MDM2 and MDMX.A. Full-length MDM2 is encoded by exons 3 to 12 of the MDM2 gene and consists of the N-terminal p53-binding domain, the nuclear localization (NLS) and export signals (NES), the central ARF binding and Zinc finger domains and the C-terminal RING domain. MDM2-ALT1 comprises only exons 3 and 12 spliced together and the protein lacks the p53-binding domain. However, it retains the RING domain. B. Full-length MDMX, a close family member of MDM2, also comprises an N-terminal p53-binding domain, a central Zinc finger domain and a C-terminal RING domain and is encoded by exons 2 to 11 of the MDMX gene. MDMX-ALT2 consists of exons 2,3,10 and 11 and the protein is architecturally similar to MDM2-ALT1 in that it lacks the p53-binding domain but retains the RING domain. C. Myc-tagged constructs of LacZ, MDM2-ALT1 or MDMX-ALT2 were transfected into MCF7 cells. Immunoprecipitation of the myc-tagged proteins revealed the specific binding of full-length MDM2 to MDM2-ALT1 and MDMX-ALT2 and not to negative control protein myc-LacZ (compare lanes 2 and 3 to lane 1). Experiments were repeated a minimum of three times and consistent results were observed. Representative gel images are presented in the figure. D. Myc-tagged MDM2-ALT1 and MDMX-ALT2 co-immunoprecipitate with full-length MDMX while the negative control protein myc-LacZ does not interact with MDMX (compare lanes 2 and 3 to lane 1). These results were observed in two independent trials and representative images are shown.
Mentions: MDM2-ALT1, a major stress- inducible splice variant of MDM2 lacks a p53-binding domain but contains an intact RING domain ([36] and fig 1A). The RING domain facilitates the homodimerization of MDM2 and also hetero-dimerization with MDMX [14], [52]. Previous reports have demonstrated the interaction of MDM2-ALT1 with full-length MDM2, which potentially affects the functions of MDM2 [30], [32]. We wanted to test the possibility that MDM2-ALT1 could also directly form heterodimers with MDMX. To this end, we transfected MCF7 cells with plasmids expressing myc-MDM2-ALT1 or a negative control myc-GFP and performed an immunoprecipitation of the myc-tagged proteins. The immunoprecipitated samples were then probed with antibodies specific for MDM2 or MDMX to identify interaction of MDM2-ALT1 and MDMX-ALT2 with the endogenous isoforms of MDM2 and MDMX (Fig 1 and S1). Consistent with previous reports, we observed the direct interaction of MDM2-ALT1 with full-length MDM2 (Fig 1C and S1A). We additionally performed reciprocal immunoprecipitation of MDM2 to validate interaction of endogenous MDM2 with MDM2-ALT1 in cells expressing control (myc-LacZ) or myc-MDM2-ALT1. Results indicate that myc-MDM2-ALT1 interacted with endogenous MDM2 (Figure S2). We also observed that MDM2-ALT1 interacts directly with full-length MDMX (Fig 1D and S1B). The specificity of these interactions is demonstrated by the fact that myc-tagged LacZ or GFP did not co-immunoprecipitate with either endogenous MDM2 or MDMX (Fig 1C and 1D).

Bottom Line: We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2.Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21.In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response.

View Article: PubMed Central - PubMed

Affiliation: From the Center for Childhood Cancer at the Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States of America; The Department of Pediatrics, and Molecular, Cellular and Developmental Biology (MCDB) program, The Ohio State University, Columbus, Ohio, United States of America; Center for RNA Biology, Wexner Medical Center, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
MDM2 and MDMX are the chief negative regulators of the tumor-suppressor protein p53 and are essential for maintaining homeostasis within the cell. In response to genotoxic stress and also in several cancer types, MDM2 and MDMX are alternatively spliced. The splice variants MDM2-ALT1 and MDMX-ALT2 lack the p53-binding domain and are incapable of negatively regulating p53. However, they retain the RING domain that facilitates dimerization of the full-length MDM proteins. Concordantly, MDM2-ALT1 has been shown to lead to the stabilization of p53 through its interaction with and inactivation of full-length MDM2. The impact of MDM2-ALT1 expression on the p53 pathway and the nature of its interaction with MDMX remain unclear. Also, the role of the architecturally similar MDMX-ALT2 and its influence of the MDM2-MDMX-p53 axis are yet to be elucidated. We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2. Additionally, we demonstrate that MDMX-ALT2 is able to dimerize with both full-length MDMX and MDM2 and that the expression of MDM2-ALT1 and MDMX-ALT2 leads to the upregulation of p53 protein, and also of its downstream target p21. Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21. Finally we present evidence that MDM2-ALT1 and MDMX-ALT2 expression can activate subtly distinct subsets of p53-transcriptional targets implying that these splice variants can modulate the p53 tumor suppressor pathway in unique ways. In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response.

Show MeSH
Related in: MedlinePlus