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The mitotic arrest deficient protein MAD2B interacts with the small GTPase RAN throughout the cell cycle.

Medendorp K, van Groningen JJ, Vreede L, Hetterschijt L, van den Hurk WH, de Bruijn DR, Brugmans L, van Kessel AG - PLoS ONE (2009)

Bottom Line: The MAD2B-RAN interaction was found to persist throughout the cell cycle.The small GTPase RAN is a novel MAD2B binding protein.This novel protein-protein interaction may play a role in (i) the control over the spindle checkpoint during mitosis and (ii) the regulation of nucleocytoplasmic trafficking during interphase.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands.

ABSTRACT

Background: Previously, we identified the mitotic arrest deficient protein MAD2B (MAD2L2) as a bona fide interactor of the renal cell carcinoma (RCC)-associated protein PRCC. In addition, we found that fusion of PRCC with the transcription factor TFE3 in t(X;1)(p11;q21)-positive RCCs results in an impairment of this interaction and, concomitantly, an abrogation of cell cycle progression. Although MAD2B is thought to inhibit the anaphase promoting complex (APC) by binding to CDC20 and/or CDH1(FZR1), its exact role in cell cycle control still remains to be established.

Methodology/principal findings: Using a yeast two-hybrid interaction trap we identified the small GTPase RAN, a well-known cell cycle regulator, as a novel MAD2B binding protein. Endogenous interaction was established in mammalian cells via co-localization and co-immunoprecipitation of the respective proteins. The interaction domain of RAN could be assigned to a C-terminal moiety of 60 amino acids, whereas MAD2B had to be present in its full-length conformation. The MAD2B-RAN interaction was found to persist throughout the cell cycle. During mitosis, co-localization at the spindle was observed.

Conclusions/significance: The small GTPase RAN is a novel MAD2B binding protein. This novel protein-protein interaction may play a role in (i) the control over the spindle checkpoint during mitosis and (ii) the regulation of nucleocytoplasmic trafficking during interphase.

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Sub-cellular (co-)localization of MAD2B, RAN and PRCC in U2OS cells.U2OS cells were transiently (co-)transfected with MAD2B-CFP (green), PRCC-dsRed (red) and/or RAN-YFP (yellow) expression constructs. The overlay of the different signals (Merge) reveals a partial co-localization of the respective proteins. Images were captured using fluorescence microscopy.
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pone-0007020-g005: Sub-cellular (co-)localization of MAD2B, RAN and PRCC in U2OS cells.U2OS cells were transiently (co-)transfected with MAD2B-CFP (green), PRCC-dsRed (red) and/or RAN-YFP (yellow) expression constructs. The overlay of the different signals (Merge) reveals a partial co-localization of the respective proteins. Images were captured using fluorescence microscopy.

Mentions: Previously, we found that MAD2B can interact with the renal cell carcinoma associated protein PRCC [12]. To assess whether PRCC may act as an integral component of the MAD2B-RAN complex, U2OS cells were transiently (co-)transfected with MAD2B-CFP, PRCC-dsRed and/or RAN-YFP expression constructs. Subsequently, the transfected cells were assayed for the sub-cellular localization of the respective proteins. By doing so, we found that single PRCC transfectants showed a nuclear staining pattern as reported before [12]. Double MAD2B and PRCC transfectants showed a nuclear co-localization, again in accordance with our previous observations [12]. Triple MAD2B, PRCC and RAN transfectants showed a partial co-localization of MAD2B, RAN and PRCC (Fig. 5). As a control, to again exclude chromatin association, we tested whether endogenous PRCC co-localizes with histone-2B. This was not found (Figure S2). Taken together, we conclude that MAD2B, RAN and PRCC share similar sub-cellular compartments. The partial co-localization observed between these three proteins indicates that the interactions of MAD2B with either RAN and/or PRCC may be dynamic in nature. To test whether PRCC and RAN can interact directly, we performed co-immunoprecipitation analyses. By doing so, no co-immunoprecipitation between PRCC and RAN was observed, thus excluding a direct interaction between these two proteins (data not shown).


The mitotic arrest deficient protein MAD2B interacts with the small GTPase RAN throughout the cell cycle.

Medendorp K, van Groningen JJ, Vreede L, Hetterschijt L, van den Hurk WH, de Bruijn DR, Brugmans L, van Kessel AG - PLoS ONE (2009)

Sub-cellular (co-)localization of MAD2B, RAN and PRCC in U2OS cells.U2OS cells were transiently (co-)transfected with MAD2B-CFP (green), PRCC-dsRed (red) and/or RAN-YFP (yellow) expression constructs. The overlay of the different signals (Merge) reveals a partial co-localization of the respective proteins. Images were captured using fluorescence microscopy.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2737141&req=5

pone-0007020-g005: Sub-cellular (co-)localization of MAD2B, RAN and PRCC in U2OS cells.U2OS cells were transiently (co-)transfected with MAD2B-CFP (green), PRCC-dsRed (red) and/or RAN-YFP (yellow) expression constructs. The overlay of the different signals (Merge) reveals a partial co-localization of the respective proteins. Images were captured using fluorescence microscopy.
Mentions: Previously, we found that MAD2B can interact with the renal cell carcinoma associated protein PRCC [12]. To assess whether PRCC may act as an integral component of the MAD2B-RAN complex, U2OS cells were transiently (co-)transfected with MAD2B-CFP, PRCC-dsRed and/or RAN-YFP expression constructs. Subsequently, the transfected cells were assayed for the sub-cellular localization of the respective proteins. By doing so, we found that single PRCC transfectants showed a nuclear staining pattern as reported before [12]. Double MAD2B and PRCC transfectants showed a nuclear co-localization, again in accordance with our previous observations [12]. Triple MAD2B, PRCC and RAN transfectants showed a partial co-localization of MAD2B, RAN and PRCC (Fig. 5). As a control, to again exclude chromatin association, we tested whether endogenous PRCC co-localizes with histone-2B. This was not found (Figure S2). Taken together, we conclude that MAD2B, RAN and PRCC share similar sub-cellular compartments. The partial co-localization observed between these three proteins indicates that the interactions of MAD2B with either RAN and/or PRCC may be dynamic in nature. To test whether PRCC and RAN can interact directly, we performed co-immunoprecipitation analyses. By doing so, no co-immunoprecipitation between PRCC and RAN was observed, thus excluding a direct interaction between these two proteins (data not shown).

Bottom Line: The MAD2B-RAN interaction was found to persist throughout the cell cycle.The small GTPase RAN is a novel MAD2B binding protein.This novel protein-protein interaction may play a role in (i) the control over the spindle checkpoint during mitosis and (ii) the regulation of nucleocytoplasmic trafficking during interphase.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands.

ABSTRACT

Background: Previously, we identified the mitotic arrest deficient protein MAD2B (MAD2L2) as a bona fide interactor of the renal cell carcinoma (RCC)-associated protein PRCC. In addition, we found that fusion of PRCC with the transcription factor TFE3 in t(X;1)(p11;q21)-positive RCCs results in an impairment of this interaction and, concomitantly, an abrogation of cell cycle progression. Although MAD2B is thought to inhibit the anaphase promoting complex (APC) by binding to CDC20 and/or CDH1(FZR1), its exact role in cell cycle control still remains to be established.

Methodology/principal findings: Using a yeast two-hybrid interaction trap we identified the small GTPase RAN, a well-known cell cycle regulator, as a novel MAD2B binding protein. Endogenous interaction was established in mammalian cells via co-localization and co-immunoprecipitation of the respective proteins. The interaction domain of RAN could be assigned to a C-terminal moiety of 60 amino acids, whereas MAD2B had to be present in its full-length conformation. The MAD2B-RAN interaction was found to persist throughout the cell cycle. During mitosis, co-localization at the spindle was observed.

Conclusions/significance: The small GTPase RAN is a novel MAD2B binding protein. This novel protein-protein interaction may play a role in (i) the control over the spindle checkpoint during mitosis and (ii) the regulation of nucleocytoplasmic trafficking during interphase.

Show MeSH
Related in: MedlinePlus