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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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Related in: MedlinePlus

MAD2B and RAN interact throughout the cell cycle.COS-1 cells were transiently transfected with MAD2B, blocked at different phases in the cell cycle and, subsequently, released from these cell cycle blocks during different time intervals. (1) cells isolated from a confluent culture, (2) cells isolated 3 h after 1∶2 splitting of a confluent culture, (3) cells blocked with nocodazole, (4) cells blocked with nocodazole and released for 4 h, (5) cells blocked with nocodazole and released for 8 h, (6) cells blocked with hydroxyurea, and (7) cells blocked with hydroxyurea and released for 6 h. (A) FACS profiles confirming the presence of the cells in the different phases of the cell cycle are shown. (B) Western blot analysis of total lysates with anti-RAN, anti-MAD2B and anti-γ-tubulin antibodies (Input) and after anti-RAN immunoprecipitation of the respective lysates (1–7) using an anti-MAD2B antibody (IP).
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pone-0007020-g004: MAD2B and RAN interact throughout the cell cycle.COS-1 cells were transiently transfected with MAD2B, blocked at different phases in the cell cycle and, subsequently, released from these cell cycle blocks during different time intervals. (1) cells isolated from a confluent culture, (2) cells isolated 3 h after 1∶2 splitting of a confluent culture, (3) cells blocked with nocodazole, (4) cells blocked with nocodazole and released for 4 h, (5) cells blocked with nocodazole and released for 8 h, (6) cells blocked with hydroxyurea, and (7) cells blocked with hydroxyurea and released for 6 h. (A) FACS profiles confirming the presence of the cells in the different phases of the cell cycle are shown. (B) Western blot analysis of total lysates with anti-RAN, anti-MAD2B and anti-γ-tubulin antibodies (Input) and after anti-RAN immunoprecipitation of the respective lysates (1–7) using an anti-MAD2B antibody (IP).

Mentions: Since MAD2B and RAN have been proposed to regulate various cell cycle processes [3], [4], [22], we decided to assess whether the observed interactions between these two proteins may relate to different phases of the cell cycle. To this end, COS-1 cells were transfected with VSV-MAD2B and, subsequently, aliquots of these transfected cells were synchronized by adding either nocodazole (G2/M phase block) or hydroxyurea (S phase block). Subsequently, the synchronized cells were released from drug-treatment during several time intervals as indicated (Fig. 4). To confirm the efficiency of synchronization, cells were fixed and analyzed by FACS at all time intervals. After harvesting cells from confluent cultures, and cultures that were split and allowed to grow for 3 h, most cells were found to be in the G0/G1 phase of the cell cycle, as expected (Fig. 4A, panel 1 and 2, respectively; 2N). After nocodazole treatment, most cells were arrested in the G2/M phase of the cell cycle, again as expected (Fig. 4A, panel 3; 4N), whereas subsequent release from this block for 4 and 8 h resulted in passage of the cells through mitosis and re-entry into the G0/G1 phase of the cell cycle (Fig. 4A; panel 4 and 5, respectively). Also as expected, treatment of the cells with the DNA replication inhibitor hydroxyurea provoked a block in the S phase, whereas after a release of this block for 6 h most of the cells were in the G2/M phase of the cell cycle (Fig. 4A; panel 6 and 7, respectively).


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)

MAD2B and RAN interact throughout the cell cycle.COS-1 cells were transiently transfected with MAD2B, blocked at different phases in the cell cycle and, subsequently, released from these cell cycle blocks during different time intervals. (1) cells isolated from a confluent culture, (2) cells isolated 3 h after 1∶2 splitting of a confluent culture, (3) cells blocked with nocodazole, (4) cells blocked with nocodazole and released for 4 h, (5) cells blocked with nocodazole and released for 8 h, (6) cells blocked with hydroxyurea, and (7) cells blocked with hydroxyurea and released for 6 h. (A) FACS profiles confirming the presence of the cells in the different phases of the cell cycle are shown. (B) Western blot analysis of total lysates with anti-RAN, anti-MAD2B and anti-γ-tubulin antibodies (Input) and after anti-RAN immunoprecipitation of the respective lysates (1–7) using an anti-MAD2B antibody (IP).
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Related In: Results  -  Collection

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pone-0007020-g004: MAD2B and RAN interact throughout the cell cycle.COS-1 cells were transiently transfected with MAD2B, blocked at different phases in the cell cycle and, subsequently, released from these cell cycle blocks during different time intervals. (1) cells isolated from a confluent culture, (2) cells isolated 3 h after 1∶2 splitting of a confluent culture, (3) cells blocked with nocodazole, (4) cells blocked with nocodazole and released for 4 h, (5) cells blocked with nocodazole and released for 8 h, (6) cells blocked with hydroxyurea, and (7) cells blocked with hydroxyurea and released for 6 h. (A) FACS profiles confirming the presence of the cells in the different phases of the cell cycle are shown. (B) Western blot analysis of total lysates with anti-RAN, anti-MAD2B and anti-γ-tubulin antibodies (Input) and after anti-RAN immunoprecipitation of the respective lysates (1–7) using an anti-MAD2B antibody (IP).
Mentions: Since MAD2B and RAN have been proposed to regulate various cell cycle processes [3], [4], [22], we decided to assess whether the observed interactions between these two proteins may relate to different phases of the cell cycle. To this end, COS-1 cells were transfected with VSV-MAD2B and, subsequently, aliquots of these transfected cells were synchronized by adding either nocodazole (G2/M phase block) or hydroxyurea (S phase block). Subsequently, the synchronized cells were released from drug-treatment during several time intervals as indicated (Fig. 4). To confirm the efficiency of synchronization, cells were fixed and analyzed by FACS at all time intervals. After harvesting cells from confluent cultures, and cultures that were split and allowed to grow for 3 h, most cells were found to be in the G0/G1 phase of the cell cycle, as expected (Fig. 4A, panel 1 and 2, respectively; 2N). After nocodazole treatment, most cells were arrested in the G2/M phase of the cell cycle, again as expected (Fig. 4A, panel 3; 4N), whereas subsequent release from this block for 4 and 8 h resulted in passage of the cells through mitosis and re-entry into the G0/G1 phase of the cell cycle (Fig. 4A; panel 4 and 5, respectively). Also as expected, treatment of the cells with the DNA replication inhibitor hydroxyurea provoked a block in the S phase, whereas after a release of this block for 6 h most of the cells were in the G2/M phase of the cell cycle (Fig. 4A; panel 6 and 7, respectively).

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