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PLK1 and β-TrCP-dependent ubiquitination and degradation of Rap1GAP controls cell proliferation.

Wang D, Zhang P, Gao K, Tang Y, Jin X, Zhang Y, Yi Q, Wang C, Yu L - PLoS ONE (2014)

Bottom Line: We revealed that PLK1 interacts with Rap1GAP in vivo through recognition of an SSP motif within Rap1GAP.PLK1 phosphorylates Ser525 in conserved 524DSGHVS529 degron of Rap1GAP and promotes its interaction with β-TrCP.We also showed that Rap1GAP was a cell cycle regulator and that tight regulation of the Rap1GAP degradation in mitosis is required for cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, P.R. China; Department of Gastroenterology, Jiangxi Institute of Gastroenterology & Hepatology, the First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province, P. R. China.

ABSTRACT
Rap1GAP is a GTPase-activating protein (GAP) that specifically stimulates the GTP hydrolysis of Rap1 GTPase. Although Rap1GAP is recognized as a tumor suppressor gene and downregulated in various cancers, little is known regarding the regulation of Rap1GAP ubiquitination and degradation under physiological conditions. Here, we demonstrated that Rap1GAP is ubiquitinated and degraded through proteasome pathway in mitosis. Proteolysis of Rap1GAP requires the PLK1 kinase and β-TrCP ubiquitin ligase complex. We revealed that PLK1 interacts with Rap1GAP in vivo through recognition of an SSP motif within Rap1GAP. PLK1 phosphorylates Ser525 in conserved 524DSGHVS529 degron of Rap1GAP and promotes its interaction with β-TrCP. We also showed that Rap1GAP was a cell cycle regulator and that tight regulation of the Rap1GAP degradation in mitosis is required for cell proliferation.

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Rap1GAP is degraded during mitosis.(A and B) HeLa cells were synchronized by double thymidine block followed by release into nocodazole-containing media and harvested at the indicated times. Cell cycle profile was assayed by FACS with propidium iodine staining (A). Protein levels were analyzed by immunoblotting (B). (C) Nocodazole-arrested HeLa cells were released into fresh medium, and the Rap1GAP status was monitored at indicated times. (D) HeLa cells were arrested using nocodazole at the indicated times, and 20 µM MG132 was added during the last 4 h of the nocodazole treatment before the cells were harvested. (E) HeLa cells or U2OS cells were treated with 20 µM MG132 and harvested at the indicated times. Protein levels were examined by immunoblotting. (F) HeLa cells that were synchronized by double thymidine block followed by release into nocodazole-containing media, and harvested at 22 h. The cell lysates were incubated with or without λ-phosphatase (λ-PPase). The phosphorylation state of Rap1GAP was analyzed by immunoblotting.
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pone-0110296-g001: Rap1GAP is degraded during mitosis.(A and B) HeLa cells were synchronized by double thymidine block followed by release into nocodazole-containing media and harvested at the indicated times. Cell cycle profile was assayed by FACS with propidium iodine staining (A). Protein levels were analyzed by immunoblotting (B). (C) Nocodazole-arrested HeLa cells were released into fresh medium, and the Rap1GAP status was monitored at indicated times. (D) HeLa cells were arrested using nocodazole at the indicated times, and 20 µM MG132 was added during the last 4 h of the nocodazole treatment before the cells were harvested. (E) HeLa cells or U2OS cells were treated with 20 µM MG132 and harvested at the indicated times. Protein levels were examined by immunoblotting. (F) HeLa cells that were synchronized by double thymidine block followed by release into nocodazole-containing media, and harvested at 22 h. The cell lysates were incubated with or without λ-phosphatase (λ-PPase). The phosphorylation state of Rap1GAP was analyzed by immunoblotting.

Mentions: We examined the Rap1GAP protein level through the cell cycle and compared it to other key cell cycle regulators. HeLa cells were arrested at the G1/S boundary by a double thymidine treatment and then released into nocodazole-containing media. The cell cycle profile of released cells was analyzed by FACS (Fig. 1A). The mitotic time-points were determined by phosphorylated histone H3 (p-histone H3) level. As shown in Fig. 1B, p-histone H3 was not detectable at the G1-S boundary but gradually accumulated as cells progressed into S and G2, peaking at 21 hours after release, a time at which the majority of cells had entered mitosis (Fig. 1B). PLK1 and Aurora B, two key mitotic kinase levels were low during interphase but high during mitosis (Fig. 1B). In contrast, the Rap1GAP protein level was consistent at the G1/S and S phase, and then gradually showed motility shifts, peaking at 15 h after release, and gradually decreased in mitosis (Fig. 1B). When cells were released from mitotic arrest, the Rap1GAP level was restored by the time the cells exited mitosis (Fig. 1C). Furthermore, a significant fraction of Rap1GAP was restored following exposure of the arrested cells to the proteasome inhibitor MG132, suggesting that the proteasome pathway is involved in Rap1GAP degradation in mitosis (Fig. 1D). To determine whether Rap1GAP was specifically degraded in mitosis, we treated the unsynchronized HeLa cells (91% in interphase) with MG132. p53, a short-lived tumor suppressor protein, was rapidly stabilized with MG132 treatment. In contrast, the Rap1GAP protein level remained constant (Fig. 1E). Similar results were obtained in U2OS cells (Fig. 1E). Finally, the Rap1GAP mobility shifts were observed throughout the cell cycle, and these mobility shifts were abolished with λ-phosphatase treatment (Figure 1F). This loss suggests that the post-translational modification of Rap1GAP resulted from phosphorylation. Taken together, these data demonstrated that Rap1GAP was phosphorylated and degraded by the proteasome pathway during mitosis.


PLK1 and β-TrCP-dependent ubiquitination and degradation of Rap1GAP controls cell proliferation.

Wang D, Zhang P, Gao K, Tang Y, Jin X, Zhang Y, Yi Q, Wang C, Yu L - PLoS ONE (2014)

Rap1GAP is degraded during mitosis.(A and B) HeLa cells were synchronized by double thymidine block followed by release into nocodazole-containing media and harvested at the indicated times. Cell cycle profile was assayed by FACS with propidium iodine staining (A). Protein levels were analyzed by immunoblotting (B). (C) Nocodazole-arrested HeLa cells were released into fresh medium, and the Rap1GAP status was monitored at indicated times. (D) HeLa cells were arrested using nocodazole at the indicated times, and 20 µM MG132 was added during the last 4 h of the nocodazole treatment before the cells were harvested. (E) HeLa cells or U2OS cells were treated with 20 µM MG132 and harvested at the indicated times. Protein levels were examined by immunoblotting. (F) HeLa cells that were synchronized by double thymidine block followed by release into nocodazole-containing media, and harvested at 22 h. The cell lysates were incubated with or without λ-phosphatase (λ-PPase). The phosphorylation state of Rap1GAP was analyzed by immunoblotting.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4201484&req=5

pone-0110296-g001: Rap1GAP is degraded during mitosis.(A and B) HeLa cells were synchronized by double thymidine block followed by release into nocodazole-containing media and harvested at the indicated times. Cell cycle profile was assayed by FACS with propidium iodine staining (A). Protein levels were analyzed by immunoblotting (B). (C) Nocodazole-arrested HeLa cells were released into fresh medium, and the Rap1GAP status was monitored at indicated times. (D) HeLa cells were arrested using nocodazole at the indicated times, and 20 µM MG132 was added during the last 4 h of the nocodazole treatment before the cells were harvested. (E) HeLa cells or U2OS cells were treated with 20 µM MG132 and harvested at the indicated times. Protein levels were examined by immunoblotting. (F) HeLa cells that were synchronized by double thymidine block followed by release into nocodazole-containing media, and harvested at 22 h. The cell lysates were incubated with or without λ-phosphatase (λ-PPase). The phosphorylation state of Rap1GAP was analyzed by immunoblotting.
Mentions: We examined the Rap1GAP protein level through the cell cycle and compared it to other key cell cycle regulators. HeLa cells were arrested at the G1/S boundary by a double thymidine treatment and then released into nocodazole-containing media. The cell cycle profile of released cells was analyzed by FACS (Fig. 1A). The mitotic time-points were determined by phosphorylated histone H3 (p-histone H3) level. As shown in Fig. 1B, p-histone H3 was not detectable at the G1-S boundary but gradually accumulated as cells progressed into S and G2, peaking at 21 hours after release, a time at which the majority of cells had entered mitosis (Fig. 1B). PLK1 and Aurora B, two key mitotic kinase levels were low during interphase but high during mitosis (Fig. 1B). In contrast, the Rap1GAP protein level was consistent at the G1/S and S phase, and then gradually showed motility shifts, peaking at 15 h after release, and gradually decreased in mitosis (Fig. 1B). When cells were released from mitotic arrest, the Rap1GAP level was restored by the time the cells exited mitosis (Fig. 1C). Furthermore, a significant fraction of Rap1GAP was restored following exposure of the arrested cells to the proteasome inhibitor MG132, suggesting that the proteasome pathway is involved in Rap1GAP degradation in mitosis (Fig. 1D). To determine whether Rap1GAP was specifically degraded in mitosis, we treated the unsynchronized HeLa cells (91% in interphase) with MG132. p53, a short-lived tumor suppressor protein, was rapidly stabilized with MG132 treatment. In contrast, the Rap1GAP protein level remained constant (Fig. 1E). Similar results were obtained in U2OS cells (Fig. 1E). Finally, the Rap1GAP mobility shifts were observed throughout the cell cycle, and these mobility shifts were abolished with λ-phosphatase treatment (Figure 1F). This loss suggests that the post-translational modification of Rap1GAP resulted from phosphorylation. Taken together, these data demonstrated that Rap1GAP was phosphorylated and degraded by the proteasome pathway during mitosis.

Bottom Line: We revealed that PLK1 interacts with Rap1GAP in vivo through recognition of an SSP motif within Rap1GAP.PLK1 phosphorylates Ser525 in conserved 524DSGHVS529 degron of Rap1GAP and promotes its interaction with β-TrCP.We also showed that Rap1GAP was a cell cycle regulator and that tight regulation of the Rap1GAP degradation in mitosis is required for cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, P.R. China; Department of Gastroenterology, Jiangxi Institute of Gastroenterology & Hepatology, the First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province, P. R. China.

ABSTRACT
Rap1GAP is a GTPase-activating protein (GAP) that specifically stimulates the GTP hydrolysis of Rap1 GTPase. Although Rap1GAP is recognized as a tumor suppressor gene and downregulated in various cancers, little is known regarding the regulation of Rap1GAP ubiquitination and degradation under physiological conditions. Here, we demonstrated that Rap1GAP is ubiquitinated and degraded through proteasome pathway in mitosis. Proteolysis of Rap1GAP requires the PLK1 kinase and β-TrCP ubiquitin ligase complex. We revealed that PLK1 interacts with Rap1GAP in vivo through recognition of an SSP motif within Rap1GAP. PLK1 phosphorylates Ser525 in conserved 524DSGHVS529 degron of Rap1GAP and promotes its interaction with β-TrCP. We also showed that Rap1GAP was a cell cycle regulator and that tight regulation of the Rap1GAP degradation in mitosis is required for cell proliferation.

Show MeSH
Related in: MedlinePlus