Limits...
Aurora-A controls pre-replicative complex assembly and DNA replication by stabilizing geminin in mitosis.

Tsunematsu T, Takihara Y, Ishimaru N, Pagano M, Takata T, Kudo Y - Nat Commun (2013)

Bottom Line: However, mitotic regulation of geminin has hitherto not been described.Here we show that Aurora-A phosphorylates geminin on Thr25 during M phase, and this event induces geminin stabilization by preventing its APC/C ubiquitin ligase complex-mediated degradation during mitosis.In turn, stabilized geminin inhibits SCF(Skp2)-mediated degradation of Cdt1 to ensure pre-replicative complex formation in the ensuing S phase.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral and Maxillofacial Pathobiology, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.

ABSTRACT
Geminin, an essential factor for DNA replication, directly binds to the licensing factor Cdt1 and inhibits pre-replicative complex formation to prevent re-replication. In G1, geminin levels are controlled by the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase complex, which targets geminin for proteasomal degradation to allow pre-replicative complex formation. Conversely, from S to G2, geminin is stabilized due to APC/C ubiquitin ligase complex inhibition, ensuring the inhibition of pre-replicative complex formation. However, mitotic regulation of geminin has hitherto not been described. Here we show that Aurora-A phosphorylates geminin on Thr25 during M phase, and this event induces geminin stabilization by preventing its APC/C ubiquitin ligase complex-mediated degradation during mitosis. In turn, stabilized geminin inhibits SCF(Skp2)-mediated degradation of Cdt1 to ensure pre-replicative complex formation in the ensuing S phase. The Aurora-A-geminin-Cdt1 axis therefore represents a critical regulator of proper DNA replication.

Show MeSH

Related in: MedlinePlus

Geminin or Aurora-A depletion during mitosis induces SCFSkp2-dependent proteolysis of soluble Cdt1 protein.(a) Geminin depletion during mitosis impaired licensing at mitotic exit. U2OS cells were treated with geminin and/or Skp2 siRNA during release from the second thymidine block and collected at 0 and 2 h after release from the Noc block as described in Fig. 4a. The soluble and chromatin fractions were separated and immunoblotted for the indicated proteins. MW, molecular weight in kDa; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; pHH, phospho-histone H3 (pHH3). (b) Aurora-A depletion during mitosis impaired licensing at mitotic exit. U2OS cells were treated with Aurora-A and/or Skp2 siRNA during release from the second thymidine block and collected at 0 and 2 h after release from the Noc block. Soluble and chromatin fractions were separated and immunoblotted for the indicated proteins. (c) Cells were treated with the geminin siRNA during release from second thymidine block and collected immediately after mitotic shake-off. After release from the first thymidine block, cells were transfected with 0.5, 1 or 2 μg of FLAG-tagged gemininT25D. Cdt1, FLAG-tagged geminin, Skp2 and Cul1 expression was examined by immunoblotting. (d) An in vitro ubiquitylation assay was performed using wild-type (WT) or T29A mutant IVT-HA-tagged Cdt1 and immunopurified SCFSkp2 from 293T cells. WT IVT-HA-tagged Cdt1 protein was incubated with or without IVT-myc-tagged gemininT25D protein for the indicated times. As a negative control, IVT-HA-tagged Cdt1T29A protein known as a non-degradable mutant for SCFSkp2-mediated degradation was also used. The abundance of IVT-HA-tagged Cdt1 was detected by SDS–PAGE with a HA antibody. (e) Schematic model for pre-RC formation regulated by the Aurora-A–geminin–Cdt1 axis. Aurora-A protects geminin from APC/CCdc20- and APC/CCdh1-mediated proteolysis through phosphorylation. Phosphorylated geminin may stabilize soluble Cdt1 protein by inhibiting SCFSkp2-mediated proteolysis. Stabilized soluble Cdt1 may load onto chromatin after dephosphorylation and then form the pre-RC. MW, molecular weight in kDa.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3675325&req=5

f7: Geminin or Aurora-A depletion during mitosis induces SCFSkp2-dependent proteolysis of soluble Cdt1 protein.(a) Geminin depletion during mitosis impaired licensing at mitotic exit. U2OS cells were treated with geminin and/or Skp2 siRNA during release from the second thymidine block and collected at 0 and 2 h after release from the Noc block as described in Fig. 4a. The soluble and chromatin fractions were separated and immunoblotted for the indicated proteins. MW, molecular weight in kDa; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; pHH, phospho-histone H3 (pHH3). (b) Aurora-A depletion during mitosis impaired licensing at mitotic exit. U2OS cells were treated with Aurora-A and/or Skp2 siRNA during release from the second thymidine block and collected at 0 and 2 h after release from the Noc block. Soluble and chromatin fractions were separated and immunoblotted for the indicated proteins. (c) Cells were treated with the geminin siRNA during release from second thymidine block and collected immediately after mitotic shake-off. After release from the first thymidine block, cells were transfected with 0.5, 1 or 2 μg of FLAG-tagged gemininT25D. Cdt1, FLAG-tagged geminin, Skp2 and Cul1 expression was examined by immunoblotting. (d) An in vitro ubiquitylation assay was performed using wild-type (WT) or T29A mutant IVT-HA-tagged Cdt1 and immunopurified SCFSkp2 from 293T cells. WT IVT-HA-tagged Cdt1 protein was incubated with or without IVT-myc-tagged gemininT25D protein for the indicated times. As a negative control, IVT-HA-tagged Cdt1T29A protein known as a non-degradable mutant for SCFSkp2-mediated degradation was also used. The abundance of IVT-HA-tagged Cdt1 was detected by SDS–PAGE with a HA antibody. (e) Schematic model for pre-RC formation regulated by the Aurora-A–geminin–Cdt1 axis. Aurora-A protects geminin from APC/CCdc20- and APC/CCdh1-mediated proteolysis through phosphorylation. Phosphorylated geminin may stabilize soluble Cdt1 protein by inhibiting SCFSkp2-mediated proteolysis. Stabilized soluble Cdt1 may load onto chromatin after dephosphorylation and then form the pre-RC. MW, molecular weight in kDa.

Mentions: We investigated why soluble Cdt1 protein becomes unstable when geminin is depleted in mitosis. Treatment with MG132 or the neddylation inhibitor MLN4924 increased the soluble Cdt1 levels in mitotic geminin- or Aurora-A-depleted cells (Fig. 6a), suggesting that soluble Cdt1 downregulation in response to the mitotic depletion of geminin or Aurora-A may be caused by ubiquitin-dependent proteolysis. During S and G2, human Cdt1 is recognized for proteolysis by two distinct E3 ubiquitin ligases, SCFSkp2 and CRL4Cdt2 (ref. 20). It has been demonstrated that Cdt1 is degraded via CRL4Cdt2 in geminin-depleted cells because of the DNA damage response21. We also found that geminin depletion induced Thr68 phosphorylation of Chk2, which is phosphorylated by ataxia telangiectasia mutated (ATM)/ATM and Rad3-related (ATR) after DNA damage (Supplementary Fig. S7). However, geminin depletion in mitosis did not induce DNA damage (Supplementary Fig. S7). These findings suggest that Cdt1 may not be ubiquitylated by CRL4Cdt2 in mitotic geminin-depleted cells. Indeed, Skp2 depletion, but not Cdt2 depletion, remarkably increased the soluble Cdt1 protein levels (Fig. 6c). Accordingly, Skp2 depletion rescued the levels of both soluble and chromatin-associated Cdt1 due to the mitotic depletion of geminin or Aurora-A (Fig. 7a and b). In mitotic geminin-depleted cells, Cdt1 expression was increased by the introduction of ectopic FLAG-geminin in an amount-dependent manner (Fig. 7c). SCFSkp2-dependent in vitro ubiquitylation of Cdt1 was inhibited by adding in vitro-translated geminin protein (Fig. 7d). Cdt1T29A, in the presence of which CDK phosphorylation and Skp2 binding are impaired22, was not ubiquitylated by SCFSkp2 (Fig. 7d). These findings indicate that in mitosis, geminin may protect soluble Cdt1 from SCFSkp2-dependent degradation for pre-RC formation.


Aurora-A controls pre-replicative complex assembly and DNA replication by stabilizing geminin in mitosis.

Tsunematsu T, Takihara Y, Ishimaru N, Pagano M, Takata T, Kudo Y - Nat Commun (2013)

Geminin or Aurora-A depletion during mitosis induces SCFSkp2-dependent proteolysis of soluble Cdt1 protein.(a) Geminin depletion during mitosis impaired licensing at mitotic exit. U2OS cells were treated with geminin and/or Skp2 siRNA during release from the second thymidine block and collected at 0 and 2 h after release from the Noc block as described in Fig. 4a. The soluble and chromatin fractions were separated and immunoblotted for the indicated proteins. MW, molecular weight in kDa; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; pHH, phospho-histone H3 (pHH3). (b) Aurora-A depletion during mitosis impaired licensing at mitotic exit. U2OS cells were treated with Aurora-A and/or Skp2 siRNA during release from the second thymidine block and collected at 0 and 2 h after release from the Noc block. Soluble and chromatin fractions were separated and immunoblotted for the indicated proteins. (c) Cells were treated with the geminin siRNA during release from second thymidine block and collected immediately after mitotic shake-off. After release from the first thymidine block, cells were transfected with 0.5, 1 or 2 μg of FLAG-tagged gemininT25D. Cdt1, FLAG-tagged geminin, Skp2 and Cul1 expression was examined by immunoblotting. (d) An in vitro ubiquitylation assay was performed using wild-type (WT) or T29A mutant IVT-HA-tagged Cdt1 and immunopurified SCFSkp2 from 293T cells. WT IVT-HA-tagged Cdt1 protein was incubated with or without IVT-myc-tagged gemininT25D protein for the indicated times. As a negative control, IVT-HA-tagged Cdt1T29A protein known as a non-degradable mutant for SCFSkp2-mediated degradation was also used. The abundance of IVT-HA-tagged Cdt1 was detected by SDS–PAGE with a HA antibody. (e) Schematic model for pre-RC formation regulated by the Aurora-A–geminin–Cdt1 axis. Aurora-A protects geminin from APC/CCdc20- and APC/CCdh1-mediated proteolysis through phosphorylation. Phosphorylated geminin may stabilize soluble Cdt1 protein by inhibiting SCFSkp2-mediated proteolysis. Stabilized soluble Cdt1 may load onto chromatin after dephosphorylation and then form the pre-RC. MW, molecular weight in kDa.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675325&req=5

f7: Geminin or Aurora-A depletion during mitosis induces SCFSkp2-dependent proteolysis of soluble Cdt1 protein.(a) Geminin depletion during mitosis impaired licensing at mitotic exit. U2OS cells were treated with geminin and/or Skp2 siRNA during release from the second thymidine block and collected at 0 and 2 h after release from the Noc block as described in Fig. 4a. The soluble and chromatin fractions were separated and immunoblotted for the indicated proteins. MW, molecular weight in kDa; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; pHH, phospho-histone H3 (pHH3). (b) Aurora-A depletion during mitosis impaired licensing at mitotic exit. U2OS cells were treated with Aurora-A and/or Skp2 siRNA during release from the second thymidine block and collected at 0 and 2 h after release from the Noc block. Soluble and chromatin fractions were separated and immunoblotted for the indicated proteins. (c) Cells were treated with the geminin siRNA during release from second thymidine block and collected immediately after mitotic shake-off. After release from the first thymidine block, cells were transfected with 0.5, 1 or 2 μg of FLAG-tagged gemininT25D. Cdt1, FLAG-tagged geminin, Skp2 and Cul1 expression was examined by immunoblotting. (d) An in vitro ubiquitylation assay was performed using wild-type (WT) or T29A mutant IVT-HA-tagged Cdt1 and immunopurified SCFSkp2 from 293T cells. WT IVT-HA-tagged Cdt1 protein was incubated with or without IVT-myc-tagged gemininT25D protein for the indicated times. As a negative control, IVT-HA-tagged Cdt1T29A protein known as a non-degradable mutant for SCFSkp2-mediated degradation was also used. The abundance of IVT-HA-tagged Cdt1 was detected by SDS–PAGE with a HA antibody. (e) Schematic model for pre-RC formation regulated by the Aurora-A–geminin–Cdt1 axis. Aurora-A protects geminin from APC/CCdc20- and APC/CCdh1-mediated proteolysis through phosphorylation. Phosphorylated geminin may stabilize soluble Cdt1 protein by inhibiting SCFSkp2-mediated proteolysis. Stabilized soluble Cdt1 may load onto chromatin after dephosphorylation and then form the pre-RC. MW, molecular weight in kDa.
Mentions: We investigated why soluble Cdt1 protein becomes unstable when geminin is depleted in mitosis. Treatment with MG132 or the neddylation inhibitor MLN4924 increased the soluble Cdt1 levels in mitotic geminin- or Aurora-A-depleted cells (Fig. 6a), suggesting that soluble Cdt1 downregulation in response to the mitotic depletion of geminin or Aurora-A may be caused by ubiquitin-dependent proteolysis. During S and G2, human Cdt1 is recognized for proteolysis by two distinct E3 ubiquitin ligases, SCFSkp2 and CRL4Cdt2 (ref. 20). It has been demonstrated that Cdt1 is degraded via CRL4Cdt2 in geminin-depleted cells because of the DNA damage response21. We also found that geminin depletion induced Thr68 phosphorylation of Chk2, which is phosphorylated by ataxia telangiectasia mutated (ATM)/ATM and Rad3-related (ATR) after DNA damage (Supplementary Fig. S7). However, geminin depletion in mitosis did not induce DNA damage (Supplementary Fig. S7). These findings suggest that Cdt1 may not be ubiquitylated by CRL4Cdt2 in mitotic geminin-depleted cells. Indeed, Skp2 depletion, but not Cdt2 depletion, remarkably increased the soluble Cdt1 protein levels (Fig. 6c). Accordingly, Skp2 depletion rescued the levels of both soluble and chromatin-associated Cdt1 due to the mitotic depletion of geminin or Aurora-A (Fig. 7a and b). In mitotic geminin-depleted cells, Cdt1 expression was increased by the introduction of ectopic FLAG-geminin in an amount-dependent manner (Fig. 7c). SCFSkp2-dependent in vitro ubiquitylation of Cdt1 was inhibited by adding in vitro-translated geminin protein (Fig. 7d). Cdt1T29A, in the presence of which CDK phosphorylation and Skp2 binding are impaired22, was not ubiquitylated by SCFSkp2 (Fig. 7d). These findings indicate that in mitosis, geminin may protect soluble Cdt1 from SCFSkp2-dependent degradation for pre-RC formation.

Bottom Line: However, mitotic regulation of geminin has hitherto not been described.Here we show that Aurora-A phosphorylates geminin on Thr25 during M phase, and this event induces geminin stabilization by preventing its APC/C ubiquitin ligase complex-mediated degradation during mitosis.In turn, stabilized geminin inhibits SCF(Skp2)-mediated degradation of Cdt1 to ensure pre-replicative complex formation in the ensuing S phase.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral and Maxillofacial Pathobiology, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.

ABSTRACT
Geminin, an essential factor for DNA replication, directly binds to the licensing factor Cdt1 and inhibits pre-replicative complex formation to prevent re-replication. In G1, geminin levels are controlled by the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase complex, which targets geminin for proteasomal degradation to allow pre-replicative complex formation. Conversely, from S to G2, geminin is stabilized due to APC/C ubiquitin ligase complex inhibition, ensuring the inhibition of pre-replicative complex formation. However, mitotic regulation of geminin has hitherto not been described. Here we show that Aurora-A phosphorylates geminin on Thr25 during M phase, and this event induces geminin stabilization by preventing its APC/C ubiquitin ligase complex-mediated degradation during mitosis. In turn, stabilized geminin inhibits SCF(Skp2)-mediated degradation of Cdt1 to ensure pre-replicative complex formation in the ensuing S phase. The Aurora-A-geminin-Cdt1 axis therefore represents a critical regulator of proper DNA replication.

Show MeSH
Related in: MedlinePlus