Geminin is partially localized to the centrosome and plays a role in proper centrosome duplication.
BACKGROUND
Centrosome duplication normally parallels with DNA replication and is responsible for correct segregation of replicated DNA into the daughter cells. Although geminin interacts with Cdt1 to prevent loading of MCMs (minichromosome maintenance proteins) on to the replication origins, inactivation of geminin nevertheless causes centrosome over-duplication in addition to the re-replication of the genome, suggesting that geminin may play a role in centrosome duplication. However, the exact mechanism by which loss of geminin affects centrosomal duplication remains unclear and the possible direct interaction of geminin with centrosomal-localized proteins is still unidentified.


RESULTS
We report in the present study that geminin is physically localized to the centrosome. This unexpected geminin localization is cell-cycle dependent and mediated by the actin-related protein, Arp1, one subunit of the dynein-dynactin complex. Disruption of the integrity of the dynein-dynactin complex by overexpression of dynamitin/p50, a well-characterized inhibitor of dynactin, reduces the centrosomal localization of both geminin and Arp1. Enrichment of geminin on centrosomes was enhanced when cellular ATP production was suppressed in the ATP-inhibitor assay, whereas the accumulation of geminin on the centrosome was disrupted by depolymerization of the microtubules using nocodazole. We further demonstrate that the coiled-coil motif of geminin is required for its centrosomal localization and the interaction of geminin with Arp1. Depletion of geminin by siRNA (small interfering RNA) in MDA-MB-231 cells led to centrosome over-duplication. Conversely, overexpression of geminin inhibits centrosome over-duplication induced by HU in S-phase-arrested cells, and the coiled-coil-motif-mediated centrosomal localization of geminin is required for its inhibition of centrosome over-duplication. Centrosomal localization of geminin is conserved among mammalian cells and geminin might perform as an inhibitor of centrosome duplication.


CONCLUSIONS
The results of the present study demonstrate that a fraction of geminin is localized on the centrosome, and the centrosomal localization of geminin is Arp1-mediated and dynein-dynactin-dependent. The coiled-coil motif of geminin is required for its targeting to the centrosome and inhibition of centrosome duplication. Thus the centrosomal localization of geminin might perform an important role in regulation of proper centrosome duplication.
Lu
Fei
F
The MOE Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking University, Beijing 100871, China.
Lan
Rongfeng
R
Zhang
Hongyin
H
Jiang
Qing
Q
Zhang
Chuanmao
C
D051837:COUP Transcription Factor II / Q000378:metabolism*; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D018385:Centrosome / Q000737:chemistry / Q000378:metabolism*; D000072159:Dynactin Complex; D004398:Dyneins / Q000378:metabolism*; D064248:Geminin; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D006918:Hydroxyurea / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism
D051837:COUP Transcription Factor II; D018797:Cell Cycle Proteins; D000072159:Dynactin Complex; D004398:Dyneins; D064248:Geminin; D006918:Hydroxyurea; D008869:Microtubule-Associated Proteins; C000606361:DCTN2 protein, human; C577995:GMNN protein, human; C095569:NR2F2 protein, human
Nature. 1992 Sep 17;359(6392):246-50
1356230
Nat Cell Biol. 2000 Jan;2(1):20-4
10620802
Science. 2000 Dec 22;290(5500):2309-12
11125146
Nature. 2000 Apr 6;404(6778):622-5
10766247
Oncogene. 2004 May 20;23(23):4068-75
15064746
Eur J Cell Biol. 2007 Jan;86(1):37-50
17157410
Cell Cycle. 2006 Jan;5(2):151-4
16397411
Trends Cell Biol. 1996 Jun;6(6):212-5
15157458
Mol Cell Biol. 1999 Sep;19(9):6154-63
10454562
J Cell Biol. 2004 May 24;165(4):473-82
15159417
Mol Cell Biol. 2004 Aug;24(16):7140-50
15282313
Nature. 1995 Jun 1;375(6530):418-21
7760937
J Cell Sci. 2000 Mar;113 ( Pt 5):877-86
10671377
Cell. 1997 Aug 22;90(4):649-60
9288745
Annu Rev Biochem. 2002;71:333-74
12045100
Genes Dev. 2001 May 15;15(10):1167-81
11358861
Methods Enzymol. 1986;134:261-8
3821566
Nature. 1987 Nov 12-18;330(6144):181-3
3670402
J Biol Chem. 2004 Oct 29;279(44):45957-68
15308655
J Biol Chem. 2004 May 7;279(19):19691-7
14993212
Nat Rev Cancer. 2002 Nov;2(11):815-25
12415252
Cell. 1998 Jun 12;93(6):1043-53
9635433
Mol Cell. 2004 Jul 23;15(2):245-58
15260975
J Cell Biol. 2000 May 15;149(4):851-62
10811826
J Biol Chem. 2001 Nov 30;276(48):44905-11
11555648
Genes Dev. 2007 Jan 15;21(2):184-94
17234884
EMBO J. 2007 Mar 7;26(5):1303-14
17318181
EMBO Rep. 2005 Nov;6(11):1052-7
16179947
J Biol Chem. 2002 Oct 25;277(43):40871-80
12192004
J Biol Chem. 2004 Apr 23;279(17):17283-8
15004027
J Cell Biol. 2001 Dec 24;155(7):1159-72
11756470
Curr Biol. 2000 May 18;10(10):603-6
10837229