Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Nationwide Children's Hospital

Nanjing University

We identified a novel splice variant of human SGT1 (SUGT1), a suppressor of the G2 allele of SKP1, by analysis of 8 human EST clones whose open reading frame encoded 365 amino acids. We termed this variant SGT1B (SUGT1B) and the original SGT1A (SUGT1A). The putative SGT1B and SGT1A proteins are 91% identical, and both contain a tetratricopeptide repeat (TPR) domain, two variable regions, a CS domain, and a SGS domain. The NCBI human genome database showed that SGT1B and SGT1A are located on chromosome band 13q14.13. SGT1B contains an additional 33 amino acids encoded by a region between exons 5 and 6 of SGT1A and lacks Ser110 of SGT1A. Immunoblotting using antibodies to N-terminal (amino acids 1-157) and C-terminal (amino acids 182-333) regions of SGT1A detected 2 bands whose sizes corresponded to those predicted for SGT1A and SGT1B. Overexpression experiments confirmed this finding. Additional immunoblot analysis demonstrated that both are highly expressed in human brain, liver, lung, and testis.

Identification of a novel splice variant: human SGT1B (SUGT1B).

The spindle checkpoint that monitors kinetochore-microtubule attachment has been implicated in tumorigenesis; however, the relation between the spindle checkpoint and cell death remains obscure. In BUB1-deficient (but not MAD2-deficient) cells, conditions that activate the spindle checkpoint (i.e., cold shock or treatment with nocodazole, paclitaxel, or 17-AAG) induced DNA fragmentation during early mitosis. This mitotic cell death was independent of caspase activation; therefore, we named it caspase-independent mitotic death (CIMD). CIMD depends on p73, a homologue of p53, but not on p53. CIMD also depends on apoptosis-inducing factor and endonuclease G, which are effectors of caspase-independent cell death. Treatment with nocodazole, paclitaxel, or 17-AAG induced CIMD in cell lines derived from colon tumors with chromosome instability, but not in cells from colon tumors with microsatellite instability. This result was due to low BUB1 expression in the former cell lines. When BUB1 is completely depleted, aneuploidy rather than CIMD occurs. These results suggest that cells prone to substantial chromosome missegregation might be eliminated via CIMD.

BUB1 mediation of caspase-independent mitotic death determines cell fate.

The spindle checkpoint, which monitors kinetochore-microtubule attachment, is required for high fidelity of chromosome transmission. A failure in this mechanism causes aneuploidy, thereby promoting progression to tumorigenesis. However, the cell death mechanism that prevents the aneuploidy caused by failure of the spindle checkpoint is yet unknown. We have recently identified a novel type of mitotic cell death, which we term caspase-independent mitotic death (CIMD). In BUB1-deficient (but not MAD2-deficient) cells, CIMD is induced by conditions that activate the spindle checkpoint (i.e., cold shock or treatment with nocodazole, paclitaxel or 17-AAG [17-allylaminogeldanamycin]). CIMD depends on p73, a homolog of p53, but not on p53. It also depends on the apoptosis-inducing factor (AIF) and endonuclease G (Endo G), which are effectors of caspase-independent cell death. When BUB1 is completely depleted, aneuploidy occurs instead of CIMD. We propose that CIMD can be the cell death mechanism that protects cells from aneuploidy by inducing the death of cells prone to substantial chromosome missegregation. Our study also shows that previous evaluations of the spindle checkpoint activity in mutant or cancer cells by monitoring mitotic index could be misleading.

Caspase-independent mitotic death (CIMD).

Wnt signalling is known to promote G1/S progression through the stimulation of gene expression, but whether this signalling regulates mitotic progression is not clear. Here, the function of dishevelled 2 (Dvl2), which transmits the Wnt signal, in mitosis was examined. Dvl2 localized to the spindles and spindle poles during mitosis. When cells were treated with nocodazole, Dvl2 was observed at the kinetochores (KTs). Dvl2 bound to and was phosphorylated at Thr206 by a mitotic kinase, Polo-like kinase 1 (Plk1), and this phosphorylation was required for spindle orientation and stable microtubule (MT)-KT attachment. Dvl2 was also found to be involved in the activation of a spindle assembly checkpoint (SAC) kinase, Mps1, and the recruitment of other SAC components, Bub1 and BubR1, to the KTs. However, the phosphorylation of Dvl2 by Plk1 was dispensable for SAC. Furthermore, Wnt receptors were involved in spindle orientation, but not in MT-KT attachment or SAC. These results suggested that Dvl2 is involved in mitotic progression by regulating the dynamics of MT plus-ends and the SAC in Plk1-dependent and -independent manners.

Dishevelled, a Wnt signalling component, is involved in mitotic progression in cooperation with Plk1.

ATM &#x6FC0;&#x9176;&#x5728;&#x9057;&#x4F20;&#x7A33;&#x5B9A;&#x6027;&#x7684;&#x7EF4;&#x6301;&#x5177;&#x6709;&#x91CD;&#x8981;&#x7684;&#x4F5C;&#x7528;&#x3002;ATM &#x5728; DNA &#x635F;&#x4F24;&#x53CD;&#x5E94;&#x4E2D;&#x6FC0;&#x6D3B;&#x548C;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x68C0;&#x67E5;&#x70B9;&#x81F3;&#x5173;&#x91CD;&#x8981;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x62A5;&#x544A; ATM &#x5728;&#x7EC6;&#x80DE;&#x6709;&#x4E1D;&#x5206;&#x88C2; DNA &#x635F;&#x4F24;&#x7684;&#x7F3A;&#x5E2D;&#x4E2D;&#x6FC0;&#x6D3B;&#x3002;&#x6211;&#x4EEC;&#x8BC1;&#x660E;&#x6709;&#x4E1D;&#x5206;&#x88C2;&#x7684; ATM &#x6FC0;&#x6D3B;&#x662F;&#x4F9D;&#x8D56; Aurora B &#x6FC0;&#x9176;&#x548C;&#x6781;&#x5149; B &#x4E0A;&#x4E1D;&#x6C28;&#x9178; 1403 phosphorylates ATM&#x3002;&#x9700;&#x8981;&#x6709;&#x4E1D;&#x5206;&#x88C2;&#x7684; ATM &#x6FC0;&#x6D3B;&#x6B64;&#x78F7;&#x9178;&#x5316;&#x4E8B;&#x4EF6;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x6211;&#x4EEC;&#x663E;&#x793A; ATM &#x673A;&#x80FD;&#x4E27;&#x5931;&#x7684;&#x7ED3;&#x679C;&#x5728;&#x7F29;&#x77ED;&#x7684;&#x6709;&#x4E1D;&#x5206;&#x88C2;&#x8BA1;&#x65F6;&#x548C;&#x6709;&#x7F3A;&#x9677;&#x7684;&#x4E3B;&#x8F74;&#x7684;&#x68C0;&#x67E5;&#x70B9;&#xFF0C;&#x5E76;&#x5BFC;&#x81F4;&#x6B64;&#x4E3B;&#x8F74;&#x68C0;&#x67E5;&#x70B9;&#x7F3A;&#x9677;&#x7684; ATM Ser1403 &#x78F7;&#x9178;&#x5316;&#xFF0C;&#x5E9F;&#x9664;&#x3002;&#x6211;&#x4EEC;&#x8FD8;&#x6F14;&#x793A; mitotically &#x6FC0;&#x6D3B;&#x7684; ATM phosphorylates Bub1&#xFF0C;&#x5173;&#x952E;&#x7EBA;&#x9524;&#x86CB;&#x767D;&#xFF0C;&#x5728; Ser314 &#x4E0A;&#x3002;ATM &#x4ECB;&#x5BFC; Bub1 Ser314 &#x78F7;&#x9178;&#x5316;&#x662F;&#x5FC5;&#x9700; Bub1 &#x6D3B;&#x52A8;&#xFF0C;&#x5E76;&#x4E14;&#x662F;&#x4E3B;&#x8F74;&#x7684;&#x68C0;&#x67E5;&#x70B9;&#x7684;&#x6FC0;&#x6D3B;&#x7684;&#x5FC5;&#x8981;&#x6761;&#x4EF6;&#x3002;&#x96C6;&#x4F53;&#xFF0C;&#x6211;&#x4EEC;&#x7684;&#x6570;&#x636E;&#x7A81;&#x51FA;&#x663E;&#x793A;&#x5173;&#x952E;&#x529F;&#x80FD;&#x7684; ATM &#x5728;&#x7EC6;&#x80DE;&#x6709;&#x4E1D;&#x5206;&#x88C2;&#x7684;&#x673A;&#x5236;&#x3002;

Aurora B &#x4ECB;&#x5BFC;&#x7684; ATM 1403 &#x4E1D;&#x6C28;&#x9178;&#x78F7;&#x9178;&#x5316;&#x662F;&#x6709;&#x4E1D;&#x5206;&#x88C2;&#x7684; ATM &#x6FC0;&#x6D3B;&#x548C;&#x4E3B;&#x8F74;&#x7684;&#x68C0;&#x67E5;&#x70B9;&#x6240;&#x5FC5;&#x9700;&#x7684;&#x3002;

The ATM kinase plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA damage and is essential for cell-cycle checkpoints. Here, we report that ATM is activated in mitosis in the absence of DNA damage. We demonstrate that mitotic ATM activation is dependent on the Aurora-B kinase and that Aurora-B phosphorylates ATM on serine 1403. This phosphorylation event is required for mitotic ATM activation. Further, we show that loss of ATM function results in shortened mitotic timing and a defective spindle checkpoint, and that abrogation of ATM Ser1403 phosphorylation leads to this spindle checkpoint defect. We also demonstrate that mitotically activated ATM phosphorylates Bub1, a critical kinetochore protein, on Ser314. ATM-mediated Bub1 Ser314 phosphorylation is required for Bub1 activity and is essential for the activation of the spindle checkpoint. Collectively, our data highlight mechanisms of a critical function of ATM in mitosis.

Aurora-B mediated ATM serine 1403 phosphorylation is required for mitotic ATM activation and the spindle checkpoint.

La kinase ATM joue un r&#xF4;le essentiel dans le maintien de la stabilit&#xE9; g&#xE9;n&#xE9;tique. ATM est activ&#xE9;e en r&#xE9;ponse aux dommages de l&#x26;#39;ADN et est essentielle pour le cycle cellulaire des points de contr&#xF4;le. Ici, nous rapportons que l&#x26;#39;ATM est activ&#xE9; lors de la mitose en l&#x26;#39;absence de l&#xE9;sions de l&#x26;#39;ADN. On d&#xE9;montrer que l&#x26;#39;activation ATM mitotique d&#xE9;pend de la kinase Aurora-B et que l&#x26;#39;Aurora-B phosphoryle ATM sur la s&#xE9;rine 1403. Cet &#xE9;v&#xE9;nement est la phosphorylation requise pour l&#x26;#39;activation mitotique ATM. En outre, nous montrons que la perte de l&#x26;#39;ATM r&#xE9;sultats de la fonction de synchronisation mitotique r&#xE9;duite et un checkpoint du fuseau d&#xE9;fectueux, et que l&#x26;#39;abrogation de l&#x26;#39;ATM phosphorylation Ser1403 conduit &#xE0; ce d&#xE9;faut checkpoint du fuseau. Nous d&#xE9;montrons &#xE9;galement que la mitose activ&#xE9; ATM phosphoryle BUB1, une prot&#xE9;ine du kin&#xE9;tochore critique, sur la Ser314. ATM &#xE0; m&#xE9;diation BUB1 Ser314 est n&#xE9;cessaire pour la phosphorylation BUB1 activit&#xE9; et est essentiel pour l&#x26;#39;activation du poste de contr&#xF4;le de la broche. Collectivement, nos donn&#xE9;es mettent en &#xE9;vidence les m&#xE9;canismes d&#x26;#39;une fonction critique de l&#x26;#39;ATM dans la mitose.

ATM m&#xE9;diation Aurora-B Serine 1403 phosphorylation est requise pour l&#x26;#39;activation mitotique ATM et le checkpoint du fuseau

Das ATM-Kinase spielt eine wichtige Rolle bei der Aufrechterhaltung der genetische Stabilit&auml;t. ATM wird aktiviert, als Antwort auf DNA-Sch&auml;den und essentiell f&uuml;r Zellzyklus Kontrollpunkte. Hier berichten wir, dass ATM in Mitose in Ermangelung von DNA-Sch&auml;den aktiviert ist. Wir zeigen, dass die mitotische ATM-Aktivierung ist abh&auml;ngig von der Aurora-B-Kinase und Aurora-B ATM auf Serin 1403 phosphorylates. Diese Phosphorylierung-Veranstaltung ist f&uuml;r die mitotische ATM-Aktivierung erforderlich. Dar&uuml;ber hinaus zeigen wir, dass ATM Funktionsverlust f&uuml;hrt zu verk&uuml;rzten mitotische Timing und einen defekten Spindel-Checkpoint und die Aufhebung der ATM Ser1403 Phosphorylierung zu diesen Spindel Pr&uuml;fpunkt defekt f&uuml;hrt. Wir zeigen auch, dass mitotically aktivierte ATM Bub1, eine kritische Kinetochor-Protein, auf Ser314 phosphorylates. ATM-vermittelte Bub1 Ser314 Phosphorylierung ist erforderlich f&uuml;r Bub1 Aktivit&auml;t und ist wichtig f&uuml;r die Aktivierung der Spindel-Checkpoint. Gemeinsam unterstreichen unsere Daten Mechanismen der eine kritische Funktion von ATM in Mitose.

Aurora-B vermittelte ATM Serin 1403 Phosphorylierung ist erforderlich f&uuml;r die mitotische ATM-Aktivierung und die Spindel-Checkpoint.

La chinasi ATM svolge un ruolo critico nel mantenimento della stabilit&agrave; genetica. ATM &egrave; attivato in risposta a danni del DNA ed &egrave; essenziale per i checkpoint del ciclo cellulare. Qui, segnaliamo che ATM &egrave; attivato nella mitosi in assenza di danni del DNA. Dimostriamo che mitotica attivazione ATM &egrave; dipendente della chinasi Aurora-B e che Aurora-B tirosine ATM su serina 1403. Questo evento di fosforilazione &egrave; richiesto per l&#39;attivazione di ATM mitotica. Ulteriormente, vi mostriamo la perdita della funzione di ATM si traduce in un checkpoint mandrino difettoso e temporizzazione mitotica accorciato, che tale abrogazione della fosforilazione ATM Ser1403 conduce a questo difetto di checkpoint mandrino. Abbiamo anche dimostrare che ATM mitoticamente attivato fosforila Bub1, una proteina fondamentale cinetocore, su Ser314. ATM-mediata Bub1 Ser314 fosforilazione &egrave; necessaria per attivit&agrave; Bub1 ed &egrave; essenziale per l&#39;attivazione del punto di arresto mandrino. Collettivamente, i nostri dati evidenziano meccanismi di una funzione critica dell&#39;ATM nella mitosi.

Aurora-B mediata ATM fosforilazione della serina 1403 &egrave; necessaria per attivazione ATM mitotica e il punto di arresto del mandrino.

ATM&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x306F;&#x3001;&#x907A;&#x4F1D;&#x7684;&#x5B89;&#x5B9A;&#x6027;&#x306E;&#x7DAD;&#x6301;&#x306B;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3057;&#x307E;&#x3059;&#x3002; ATM&#x306F;DNA&#x640D;&#x50B7;&#x306B;&#x5FDC;&#x7B54;&#x3057;&#x3066;&#x6D3B;&#x6027;&#x5316;&#x3068;&#x7D30;&#x80DE;&#x5468;&#x671F;&#x30C1;&#x30A7;&#x30C3;&#x30AF;&#x30DD;&#x30A4;&#x30F3;&#x30C8;&#x306B;&#x5FC5;&#x9808;&#x3067;&#x3042;&#x308B;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;ATM&#x306F;DNA&#x640D;&#x50B7;&#x306E;&#x306A;&#x3044;&#x72B6;&#x614B;&#x3067;&#x6709;&#x7CF8;&#x5206;&#x88C2;&#x3067;&#x6D3B;&#x6027;&#x5316;&#x3055;&#x308C;&#x308B;&#x3053;&#x3068;&#x3092;&#x5831;&#x544A;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x6211;&#x3005;&#x306F;&#x3001;&#x5206;&#x88C2;ATM&#x306E;&#x6D3B;&#x6027;&#x5316;&#x304C;&#x30AA;&#x30FC;&#x30ED;&#x30E9;B&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x3066;&#x304A;&#x308A;&#x3001;&#x30AA;&#x30FC;&#x30ED;&#x30E9;-B&#x306F;1403&#x5E74;&#x30BB;&#x30EA;&#x30F3;&#x3067;ATM&#x3092;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x3053;&#x306E;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x30A4;&#x30D9;&#x30F3;&#x30C8;&#x304C;&#x6709;&#x7CF8;&#x5206;&#x88C2;&#x306E;ATM&#x6D3B;&#x6027;&#x5316;&#x306E;&#x305F;&#x3081;&#x306B;&#x5FC5;&#x8981;&#x3068;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x6211;&#x3005;&#x306F;&#x77ED;&#x7E2E;&#x6709;&#x7CF8;&#x5206;&#x88C2;&#x306E;&#x30BF;&#x30A4;&#x30DF;&#x30F3;&#x30B0;&#x3068;&#x6B20;&#x9665;&#x306E;&#x3042;&#x308B;&#x7D21;&#x9318;&#x4F53;&#x30C1;&#x30A7;&#x30C3;&#x30AF;&#x30DD;&#x30A4;&#x30F3;&#x30C8;&#x306E;ATM&#x95A2;&#x6570;&#x306E;&#x7D50;&#x679C;&#x306E;&#x640D;&#x5931;&#x3092;&#x793A;&#x3057;&#x3001;ATM Ser1403&#x306E;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x306E;&#x305D;&#x306E;&#x5EC3;&#x6B62;&#x306F;&#x3001;&#x3053;&#x306E;&#x7D21;&#x9318;&#x4F53;&#x30C1;&#x30A7;&#x30C3;&#x30AF;&#x30DD;&#x30A4;&#x30F3;&#x30C8;&#x306E;&#x6B20;&#x9665;&#x306B;&#x3064;&#x306A;&#x304C;&#x308A;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005;&#x306F;&#x307E;&#x305F;&#x3001;&#x6709;&#x7CF8;&#x5206;&#x88C2;&#x6D3B;&#x6027;&#x5316;ATM&#x306F;Ser314&#x306B;&#x3001;Bub1&#x3001;&#x91CD;&#x8981;&#x306A;&#x52D5;&#x539F;&#x4F53;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3092;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002; ATM-&#x5A92;&#x4ECB;Bub1 Ser314&#x30EA;&#x30F3;&#x9178;&#x5316;&#x306F;Bub1&#x6D3B;&#x6027;&#x306B;&#x5FC5;&#x8981;&#x3068;&#x30B9;&#x30D4;&#x30F3;&#x30C9;&#x30EB;&#x30C1;&#x30A7;&#x30C3;&#x30AF;&#x30DD;&#x30A4;&#x30F3;&#x30C8;&#x306E;&#x6D3B;&#x6027;&#x5316;&#x306B;&#x5FC5;&#x9808;&#x3067;&#x3042;&#x308B;&#x3002;&#x7DCF;&#x79F0;&#x3057;&#x3066;&#x3001;&#x6211;&#x3005;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x3001;&#x6709;&#x7CF8;&#x5206;&#x88C2;&#x306E;ATM&#x306E;&#x91CD;&#x8981;&#x306A;&#x6A5F;&#x80FD;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x5F37;&#x8ABF;&#x8868;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;

1403&#x30EA;&#x30F3;&#x9178;&#x5316;&#x30BB;&#x30EA;&#x30F3;&#x30AA;&#x30FC;&#x30ED;&#x30E9;-B&#x3092;&#x4ECB;&#x3057;&#x305F;ATM&#x304C;&#x5206;&#x88C2;ATM&#x306E;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D9;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3068;&#x30B9;&#x30D4;&#x30F3;&#x30C9;&#x30EB;&#x30C1;&#x30A7;&#x30C3;&#x30AF;&#x30DD;&#x30A4;&#x30F3;&#x30C8;&#x304C;&#x5FC5;&#x8981;&#x3067;&#x3059;

ATM &#xB2C8; &#xC720;&#xC804;&#xC801; &#xC548;&#xC815;&#xC131;&#xC758; &#xC720;&#xC9C0; &#xBCF4;&#xC218;&#xC5D0; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xD55C;&#xB2E4;. ATM DNA &#xC190;&#xC0C1;&#xC5D0; &#xB300; &#xD55C; &#xC751;&#xB2F5;&#xC73C;&#xB85C; &#xD65C;&#xC131;&#xD654; &#xD558; &#xACE0; &#xC138;&#xD3EC; &#xC8FC;&#xAE30; &#xAC80;&#xC0AC;&#xC810;&#xC744; &#xC704;&#xD574; &#xD544;&#xC218;&#xC801; &#xC774;&#xB2E4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC; &#xBCF4;&#xACE0; ATM DNA &#xC190;&#xC0C1; &#xC5C6;&#xC744; &#xACBD;&#xC6B0;&#xC5D0;&#xC11C; &#xC720;&#xC0AC; &#xBD84;&#xC5F4;&#xC5D0; &#xD65C;&#xC131;&#xD654; &#xB429;&#xB2C8;&#xB2E4;. Mitotic ATM &#xD65C;&#xC131;&#xD654; &#xC624;&#xB85C;&#xB77C; B &#xD0A4;&#xC5D0; &#xC758;&#xC874; &#xD558; &#xACE0; &#xC624;&#xB85C;&#xB77C; B &#xB5A0;&#xB4E4;&#xACE0; 1403&#xC5D0; ATM&#xC744; phosphorylates &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. &#xC774; &#xC778; &#xC0B0;&#xD654; &#xC774;&#xBCA4;&#xD2B8;&#xB294; mitotic ATM &#xD65C;&#xC131;&#xD654;&#xB97C; &#xC704;&#xD574; &#xD544;&#xC694; &#xD569;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, &#xC6B0;&#xB9AC;&#xB294; &#xD604;&#xAE08; &#xAE30;&#xB2A5;&#xC758; &#xC190;&#xC2E4; &#xACB0;&#xACFC; &#xB2E8;&#xCD95;&#xB41C; mitotic &#xD0C0;&#xC774;&#xBC0D;&#xC640; &#xACB0;&#xD568;&#xC774; &#xC788;&#xB294; &#xC2A4;&#xD540; &#xB4E4; &#xAC80;&#xC0AC;&#xC810; &#xADF8;&#xB9AC;&#xACE0; ATM Ser1403 &#xC778; &#xC0B0;&#xD654;&#xC758; &#xADF8; &#xD3D0;&#xC9C0;&#xAC00; &#xC2A4;&#xD540; &#xB4E4; &#xAC80;&#xC0AC;&#xC810; &#xACB0;&#xD568;&#xC5D0; &#xC774;&#xB974;&#xAC8C; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xB610;&#xD55C; mitotically &#xD65C;&#xC131;&#xD654; &#xB41C; ATM phosphorylates Bub1, ser314&#xC5D0; &#xC911;&#xC694; &#xD55C; kinetochore &#xB2E8;&#xBC31;&#xC9C8;&#xC744; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. Bub1 Ser314 &#xC778; &#xC0B0;&#xD654; ATM &#xC911;&#xC7AC; Bub1 &#xD65C;&#xB3D9;&#xC5D0; &#xD544;&#xC694;&#xD55C; &#xACE0; &#xC2A4;&#xD540; &#xB4E4; &#xCCB4;&#xD06C; &#xD3EC;&#xC778;&#xD2B8;&#xC758; &#xD65C;&#xC131;&#xD654;&#xB97C; &#xC704;&#xD574; &#xD544;&#xC218;&#xC801; &#xC774;&#xB2E4;. &#xD1B5;&#xCE6D; &#xD558; &#xC5EC; &#xC6B0;&#xB9AC;&#xC758; &#xB370;&#xC774;&#xD130; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC758; &#xC720;&#xC0AC; &#xBD84;&#xC5F4;&#xC5D0;&#xB294; &#xD604;&#xAE08; &#xC778;&#xCD9C;&#xAE30;&#xC758; &#xC911;&#xC694; &#xD55C; &#xAE30;&#xB2A5;&#xC744; &#xAC15;&#xC870; &#xD45C;&#xC2DC;&#xD569;&#xB2C8;&#xB2E4;.

&#xC624;&#xB85C;&#xB77C; B &#xC911;&#xC7AC; ATM &#xB5A0;&#xB4E4;&#xACE0; 1403 &#xC778; &#xC0B0;&#xD654;&#xB294; mitotic ATM &#xD65C;&#xC131;&#xD654; &#xBC0F; &#xC2A4;&#xD540; &#xB4E4; &#xAC80;&#xC0AC;&#xC810;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC694; &#xD569;&#xB2C8;&#xB2E4;.

La kinasa ATM juega un papel cr&#xED;tico en el mantenimiento de la estabilidad gen&#xE9;tica. ATM se activa en respuesta al da&#xF1;o del ADN y es esencial para los puestos de control del ciclo celular. En este caso, nos informan de que ATM se activa en la mitosis, en ausencia de da&#xF1;o en el ADN. Se demuestra que la activaci&#xF3;n mit&#xF3;tica ATM es dependiente de la quinasa Aurora-B y que Aurora B-ATM fosforila en serina 1403. Este evento fosforilaci&#xF3;n es necesaria para la activaci&#xF3;n mit&#xF3;tica cajero autom&#xE1;tico. Adem&#xE1;s, nos muestran que la p&#xE9;rdida de la ATM resultados de la funci&#xF3;n en el momento mit&#xF3;tico acortado y un puesto de control del eje defectuoso, y que la derogaci&#xF3;n de la ATM fosforilaci&#xF3;n Ser1403 conduce a este defecto puesto de control del husillo. Tambi&#xE9;n demuestran que la mitosis activa ATM fosforila Bub1, una prote&#xED;na esencial cinetocoro, en Ser314. ATM mediada Bub1 Ser314 fosforilaci&#xF3;n es necesaria para Bub1 actividad y es esencial para la activaci&#xF3;n del puesto de control del husillo. En conjunto, nuestros datos ponen de relieve los mecanismos de una funci&#xF3;n cr&#xED;tica de la ATM en la mitosis.

Aurora B ATM mediada Serina 1403 fosforilaci&#xF3;n es necesaria para la activaci&#xF3;n mit&#xF3;tica ATM y el puesto de control del eje

CENP-A is a centromere-specific histone H3 variant that epigenetically determines centromere identity to ensure kinetochore assembly and proper chromosome segregation, but the precise mechanism of its specific localization within centromeric heterochromatin remains obscure. We have discovered that CUL4A-RBX1-COPS8 E3 ligase activity is required for CENP-A ubiquitylation on lysine 124 (K124) and CENP-A centromere localization. A mutation of CENP-A, K124R, reduces interaction with HJURP (a CENP-A-specific histone chaperone) and abrogates localization of CENP-A to the centromere. Addition of monoubiquitin is sufficient to restore CENP-A K124R to centromeres and the interaction with HJURP, indicating that "signaling" ubiquitylation is required for CENP-A loading at centromeres. The CUL4A-RBX1 complex is required for loading newly synthesized CENP-A and maintaining preassembled CENP-A at centromeres. Thus, CENP-A K124R ubiquitylation, mediated by the CUL4A-RBX1-COPS8 complex, is essential for CENP-A deposition at the centromere.

CENP-A K124 Ubiquitylation Is Required for CENP-A Deposition at the Centromere.

The centromere plays an essential role in accurate chromosome segregation, and the chromosomal location of the centromere is determined by the presence of a histone H3 variant, centromere protein A (CENP-A), in centromeric nucleosomes. However, the precise mechanisms of deposition, maintenance, and inheritance of CENP-A at centromeres are unclear. We have reported that CENP-A deposition requires ubiquitylation of CENP-A lysine 124 mediated by the E3 ligase activity of Cullin 4A (CUL4A)-RING-box protein 1 (RBX1)-COP9 signalsome complex subunit 8 (COPS8). We have proposed a model of inheritance for CENP-A ubiquitylation, through dimerization between rounds of cell divisions, that maintains the position of centromeres.

CENP-A Ubiquitylation Contributes to Maintaining the Chromosomal Location of the Centromere.

CENP-A is a centromere-specific histone H3 variant that epigenetically determines centromere identity, but how CENP-A is deposited at the centromere remains obscure. We previously reported that CENP-A K124 ubiquitylation, mediated by the CUL4A-RBX1-COPS8 complex, is essential for CENP-A deposition at the centromere. However, a recent report stated that CENP-A K124R mutants show no defects in centromere localization and cell viability. In the present study, we found that EYFP tagging induces additional ubiquitylation of EYFP-CENP-A K124R, which allows the mutant protein to bind to HJURP. Using a previously developed conditional CENP-A knockout system and our CENP-A K124R knockin mutant created by the CRISPR-Cas9 system, we show that the Flag-tagged or untagged CENP-A K124R mutant is lethal. This lethality is rescued by monoubiquitin fusion, indicating that CENP-A ubiquitylation is essential for viability.

Yohei Niikura

Center for Childhood Cancer and Blood Diseases

Nationwide Children's Hospital

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Yohei Niikura

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Center for Childhood Cancer and Blood Diseases

Nationwide Children's Hospital

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Center for Childhood Cancer and Blood Diseases