AbstractCDK1 has been known to be the sole cyclin-dependent kinase (CDK) partner of cyclin B1 to drive mitotic progression1. Here we demonstrate that CDK5 is active during mitosis and is necessary for maintaining mitotic fidelity. CDK5 is an atypical CDK owing to its high expression in post-mitotic neurons and activation by non-cyclin proteins p35 and p392.

摘要已知CDK1是细胞周期蛋白B1驱动有丝分裂进程的唯一细胞周期蛋白依赖性激酶（CDK）伴侣1。在这里，我们证明CDK5在有丝分裂期间是活跃的，并且对于维持有丝分裂保真度是必需的。CDK5是一种非典型CDK，因为它在有丝分裂后神经元中高表达，并被非细胞周期蛋白p35和p392激活。

Here, using independent chemical genetic approaches, we specifically abrogated CDK5 activity during mitosis, and observed mitotic defects, nuclear atypia and substantial alterations in the mitotic phosphoproteome. Notably, cyclin B1 is a mitotic co-factor of CDK5. Computational modelling, comparison with experimentally derived structures of CDK–cyclin complexes and validation with mutational analysis indicate that CDK5–cyclin B1 can form a functional complex.

。值得注意的是，细胞周期蛋白B1是CDK5的有丝分裂辅因子。计算建模，与实验得出的CDK–细胞周期蛋白复合物结构的比较以及突变分析的验证表明，CDK5–细胞周期蛋白B1可以形成功能复合物。

Disruption of the CDK5–cyclin B1 complex phenocopies CDK5 abrogation in mitosis. Together, our results demonstrate that cyclin B1 partners with both CDK5 and CDK1, and CDK5–cyclin B1 functions as a canonical CDK–cyclin complex to ensure mitotic fidelity..

。总之，我们的结果表明，细胞周期蛋白B1与CDK5和CDK1都有伴侣，而CDK5–细胞周期蛋白B1作为典型的CDK–细胞周期蛋白复合物起作用，以确保有丝分裂的保真度。。

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Fig. 1: Abrogation of CDK5 leads to abnormal nuclear morphology, lagging chromosomes and micronuclei.Fig. 2: Abrogation of CDK5 leads to chromosome alignment defects and abnormal spindle architecture.Fig. 3: Abrogation of CDK5 is associated with reduced phosphorylation of spindle regulators.Fig. 4: Cyclin B1 forms a complex with and activates CDK5 in mitosis.Fig.

图1:CDK5的废除导致核形态异常，染色体滞后和微核。图2:CDK5的废除导致染色体比对缺陷和纺锤体结构异常。图3:CDK5的消除与纺锤体调节剂的磷酸化减少有关。图4：细胞周期蛋白B1与有丝分裂中的CDK5形成复合物并激活CDK5。图。

5: Disruption of the CDK5–cyclin B1 complex phenotypically recapitulates CDK5 abrogation..

5： CDK5-cyclin B1复合物的破坏在表型上概括了CDK5的废除。。

Data availability

数据可用性

All data supporting the findings of this study are available in the Article and its Supplementary Information. The LC–MS/MS proteomics data have been deposited to the ProteomeXchange Consortium60 via the PRIDE61 partner repository under dataset identifier PXD038386. Correspondence regarding experiments and requests for materials should be addressed to the corresponding authors..

本文及其补充信息中提供了支持本研究结果的所有数据。LC-MS/MS蛋白质组学数据已通过PRIDE61合作伙伴存储库以数据集标识符PXD038386保存到ProteomeXchange Consortium60。有关实验和材料要求的信件应寄给通讯作者。。

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Download referencesAcknowledgementsWe thank D. Pellman for comments on the manuscript; W. Michowski, S. Sharma, P. Sicinski, B. Nabet and N. Gray for the reagents; J. A. Tainer for providing access to software used for structural analysis; and S. Gerber for sharing unpublished results.

下载参考文献致谢我们感谢D.Pellman对稿件的评论；W、 Michowski，S。Sharma，P。Sicinski，B。Nabet和N。Gray的试剂；J、 A.Tainer，用于访问用于结构分析的软件；和S.Gerber分享未发表的结果。

D.C. is supported by grants R01 CA208244 and R01 CA264900, DOD Ovarian Cancer Award W81XWH-15-0564/OC140632, Tina’s Wish Foundation, Detect Me If You Can, a V Foundation Award, a Gray Foundation grant and the Claudia Adams Barr Program in Innovative Basic Cancer Research. A. Spektor would like to acknowledge support from K08 CA208008, the Burroughs Wellcome Fund Career Award for Medical Scientists, Saverin Breast Cancer Research Fund and the Claudia Adams Barr Program in Innovative Basic Cancer Research.

D、 C.得到了资助R01 CA208244和R01 CA264900，国防部卵巢癌奖W81XWH-15-0564/OC140632，蒂娜的愿望基金会，如果可以的话检测我，V基金会奖，灰色基金会奖和克劳迪娅·亚当斯·巴尔创新基础癌症研究计划的支持。A、 Spektor要感谢K08 CA208008，Burroughs Wellcome基金医学科学家职业奖，Saverin乳腺癌研究基金和Claudia Adams Barr创新基础癌症研究计划的支持。

X.-F.Z. was an American Cancer Society Fellow and is supported by the Breast and Gynecologic Cancer Innovation Award from Susan F. Smith Center for Women’s Cancers at Dana-Farber Cancer Institute. A. Syed is supported by the Claudia Adams Barr Program in Innovative Basic Cancer Research. B.T. was supported by the Polish National Agency for Academic Exchange (grant PPN/WAL/2019/1/00018) and by the Foundation for Polish Science (START Program).

十、 -F.Z.是美国癌症协会研究员，并获得了达纳法伯癌症研究所苏珊·F·史密斯女性癌症中心乳腺癌和妇科癌症创新奖的支持。A、 Syed在创新的基础癌症研究中得到了Claudia Adams Barr计划的支持。B。T、 得到了波兰国家学术交流局（grant PPN/WAL/2019/1/00018）和波兰科学基金会（START计划）的支持。

A.D.D is supported by NIH grant R01 HL52725. A.G.P. by National Cancer Institute grants U01CA214114 and U01CA271407, as well as a donation from the Aven Foundation; J.R.W. by National Cancer Institute grant R50CA211499; and K.S. by NIH awards 1R01-CA237660 and 1RF1NS124779.Author informationAuthor notesBartłomiej TomasikPresent address: Department of Oncology and Radiotherapy, Medical University of Gdańsk, Faculty of Medicine, Gdańsk, PolandThese authors contributed equally: Xiao-Feng Zheng, Aniruddha SarkarAuthors and AffiliationsDiv.

A、 D.D得到了NIH拨款R01 HL52725.A的支持。G、 P.由国家癌症研究所资助U01CA214114和U01CA271407，以及Aven基金会的捐赠；J、 R.W.由国家癌症研究所资助R50CA211499；和美国国立卫生研究院奖1R01-CA237660和1RF1NS124779的K.S.作者信息作者notesBartłomiej Tomasik目前的地址：格但斯克医科大学肿瘤与放射治疗系，格但斯克医学院，波兰这些作者做出了同样的贡献：郑晓峰，Aniruddha SarkarAuthors和附属机构IV。

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PubMed Google ScholarContributionsX.-F.Z., A. Sarkar., A. Spektor. and D.C. conceived the project and designed the experiments. X.-F.Z. and A. Sarkar performed the majority of experiments and associated analyses except as listed below. H.L. expressed relevant proteins and conducted the kinase activity assays for CDK5–cyclin B1, CDK5–p35 and CDK5(S46) variant complexes under the guidance of K.S.; A.

PubMed谷歌学术贡献x-F、 Z.，A.Sarkar。，A、 扬声器。华盛顿特区构思了这个项目并设计了实验。十、 -F.Z.和A.Sarkar进行了大多数实验和相关分析，以下列出的除外。H、 L.表达相关蛋白，并在K.S.的指导下对CDK5–细胞周期蛋白B1，CDK5–p35和CDK5（S46）变异复合物进行激酶活性测定。；答：。

Syed performed structural modelling and analysis. R.G.I., J.J.K. and J.R.W. performed MS and analysis. B.T. and H.N. performed MS data analyses. K.H. provided guidance to screen CDK5(as) knocked-in clones and performed sequence analysis to confirm CDK5(as) knock-in. F.L. and A.D.D. provided reagents and discussion on CDK5 substrates analyses.

Syed进行了结构建模和分析。R、 G.I.，J.J.K.和J.R.W.进行了MS和分析。B、 T.和H.N.进行了MS数据分析。K、 H.提供了筛选CDK5（as）敲入克隆的指导，并进行了序列分析以确认CDK5（as）敲入。F.L.和A.D.D.提供了试剂并讨论了CDK5底物分析。

X.-F.Z., A. Sarkar, A. Spektor and D.C. wrote the manuscript with inputs and edits from all of the other authors.Corresponding authorsCorrespondence to.

十、 -F.Z.，A.Sarkar，A.Spektor和D.C.用所有其他作者的输入和编辑撰写了手稿。通讯作者通讯。

Alexander Spektor or Dipanjan Chowdhury.Ethics declarations

Alexander Spektor或Dipanjan Chowdhury。道德宣言

Competing interests

相互竞争的利益

A.D.D. reports consulting for AstraZeneca, Bayer AG, Blacksmith/Lightstone Ventures, Bristol Myers Squibb, Cyteir Therapeutics, EMD Serono, Impact Therapeutics, PrimeFour Therapeutics, Pfizer, Tango Therapeutics and Zentalis Pharmaceuticals/Zeno Management; is an advisory board member for Cyteir and Impact Therapeutics; a stockholder in Cedilla Therapeutics, Cyteir, Impact Therapeutics and PrimeFour Therapeutics; and reports receiving commercial research grants from Bristol Myers Squibb, EMD Serono, Moderna and Tango Therapeutics.

A、 D.D.报告咨询阿斯利康，拜耳公司，铁匠/光石风险投资公司，百时美施贵宝，Cyteir Therapeutics，EMD Serono，Impact Therapeutics，PrimeFour Therapeutics，辉瑞，Tango Therapeutics和Zentalis Pharmaceuticals/Zeno Management；是Cyteir和Impact Therapeutics的顾问委员会成员；Cedilla Therapeutics、Cyteir、Impact Therapeutics和PrimeFour Therapeutics的股东；并报告获得了百时美施贵宝，EMD Serono，Moderna和Tango Therapeutics的商业研究资助。

The other authors declare no competing interests..

其他作者声明没有利益冲突。。

Peer review

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Peer review information

同行评审信息

Nature thanks Yibing Shan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

《自然》杂志感谢单一冰和另一位匿名审稿人对这项工作的同行评审所做的贡献。同行评审报告可供查阅。

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended data figures and tablesExtended Data Fig. 1 Inhibition of CDK5 in analogue-sensitive (CDK5-as) system.a, Schematics depicting specific inhibition of the CDK5 analogue-sensitive (as) variant.

Additional informationPublisher的注释Springer Nature在已发布的地图和机构隶属关系中的管辖权主张方面保持中立。扩展数据图和表扩展数据图1在类似物敏感（CDK5-as）系统中抑制CDK5。a，描绘CDK5类似物敏感（as）变体的特异性抑制的示意图。

Canonical ATP-analogue inhibitor (In, yellow) targets endogenous CDK5 (dark green) at its ATP-binding catalytic site nonspecifically since multiple kinases share structurally similar catalytic sites (left panel). The analogue-sensitive (as, light green) phenylalanine-to-glycine (F80G) mutation confers a structural change adjacent to the catalytic site of CDK5 that does not impact its catalysis but accommodates the specific binding of a non-hydrolysable bulky orthogonal inhibitor 1NM-PP1(In*, orange).

典型的ATP类似物抑制剂（In，黄色）非特异性地靶向其ATP结合催化位点的内源性CDK5（深绿色），因为多种激酶共享结构相似的催化位点（左图）。类似物敏感的（as，浅绿色）苯丙氨酸-甘氨酸（F80G）突变赋予CDK5催化位点附近的结构变化，其不影响其催化作用，但适应不可水解的庞大正交抑制剂1NM-PP1的特异性结合*，橙色）。

Introduction of 1NM-PP1 thus selectively inhibits CDK5-as variant (right panel). b, Immunoblots showing two clones (Cl 23 and Cl 50) of RPE-1 cells expressing FLAG-HA-CDK5-as in place of endogenous CDK5. Representative results are shown from three independent repeats. c, Proliferation curve of parental RPE-1 and RPE-1 CDK5-as cells.

因此，引入1NM-PP1选择性地抑制CDK5作为变体（右图）。b、 免疫印迹显示表达FLAG-HA-CDK5-as代替内源性CDK5的RPE-1细胞的两个克隆（Cl 23和Cl 50）。三个独立的重复显示了代表性的结果。c、 亲本RPE-1和RPE-1 CDK5 as细胞的增殖曲线。

Data represent mean ± s.d. from three independent repeats. p-value was determined by Mann Whitney U test. d, Immunoblots showing immunoprecipitated CDK1-cyclin B1 complex or CDK5-as-cyclin B1 complex by the indicated antibody-coupled agarose, from nocodazole arrested RPE-1 CDK5-as cells with treated with or without 1NM-PP1 for inhibition of CDK5-as, from three independent replicate experiments.

数据代表来自三个独立重复的平均值±标准差。p值由Mann-Whitney U检验确定。d、 通过指定的抗体偶联琼脂糖显示免疫沉淀的CDK1-细胞周期蛋白B1复合物或CDK5作为细胞周期蛋白B1复合物的免疫印迹，来自诺考达唑的RPE-1 CDK5-as细胞，用或不用1NM-PP1处理以抑制CDK5-as，来自三个独立的重复实验。

e, In-vitro kinase activity quantification of immunoprecipitated complex shown in d. Data represent mean ± s.d. from three independent experiments. p-values were determined by unpaired, two-tailed student.

e、 d中显示的免疫沉淀复合物的体外激酶活性定量。数据代表来自三个独立实验的平均值±s.d。p值由不成对的双尾学生确定。

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