RESUMO
Cytokinesis partitions cytoplasmic and genomic materials at the end of cell division. Failure in this process causes polyploidy, which in turn can generate chromosomal instability, a hallmark of many cancers. Successful cytokinesis requires cooperative interaction between contractile ring and central spindle components, but how this cooperation is established is poorly understood. Here we show that Sticky (Sti), the Drosophila ortholog of the contractile ring component Citron kinase (CIT-K), interacts directly with two kinesins, Nebbish [the fly counterpart of human kinesin family member 14 (KIF14)] and Pavarotti [the Drosophila ortholog of human mitotic kinesin-like protein 1 (MKLP1)], and that in turn these kinesins interact with each other and with another central spindle protein, Fascetto [the fly ortholog of protein regulator of cytokinesis 1 (PRC1)]. Sti recruits Nebbish to the cleavage furrow, and both proteins are required for midbody formation and proper localization of Pavarotti and Fascetto. These functions require Sti kinase activity, indicating that Sti plays both structural and regulatory roles in midbody formation. Finally, we show that CIT-K's role in midbody formation is conserved in human cells. Our findings indicate that CIT-K is likely to act at the top of the midbody-formation hierarchy by connecting and regulating a molecular network of contractile ring components and microtubule-associated proteins.
Assuntos
Citocinese/fisiologia , Proteínas de Drosophila/metabolismo , Redes Reguladoras de Genes/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Sítios de Ligação/genética , Western Blotting , Linhagem Celular , Citocinese/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Redes Reguladoras de Genes/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinesinas/genética , Cinesinas/metabolismo , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Interferência de RNARESUMO
The midbody is an organelle assembled at the intercellular bridge between the two daughter cells at the end of mitosis. It controls the final separation of the daughter cells and has been involved in cell fate, polarity, tissue organization, and cilium and lumen formation. Here, we report the characterization of the intricate midbody protein-protein interaction network (interactome), which identifies many previously unknown interactions and provides an extremely valuable resource for dissecting the multiple roles of the midbody. Initial analysis of this interactome revealed that PP1ß-MYPT1 phosphatase regulates microtubule dynamics in late cytokinesis and de-phosphorylates the kinesin component MKLP1/KIF23 of the centralspindlin complex. This de-phosphorylation antagonizes Aurora B kinase to modify the functions and interactions of centralspindlin in late cytokinesis. Our findings expand the repertoire of PP1 functions during mitosis and indicate that spatiotemporal changes in the distribution of kinases and counteracting phosphatases finely tune the activity of cytokinesis proteins.
Assuntos
Citocinese/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Mapas de Interação de Proteínas/fisiologia , Proteína Fosfatase 1/metabolismo , Aurora Quinase B/metabolismo , Sítios de Ligação/genética , Células HeLa , Humanos , Microscopia Intravital , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Mitose/fisiologia , Mutagênese Sítio-Dirigida , Fosforilação/fisiologia , Proteína Fosfatase 1/genética , RNA Interferente Pequeno/metabolismo , Fuso Acromático/metabolismo , Imagem com Lapso de TempoRESUMO
The impact of covalent binding on PROTAC-mediated degradation of BTK was investigated through the preparation of both covalent binding and reversible binding PROTACs derived from the covalent BTK inhibitor ibrutinib. It was determined that a covalent binding PROTAC inhibited BTK degradation despite evidence of target engagement, while BTK degradation was observed with a reversible binding PROTAC. These observations were consistently found when PROTACs that were able to recruit either IAP or cereblon E3 ligases were employed. Proteomics analysis determined that the use of a covalently bound PROTAC did not result in the degradation of covalently bound targets, while degradation was observed for some reversibly bound targets. This observation highlights the importance of catalysis for successful PROTAC-mediated degradation and highlights a potential caveat for the use of covalent target binders in PROTAC design.
Assuntos
Tirosina Quinase da Agamaglobulinemia , Inibidores de Proteínas Quinases , Pirazóis , Pirimidinas , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Piperidinas , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteólise , Pirazóis/química , Pirazóis/metabolismo , Pirimidinas/química , Pirimidinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
P300/CBP-associated factor (PCAF) and general control nonderepressible 5 (GCN5) are closely related epigenetic proteins, each containing an acetyltransferase domain and a bromodomain. Consistent with reported roles for these proteins in immune function, we find that PCAF-deficient macrophages exhibit a markedly reduced ability to produce cytokines upon stimulation with lipopolysaccharide (LPS). Investigating the potential to target this pathway pharmacologically, we show that chemical inhibition of the PCAF/GCN5 bromodomains is insufficient to recapitulate the diminished inflammatory response of PCAF-deficient immune cells. However, by generating the first PCAF/GCN5 proteolysis targeting chimera (PROTAC), we identify small molecules able to degrade PCAF/GCN5 and to potently modulate the expression of multiple inflammatory mediators in LPS-stimulated macrophages and dendritic cells. Our data illustrate the power of the PROTAC approach in the context of multidomain proteins, revealing a novel anti-inflammatory therapeutic opportunity for targeting PCAF/GCN5.
Assuntos
Benzoatos/farmacologia , Piperidinas/farmacologia , Piridazinas/farmacologia , Fatores de Transcrição de p300-CBP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Benzoatos/síntese química , Benzoatos/química , Diferenciação Celular/efeitos dos fármacos , Citocinas/metabolismo , Células Dendríticas/metabolismo , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Lipopolissacarídeos , Macrófagos/metabolismo , Camundongos , Monócitos/metabolismo , Peptídeo Hidrolases/metabolismo , Piperidinas/síntese química , Piperidinas/química , Domínios Proteicos , Proteólise , Piridazinas/síntese química , Piridazinas/química , Estereoisomerismo , Ubiquitina-Proteína Ligases , Fatores de Transcrição de p300-CBP/químicaRESUMO
Cytokinesis culminates in the final separation, or abscission, of the two daughter cells at the end of cell division. Abscission relies on an organelle, the midbody, which forms at the intercellular bridge and is composed of various proteins arranged in a precise stereotypic pattern. The molecular mechanisms controlling midbody organization and function, however, are obscure. Here we show that proper midbody architecture requires cross-regulation between two cell division kinases, Citron kinase (CIT-K) and Aurora B, the kinase component of the chromosomal passenger complex (CPC). CIT-K interacts directly with three CPC components and is required for proper midbody architecture and the orderly arrangement of midbody proteins, including the CPC. In addition, we show that CIT-K promotes Aurora B activity through phosphorylation of the INCENP CPC subunit at the TSS motif. In turn, Aurora B controls CIT-K localization and association with its central spindle partners through phosphorylation of CIT-K's coiled coil domain. Our results identify, for the first time, a cross-regulatory mechanism between two kinases during cytokinesis, which is crucial for establishing the stereotyped organization of midbody proteins.
Assuntos
Aurora Quinase B/metabolismo , Citocinese , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Aurora Quinase B/análise , Divisão Celular , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Fosforilação , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/análise , Fuso Acromático/metabolismo , Fuso Acromático/ultraestruturaRESUMO
Cytokinesis controls the proper segregation of nuclear and cytoplasmic materials at the end of cell division. The chromosomal passenger complex (CPC) has been proposed to monitor the final separation of the two daughter cells at the end of cytokinesis in order to prevent cell abscission in the presence of DNA at the cleavage site, but the precise molecular basis for this is unclear. Recent studies indicate that abscission could be mediated by the assembly of filaments comprising components of the endosomal sorting complex required for transport-III (ESCRT-III). Here, we show that the CPC subunit Borealin interacts directly with the Snf7 components of ESCRT-III in both Drosophila and human cells. Moreover, we find that the CPC's catalytic subunit, Aurora B kinase, phosphorylates one of the three human Snf7 paralogues-CHMP4C-in its C-terminal tail, a region known to regulate its ability to form polymers and associate with membranes. Phosphorylation at these sites appears essential for CHMP4C function because their mutation leads to cytokinesis defects. We propose that CPC controls abscission timing through inhibition of ESCRT-III Snf7 polymerization and membrane association using two concurrent mechanisms: interaction of its Borealin component with Snf7 proteins and phosphorylation of CHMP4C by Aurora B.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Citocinese/fisiologia , Proteínas de Drosophila/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Aurora Quinase B , Aurora Quinases , Biopolímeros , Linhagem Celular , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Genes Reporter , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação , Proteínas do Tecido Nervoso , Fosforilação , Fosfosserina/metabolismo , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
In many organisms, the small guanosine triphosphatase RhoA controls assembly and contraction of the actomyosin ring during cytokinesis by activating different effectors. Although the role of some RhoA effectors like formins and Rho kinase is reasonably understood, the functions of another putative effector, Citron kinase (CIT-K), are still debated. In this paper, we show that, contrary to previous models, the Drosophila melanogaster CIT-K orthologue Sticky (Sti) does not require interaction with RhoA to localize to the cleavage site. Instead, RhoA fails to form a compact ring in late cytokinesis after Sti depletion, and this function requires Sti kinase activity. Moreover, we found that the Sti Citron-Nik1 homology domain interacts with RhoA regardless of its status, indicating that Sti is not a canonical RhoA effector. Finally, Sti depletion caused an increase of phosphorylated myosin regulatory light chain at the cleavage site in late cytokinesis. We propose that Sti/CIT-K maintains correct RhoA localization at the cleavage site, which is necessary for proper RhoA activity and contractile ring dynamics.