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1.
J Cell Sci ; 128(24): 4526-37, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26542020

RESUMO

The serine/threonine protein phosphatase-1 (PP1) complex is a key regulator of the cell cycle. However, the redundancy of PP1 isoforms and the lack of specific inhibitors have hampered studies on the global role of PP1 in cell cycle progression in vertebrates. Here, we show that the overexpression of nuclear inhibitor of PP1 (NIPP1; also known as PPP1R8) in HeLa cells culminated in a prometaphase arrest, associated with severe spindle-formation and chromosome-congression defects. In addition, the spindle assembly checkpoint was activated and checkpoint silencing was hampered. Eventually, most cells either died by apoptosis or formed binucleated cells. The NIPP1-induced mitotic arrest could be explained by the inhibition of PP1 that was titrated away from other mitotic PP1 interactors. Consistent with this notion, the mitotic-arrest phenotype could be rescued by the overexpression of PP1 or the inhibition of the Aurora B kinase, which acts antagonistically to PP1. Finally, we demonstrate that the overexpression of NIPP1 also hampered colony formation and tumor growth in xenograft assays in a PP1-dependent manner. Our data show that the selective inhibition of PP1 can be used to induce cancer cell death through mitotic catastrophe.


Assuntos
Endorribonucleases/metabolismo , Mitose , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimologia , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 1/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Morte Celular , Endorribonucleases/genética , Células HeLa , Humanos , Proteínas de Neoplasias/genética , Neoplasias/genética , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteínas de Ligação a RNA/genética
2.
Cell Cycle ; 19(22): 3029-3041, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33054506

RESUMO

RepoMan is a chromosome-associated scaffold protein that integrates signaling of multiple kinases and phosphatases to coordinate spindle-kinetochore interactions, chromosome (de)condensation and nuclear envelope (dis)assembly during mitosis. Another key mitotic event is the assembly of a microtubule-based spindle, which involves redundant pathways emanating from the centrosomes, microtubules and chromosomes. Here we describe a novel mitotic function of RepoMan in regulating chromosome-dependent microtubule assembly. At limiting concentrations of microtubule-destabilizing agents, RepoMan-depleted cells showed enhanced chromosome clustering. This clustering was completely dependent on the partial inhibition of microtubule growth originating from the chromosome-dependent pathway. We also demonstrated that RepoMan interacts with prime regulators of the chromosome-dependent spindle assembly such as NuSAP1, NuMA, and TPX2. In addition, RepoMan was required to enable or maintain phosphorylation of NuSAP1 at CDK sites, thereby enabling activation of NuSAP1 through dissociation of inhibitory importin ß/7. Our data identify RepoMan as an enhancer of microtubule assembly at chromosomes.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromossomos Humanos/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/genética , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Centrossomo/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/genética , Fosforilação , Proteína Fosfatase 1/metabolismo , Transfecção
3.
FEBS Lett ; 591(2): 415-424, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28032891

RESUMO

The biotin identification (BioID) protocol uses a mutant of the biotin ligase BirA (BirA*) fused to a protein-of-interest to biotinylate proximate proteins in intact cells. Here, we show that two inactive halves of BirA* separately fused to a catalytic and regulatory subunit of protein phosphatase PP1 reconstitute a functional BirA* enzyme upon heterodimerization of the phosphatase subunits. We also demonstrate that this BirA* fragment complementation approach, termed split-BioID, can be used to screen for substrates and other protein interactors of PP1 holoenzymes. Split-BioID is a novel and versatile tool for the identification of (transient) interactors of protein dimers.


Assuntos
Bioensaio/métodos , Dimerização , Mapeamento de Interação de Proteínas , Biotinilação , Teste de Complementação Genética , Células HEK293 , Humanos , Reprodutibilidade dos Testes
4.
Elife ; 52016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27572260

RESUMO

Ki-67 and RepoMan have key roles during mitotic exit. Previously, we showed that Ki-67 organizes the mitotic chromosome periphery and recruits protein phosphatase 1 (PP1) to chromatin at anaphase onset, in a similar manner as RepoMan (Booth et al., 2014). Here we show how Ki-67 and RepoMan form mitotic exit phosphatases by recruiting PP1, how they distinguish between distinct PP1 isoforms and how the assembly of these two holoenzymes are dynamically regulated by Aurora B kinase during mitosis. Unexpectedly, our data also reveal that Ki-67 and RepoMan bind PP1 using an identical, yet novel mechanism, interacting with a PP1 pocket that is engaged only by these two PP1 regulators. These findings not only show how two distinct mitotic exit phosphatases are recruited to their substrates, but also provide immediate opportunities for the design of novel cancer therapeutics that selectively target the Ki-67:PP1 and RepoMan:PP1 holoenzymes.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Antígeno Ki-67/metabolismo , Proteínas Nucleares/metabolismo , Multimerização Proteica , Proteína Fosfatase 1/metabolismo , Proteínas de Transporte/química , Ciclo Celular , Proteínas de Ciclo Celular/química , Cristalografia por Raios X , Humanos , Antígeno Ki-67/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteínas Nucleares/química , Conformação Proteica , Proteína Fosfatase 1/química
5.
Nat Commun ; 6: 10215, 2015 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-26674376

RESUMO

RepoMan is a scaffold for signalling by mitotic phosphatases at the chromosomes. During (pro)metaphase, RepoMan-associated protein phosphatases PP1 and PP2A-B56 regulate the chromosome targeting of Aurora-B kinase and RepoMan, respectively. Here we show that this task division is critically dependent on the phosphorylation of RepoMan by protein kinase Cyclin-dependent kinase 1 (Cdk1), which reduces the binding of PP1 but facilitates the recruitment of PP2A-B56. The inactivation of Cdk1 in early anaphase reverses this phosphatase switch, resulting in the accumulation of PP1-RepoMan to a level that is sufficient to catalyse its own chromosome targeting in a PP2A-independent and irreversible manner. Bulk-targeted PP1-RepoMan also inactivates Aurora B and initiates nuclear-envelope reassembly through dephosphorylation-mediated recruitment of Importin ß. Bypassing the Cdk1 regulation of PP1-RepoMan causes the premature dephosphorylation of its mitotic-exit substrates in prometaphase. Hence, the regulation of RepoMan-associated phosphatases by Cdk1 is essential for the timely dephosphorylation of their mitotic substrates.


Assuntos
Aurora Quinase B/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/genética , Mitose/genética , Proteínas Nucleares/metabolismo , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/metabolismo , beta Carioferinas/metabolismo , Anáfase , Proteína Quinase CDC2 , Linhagem Celular Tumoral , Cromossomos/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Imunofluorescência , Células HEK293 , Células HeLa , Humanos , Microscopia Confocal , Membrana Nuclear/metabolismo , Monoéster Fosfórico Hidrolases , Prometáfase
6.
ACS Chem Biol ; 8(1): 36-45, 2013 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-23214403

RESUMO

Protein phosphatases have both protective and promoting roles in the etiology of diseases. A prominent example is the existence of oncogenic as well as tumor-suppressing protein phosphatases. A few protein phosphatase activity modulators are already applied in therapies. These were however not developed in target-directed approaches, and the recent discovery of phosphatase involvement followed their application in therapy. Nevertheless, these examples demonstrate that small molecules can be generated that modulate the activity of protein phosphatases and are beneficial for the treatment of protein phosphorylation diseases. We describe here strategies for the development of activators and inhibitors of protein phosphatases and clarify some long-standing misconceptions concerning the druggability of these enzymes. Recent developments suggest that it is feasible to design potent and selective protein phosphatase modulators with a therapeutic potential.


Assuntos
Sistemas de Liberação de Medicamentos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Sistemas de Liberação de Medicamentos/tendências , Humanos , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
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