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1.
PLoS One ; 12(6): e0178925, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28582471

RESUMO

We previously showed that p21Cip1 transits through the nucleolus on its way from the nucleus to the cytoplasm and that DNA damage inhibits this transit and induces the formation of p21Cip1-containing intranucleolar bodies (INoBs). Here, we demonstrate that these INoBs also contain SUMO-1 and UBC9, the E2 SUMO-conjugating enzyme. Furthermore, whereas wild type SUMO-1 localized in INoBs, a SUMO-1 mutant, which is unable to conjugate with proteins, does not, suggesting the presence of SUMOylated proteins at INoBs. Moreover, depletion of the SUMO-conjugating enzyme UBC9 or the sumo hydrolase SENP2 changed p21Cip1 intracellular distribution. In addition to SUMO-1 and p21Cip1, cell cycle regulators and DNA damage checkpoint proteins, including Cdk2, Cyclin E, PCNA, p53 and Mdm2, and PML were also detected in INoBs. Importantly, depletion of UBC9 or p21Cip1 impacted INoB biogenesis and the nucleolar accumulation of the cell cycle regulators and DNA damage checkpoint proteins following DNA damage. The impact of p21Cip1 and SUMO-1 on the accumulation of proteins in INoBs extends also to CRM1, a nuclear exportin that is also important for protein translocation from the cytoplasm to the nucleolus. Thus, SUMO and p21Cip1 regulate the transit of proteins through the nucleolus, and that disruption of nucleolar export by DNA damage induces SUMO and p21Cip1 to act as hub proteins to form a multiprotein complex in the nucleolus.


Assuntos
Nucléolo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação da Expressão Gênica , Organelas/metabolismo , Proteína SUMO-1/metabolismo , Nucléolo Celular/genética , Ciclina E/genética , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/deficiência , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Dano ao DNA , Células HCT116 , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Biogênese de Organelas , Organelas/genética , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína SUMO-1/genética , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Enzimas de Conjugação de Ubiquitina/deficiência , Enzimas de Conjugação de Ubiquitina/genética , Proteína Exportina 1
2.
Traffic ; 11(6): 743-55, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20331843

RESUMO

p21(cip1) is a protein with a dual function in oncogenesis depending mainly on its intracellular localization: tumor suppressor in the nucleus and oncogenic in the cytoplasm. After DNA damage, p21(cip1) increases and accumulates in the nucleus to ensure cell cycle arrest. We show here that the nuclear accumulation of p21(cip1) is not only a consequence of its increased levels but to a DNA damage cellular response, which is ataxia telangiectasia and Rad3 related (ATR)/ataxia telangiectasia mutated (ATM) and p53 independent. Furthermore, after DNA damage, p21(cip1) not only accumulates in the nucleoplasm but also in the disrupted nucleolus. Inside the nucleolus, it is found in spherical structures, which are not a protrusion of the nucleoplasm. The steady-state distribution of p21(cip1) in the nucleolus resulted from a highly dynamic equilibrium between nucleoplasmic and nucleolar p21(cip1) and correlated with the inhibition of p21(cip1) nuclear export. Most interestingly, inhibition of ribosomal export after expressing a dominant-negative mutant of nucleophosmin induced p21(cip1) accumulation in the nucleus and the nucleolus in the absence of DNA damage. This proved the existence of a nucleolar export route to the cytoplasm for p21(cip1) in control conditions that would be inhibited upon DNA damage leading to nuclear and nucleolar accumulation of p21(cip1).


Assuntos
Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/química , Dano ao DNA , Ciclo Celular , Linhagem Celular Tumoral , Genes Dominantes , Humanos , Imuno-Histoquímica , Microscopia de Fluorescência/métodos , Mutação , Proteínas Nucleares/química , Nucleofosmina , Fotodegradação , Plasmídeos/metabolismo , Frações Subcelulares/metabolismo
3.
Cell Cycle ; 5(1): 3-6, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16357525

RESUMO

Intracellular localization plays an important role in the functional regulation of the cyclin-dependent kinase inhibitor p21. While nuclear functions have been linked to the tumor suppressor activity of p21, cytoplasmatic functions are oncogenic. We have recently shown that Ser153 phosphorylation of p21 by PKC contributes to its cytoplasmatic accumulation, and that this phosphorylation is inhibited by Ca(2+)-dependent calmodulin binding to the C-terminal region of p21. Consequently, PKC and calmodulin/Ca(2+) play diverging roles in the regulation of p21 intracellular localization. Other kinases such as AKT and MIRK/dyrk1B also phosphorylate p21 near the nuclear localization signal, thus inhibiting its nuclear accumulation. We discuss here the effects of such phosphorylations on p21 functionality, as well as its relevance to cell cycle progression and differentiation.


Assuntos
Calmodulina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteína Quinase C/metabolismo , Animais , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/química , Citoplasma/metabolismo , Humanos , Fosforilação , Ligação Proteica , Transporte Proteico
4.
Mol Cell Biol ; 25(16): 7364-74, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16055744

RESUMO

Intracellular localization plays an important role in the functional regulation of the cell cycle inhibitor p21. We have previously shown that calmodulin binds to p21 and that calmodulin is essential for the nuclear accumulation of p21. Here, we analyze the mechanism of this regulation. We show that calmodulin inhibits in vitro phosphorylation of p21 by protein kinase C (PKC) and that this inhibition is dependent upon calmodulin binding to p21. Two-dimensional electrophoresis analysis of cells expressing the p21 wild type or p21S153A, a nonphosphorylatable mutant of p21 at position 153, indicates that Ser153 of p21 is a phosphorylable residue in vivo. Furthermore, Western blot analysis using phospho-Ser153-specific antibodies indicates that Ser153 phosphorylation in vivo is induced when PKC is activated and calmodulin is inhibited. The mutation of Ser153 to aspartate, a pseudophosphorylated residue, inhibits the nuclear accumulation of p21. Finally, whereas wild-type p21 translocates to the cytoplasm after PKC activation in the presence of calmodulin inhibitors, p21 carrying a nonphosphorylatable residue at position 153 remains in the nucleus. We propose that calmodulin binding to p21 prevents its phosphorylation by PKC at Ser153 and consequently allows its nuclear localization. When phosphorylated at Ser153, p21 is located at the cytoplasm and disrupts stress fibers.


Assuntos
Calmodulina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteína Quinase C/metabolismo , Serina/química , Transporte Ativo do Núcleo Celular , Animais , Western Blotting , Células COS , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Citoplasma/metabolismo , Eletroforese em Gel Bidimensional , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Camundongos , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Mutação , Células NIH 3T3 , Fosforilação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Frações Subcelulares
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