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1.
EMBO J ; 33(16): 1784-801, 2014 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-24975362

RESUMO

Mitogen-activated protein kinases (MAPKs) are highly conserved protein kinase modules, and they control fundamental cellular processes. While the activation of MAPKs has been well studied, little is known on the mechanisms driving their inactivation. Here we uncover a role for ubiquitination in the inactivation of a MAPK module. Extracellular-signal-regulated kinase 5 (ERK5) is a unique, conserved member of the MAPK family and is activated in response to various stimuli through a three-tier cascade constituting MEK5 and MEKK2/3. We reveal an unexpected role for Inhibitors of Apoptosis Proteins (IAPs) in the inactivation of ERK5 pathway in a bimodal manner involving direct interaction and ubiquitination. XIAP directly interacts with MEKK2/3 and competes with PB1 domain-mediated binding to MEK5. XIAP and cIAP1 conjugate predominantly K63-linked ubiquitin chains to MEKK2 and MEKK3 which directly impede MEK5-ERK5 interaction in a trimeric complex leading to ERK5 inactivation. Consistently, loss of XIAP or cIAP1 by various strategies leads to hyperactivation of ERK5 in normal and tumorigenic cells. Loss of XIAP promotes differentiation of human primary skeletal myoblasts to myocytes in a MEKK2/3-ERK5-dependent manner. Our results reveal a novel, obligatory role for IAPs and ubiquitination in the physical and functional disassembly of ERK5-MAPK module and human muscle cell differentiation.


Assuntos
Proteínas Inibidoras de Apoptose/metabolismo , MAP Quinase Quinase 5/metabolismo , MAP Quinase Quinase Quinase 3/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Ubiquitina/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA , Ativação Enzimática , Humanos , Proteínas Inibidoras de Apoptose/genética , MAP Quinase Quinase 5/genética , MAP Quinase Quinase Quinase 2 , MAP Quinase Quinase Quinase 3/genética , MAP Quinase Quinase Quinases/genética , Fatores de Transcrição MEF2/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/genética , Mioblastos/citologia , Mioblastos/metabolismo , Proteínas Nucleares/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Ubiquitinação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética
2.
EMBO J ; 31(1): 14-28, 2012 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-22117219

RESUMO

Inhibitors of apoptosis proteins (IAPs) are a highly conserved class of multifunctional proteins. Rac1 is a well-studied Rho GTPase that controls numerous basic cellular processes. While the regulation of nucleotide binding to Rac1 is well understood, the molecular mechanisms controlling Rac1 degradation are not known. Here, we demonstrate X-linked IAP (XIAP) and cellular IAP1 (c-IAP1) directly bind to Rac1 in a nucleotide-independent manner to promote its polyubiquitination at Lys147 and proteasomal degradation. These IAPs are also required for degradation of Rac1 upon CNF1 toxin treatment or RhoGDI depletion. Consistently, downregulation of XIAP or c-IAP1 by various strategies led to an increase in Rac1 protein levels in primary and tumour cells, leading to an elongated morphology and enhanced cell migration. Further, XIAP counteracts Rac1-dependent cellular polarization in the developing zebrafish hindbrain and promotes the delamination of neurons from the normal tissue architecture. These observations unveil an evolutionarily conserved role of IAPs in controlling Rac1 stability thereby regulating the plasticity of cell migration and morphogenesis.


Assuntos
Movimento Celular/fisiologia , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Apoptose , Células HeLa , Humanos , Ubiquitinação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Peixe-Zebra
3.
Exp Cell Res ; 317(4): 496-503, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21087603

RESUMO

Deleted in liver cancer 1 (DLC1) is a tumor suppressor protein that is frequently downregulated in various tumor types. DLC1 contains a Rho GTPase activating protein (GAP) domain that appears to be required for its tumor suppressive functions. Little is known about the molecular mechanisms that regulate DLC1. By mass spectrometry we have mapped a novel phosphorylation site within the DLC1 GAP domain on serine 807. Using a phospho-S807-specific antibody, our results identify protein kinase D (PKD) to phosphorylate this site in DLC1 in intact cells. Although phosphorylation on serine 807 did not directly impact on in vitro GAP activity, a DLC1 serine-to-alanine exchange mutant inhibited colony formation more potently than the wild type protein. Our results thus show that PKD-mediated phosphorylation of DLC1 on serine 807 negatively regulates DLC1 cellular function.


Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Proteína Quinase C/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Espectrometria de Massas , Fosforilação
4.
Mol Cell Biol ; 25(17): 7494-504, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16107698

RESUMO

Dbf4/Cdc7 is required for DNA replication in Saccharomyces cerevisiae and appears to be a target in the S-phase checkpoint. Previously, a 186-amino-acid Dbf4 region that mediates interactions with both the origin recognition complex and Rad53 was identified. We now show this domain also mediates the association between Dbf4 and Mcm2, a key Dbf4/Cdc7 phosphorylation target. Two conserved sequences, the N and M motifs, have been identified within this Dbf4 region. Removing motif M (Dbf4DeltaM) impairs the ability of Dbf4 to support normal cell cycle progression and abrogates the Dbf4-Mcm2 association but has no effect on the Dbf4-Rad53 interaction. In contrast, deleting motif N (Dbf4DeltaN) does not affect the essential function of Dbf4, disrupts the Dbf4-Rad53 interaction, largely preserves the Dbf4-Mcm2 association, and renders the cells hypersensitive to genotoxic agents. Surprisingly, Dbf4DeltaM interacts strongly with Orc2, while Dbf4DeltaN does not. The DBF4 allele dna52-1 was cloned and sequenced, revealing a single point mutation within the M motif. This mutant is unable to maintain interactions with either Mcm2 or Orc2 at the semipermissive temperature of 30 degrees C, while the interaction with Rad53 is preserved. Furthermore, this mutation confers increased resistance to genotoxic agents, which we propose is more likely due to a role for Dbf4 in the resumption of fork progression following checkpoint-induced arrest than prevention of late origin firing. Thus, the alteration of the M motif may facilitate the role of Dbf4 as a checkpoint target.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , DNA Fúngico/genética , Mutagênicos/farmacologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Cromossômicas não Histona , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Farmacorresistência Fúngica , Proteínas Fúngicas/metabolismo , Hidroxiureia/farmacologia , Metanossulfonato de Metila/farmacologia , Mutação/efeitos dos fármacos , Mutação/genética , Complexo de Reconhecimento de Origem , Ligação Proteica , Saccharomyces cerevisiae/efeitos dos fármacos , Técnicas do Sistema de Duplo-Híbrido
5.
Cancer Cell ; 23(5): 603-17, 2013 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-23680147

RESUMO

The human epidermal growth factor receptor (HER) family of tyrosine kinases is deregulated in multiple cancers either through amplification, overexpression, or mutation. ERBB3/HER3, the only member with an impaired kinase domain, although amplified or overexpressed in some cancers, has not been reported to carry oncogenic mutations. Here, we report the identification of ERBB3 somatic mutations in ~11% of colon and gastric cancers. We found that the ERBB3 mutants transformed colonic and breast epithelial cells in a ligand-independent manner. However, the mutant ERBB3 oncogenic activity was dependent on kinase-active ERBB2. Furthermore, we found that anti-ERBB antibodies and small molecule inhibitors effectively blocked mutant ERBB3-mediated oncogenic signaling and disease progression in vivo.


Assuntos
Neoplasias do Colo/genética , Mutação , Receptor ErbB-3/genética , Neoplasias Gástricas/genética , Sítios de Ligação , Proliferação de Células , Sobrevivência Celular/genética , Transformação Celular Neoplásica/genética , Técnicas de Silenciamento de Genes , Humanos , Modelos Moleculares , Estrutura Terciária de Proteína , Receptor ErbB-3/metabolismo , Receptor ErbB-3/fisiologia
6.
Mol Biol Cell ; 22(5): 570-80, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21209314

RESUMO

The multifunctional signal adapter protein Ras and Rab interactor 1 (RIN1) is a Ras effector protein involved in the regulation of epithelial cell processes such as cell migration and endocytosis. RIN1 signals via two downstream pathways, namely the activation of Rab5 and Abl family kinases. Protein kinase D (PKD) phosphorylates RIN1 at serine 351 in vitro, thereby regulating interaction with 14-3-3 proteins. Here, we report the identification of serine 292 in RIN1 as an in vivo PKD phosphorylation site. PKD-mediated phosphorylation at this site was confirmed with a phospho-specific antibody and by mass spectrometry. We demonstrate that phosphorylation at serine 292 controls RIN1-mediated inhibition of cell migration by modulating the activation of Abl kinases. We further provide evidence that RIN1 in vivo phosphorylation at serine 351 occurs independently of PKD. Collectively, our data identify a novel PKD signaling pathway through RIN1 and Abl kinases that is involved in the regulation of actin remodeling and cell migration.


Assuntos
Movimento Celular , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Quinase C/metabolismo , Actinas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Anticorpos/imunologia , Células HEK293 , Humanos , Dados de Sequência Molecular , Fosforilação , Fosfosserina/metabolismo , Transporte Proteico , Proteínas Tirosina Quinases/metabolismo , Frações Subcelulares/metabolismo , Proteínas Supressoras de Tumor/metabolismo
7.
J Cell Sci ; 122(Pt 1): 92-102, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19066281

RESUMO

Deleted in liver cancer 1 (DLC1) is a Rho-GTPase-activating protein (GAP) that is downregulated in various tumor types. In vitro, DLC1 specifically inactivates the small GTPases RhoA, RhoB and RhoC through its GAP domain and this appears to contribute to its tumor suppressor function in vivo. Molecular mechanisms that control DLC1 activity have not so far been investigated. Here, we show that phorbol-ester-induced activation of protein kinase C and protein kinase D stimulates association of DLC1 with the phosphoserine/phosphothreonine-binding 14-3-3 adaptor proteins via recognition motifs that involve Ser327 and Ser431. Association with 14-3-3 proteins inhibits DLC1 GAP activity and facilitates signaling by active Rho. We further show that treatment of cells with phorbol ester or coexpression of 14-3-3 proteins, blocks DLC1 nucleocytoplasmic shuttling, probably by masking a previously unrecognized nuclear localization sequence. The binding to 14-3-3 proteins is thus a newly discovered mechanism by which DLC1 activity is regulated and compartmentalized.


Assuntos
Proteínas 14-3-3/metabolismo , Núcleo Celular/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas 14-3-3/genética , Transporte Ativo do Núcleo Celular/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Ativação Enzimática , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Humanos , Dados de Sequência Molecular , Ésteres de Forbol/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Proteína Quinase C/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo , Proteínas Supressoras de Tumor/genética
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