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1.
Mol Cell Biol ; 39(11)2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31061093

RESUMO

GP78 is an autocrine motility factor (AMF) receptor (AMFR) with E3 ubiquitin ligase activity that plays a significant role in tumor cell proliferation, motility, and metastasis. Aberrant extracellular signal-regulated kinase (ERK) activation via receptor tyrosine kinases promotes tumor proliferation and invasion. The activation of GP78 leads to ERK activation, but its underlying mechanism is not fully understood. Here, we show that GP78 is required for epidermal growth factor receptor (EGFR)-mediated ERK activation. On one hand, GP78 interacts with and promotes the ubiquitination and subsequent degradation of dual-specificity phosphatase 1 (DUSP1), an endogenous negative regulator of mitogen-activated protein kinases (MAPKs), resulting in ERK activation. On the other hand, GP78 maintains the activation status of EGFR, as evidenced by the fact that EGF fails to induce EGFR phosphorylation in GP78-deficient cells. By the regulation of both EGFR and ERK activation, GP78 promotes cell proliferation, motility, and invasion. Therefore, this study identifies a previously unknown signaling pathway by which GP78 stimulates ERK activation via DUSP1 degradation to mediate EGFR-dependent cancer cell proliferation and invasion.


Assuntos
Carcinoma Hepatocelular/metabolismo , Fosfatase 1 de Especificidade Dupla/metabolismo , Neoplasias Hepáticas/metabolismo , Receptores do Fator Autócrino de Motilidade/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Fosfatase 1 de Especificidade Dupla/química , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Invasividade Neoplásica , Fosforilação , Proteólise , Ubiquitinação
2.
J Biol Chem ; 292(2): 539-550, 2017 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-27856639

RESUMO

Human YVH1 (hYVH1), also known as dual specificity phosphatase 12 (DUSP12), is a poorly characterized atypical dual specificity phosphatase widely conserved throughout evolution. Recent findings have demonstrated that hYVH1 expression affects cellular DNA content and is a novel cell survival phosphatase preventing both thermal and oxidative stress-induced cell death, whereas studies in yeast have established YVH1 as a novel 60S ribosome biogenesis factor. In this study, we have isolated novel hYVH1-associating proteins from human U2OS osteosarcoma cells using affinity chromatography coupled to mass spectrometry employing ion mobility separation. Numerous ribosomal proteins were identified, confirming the work done in yeast. Furthermore, proteins known to be present on additional RNP particles were identified, including Y box-binding protein 1 (YB-1) and fragile X mental retardation protein, proteins that function in translational repression and stress granule regulation. Follow-up studies demonstrated that hYVH1 co-localizes with YB-1 and fragile X mental retardation protein on stress granules in response to arsenic treatment. Interestingly, hYVH1-positive stress granules were significantly smaller, whereas knocking down hYVH1 expression attenuated stress granule breakdown during recovery from arsenite stress, indicating a possible role for hYVH1 in stress granule disassembly. These results propagate a role for dual specificity phosphatases at RNP particles and suggest that hYVH1 may affect a variety of fundamental cellular processes by regulating messenger ribonucleoprotein (mRNP) dynamics.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Fosfatase 1 de Especificidade Dupla/metabolismo , Ribonucleoproteínas/metabolismo , Arsenitos/farmacologia , Linhagem Celular Tumoral , Grânulos Citoplasmáticos/química , Fosfatase 1 de Especificidade Dupla/química , Fosfatase 1 de Especificidade Dupla/isolamento & purificação , Humanos , Ribonucleoproteínas/química , Ribonucleoproteínas/isolamento & purificação , Proteínas Ribossômicas/química , Proteínas Ribossômicas/isolamento & purificação , Proteínas Ribossômicas/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Proteína 1 de Ligação a Y-Box/química , Proteína 1 de Ligação a Y-Box/isolamento & purificação , Proteína 1 de Ligação a Y-Box/metabolismo
3.
Cancer Lett ; 320(2): 123-9, 2012 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-22425962

RESUMO

Protein S-nitrosylation is a covalent post-translational modification through coupling of a nitric oxide (NO) moiety with the reactive thiol group of a protein cysteine residue to form an S-nitrosothiol (SNO). S-nitrosylation is a key mechanism in the transmission of NO-based cellular signals in the vital cellular processes, including transcription regulation, DNA repair, and apoptosis. Contemporary research has implicated dysregulation of S-nitrosylation in severe pathological events, including cancer onset, progression, and treatment resistance. The S-nitrosylation status may be directly linked to many cancer therapy outcomes as well as therapeutic-resistance, emphasizing the need to develop S-nitrosylation-related anti-cancer therapeutics. The role of S-nitrosylated proteins in the development and progression of cancer are varied, generating a critical need for a thorough review of the current dynamic research in this area.


Assuntos
Neoplasias/genética , Óxido Nítrico/metabolismo , Processamento de Proteína Pós-Traducional , Progressão da Doença , Fosfatase 1 de Especificidade Dupla/química , Humanos , Neoplasias/metabolismo , S-Nitrosotióis/metabolismo
4.
J Comput Aided Mol Des ; 25(5): 469-75, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21567231

RESUMO

Mitogen-activated protein kinase phosphatase-1 (MKP-1) has proved to be an attractive target for the development of therapeutics for the treatment of cancer. We report the first example for a successful application of the structure-based virtual screening to identify the novel inhibitors of MKP-1. It is shown that the efficiency of virtual screening can be enhanced significantly by the incorporation of a new solvation energy term in the scoring function. The newly found inhibitors have desirable physicochemical properties as a drug candidate and reveal a moderate potency with IC(50) values ranging from 20 to 50 µM. Therefore, they deserve a consideration for further development by structure-activity relationship studies to optimize the inhibitory activities. Structural features relevant to the stabilization of the inhibitors in the active site of MKP-1 are discussed in detail.


Assuntos
Avaliação Pré-Clínica de Medicamentos , Fosfatase 1 de Especificidade Dupla/antagonistas & inibidores , Fosfatase 1 de Especificidade Dupla/química , Proteína Quinase 1 Ativada por Mitógeno/química , Modelos Moleculares , Sítios de Ligação , Domínio Catalítico , Desenho de Fármacos , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Concentração Inibidora 50 , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Estrutura Molecular , Relação Estrutura-Atividade
5.
J Am Chem Soc ; 132(33): 11392-4, 2010 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-20677743

RESUMO

We present a simple method by which gold nanoparticles (AuNPs) are used to simultaneously isolate and enrich for free or modified thiol-containing peptides, thus facilitating the identification of protein S-modification sites. Here, protein disulfide isomerase (PDI) and dual specificity phosphatase 12 (DUSP12 or hYVH1) were S-nitrosylated or S-glutathionylated, their free thiols differentially alkylated, and subjected to proteolysis. AuNPs were added to the digests, and the AuNP-bound peptides were isolated by centrifugation and released by thiol exchange. These AuNP-bound peptides were analyzed by MALDI-TOF mass spectrometry revealing that AuNPs result in a significant enrichment of free thiol-containing as well as S-nitrosylated, S-glutathionylated, and S-alkylated peptides, leading to the unequivocal assignment of thiols susceptible to modification.


Assuntos
Fosfatase 1 de Especificidade Dupla/metabolismo , Glutationa/metabolismo , Ouro/química , Nanopartículas Metálicas/química , Isomerases de Dissulfetos de Proteínas/metabolismo , S-Nitrosotióis/metabolismo , Alquilação , Sítios de Ligação , Fosfatase 1 de Especificidade Dupla/química , Glutationa/química , Humanos , Isomerases de Dissulfetos de Proteínas/química , S-Nitrosotióis/química
6.
J Biol Chem ; 284(41): 28292-28305, 2009 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-19667066

RESUMO

The calcium regulatory protein calmodulin (CaM) binds in a calcium-dependent manner to numerous target proteins. The calmodulin-binding domain (CaMBD) region of Nicotiana tabacum MAPK phosphatase has an amino acid sequence that does not resemble the CaMBD of any other known Ca(2+)-CaM-binding proteins. Using a unique fusion protein strategy, we have been able to obtain a high resolution solution structure of the complex of soybean Ca(2+)-CaM4 (SCaM4) and this CaMBD. Complete isotope labeling of both parts of the complex in the fusion protein greatly facilitated the structure determination by NMR. The 12-residue CaMBD region was found to bind exclusively to the C-lobe of SCaM4. A specific Trp and Leu side chain are utilized to facilitate strong binding through a novel "double anchor" motif. Moreover, the orientation of the helical peptide on the surface of Ca(2+)-SCaM4 is distinct from other known complexes. The N-lobe of Ca(2+)-SCaM4 in the complex remains free for additional interactions and could possibly act as a calcium-dependent adapter protein. Signaling through the MAPK pathway and increases in intracellular Ca(2+) are both hallmarks of the plant stress response, and our data support the notion that coordination of these responses may occur through the formation of a unique CaM-MAPK phosphatase multiprotein complex.


Assuntos
Calmodulina/química , Calmodulina/metabolismo , Fosfatase 1 de Especificidade Dupla/química , Fosfatase 1 de Especificidade Dupla/metabolismo , Glycine max/metabolismo , Nicotiana/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Animais , Calmodulina/genética , Fosfatase 1 de Especificidade Dupla/genética , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência
7.
J Biol Chem ; 284(34): 22853-64, 2009 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-19567874

RESUMO

YVH1 was one of the first eukaryotic dual specificity phosphatases cloned, and orthologues poses a unique C-terminal zinc-coordinating domain in addition to a cysteine-based phosphatase domain. Our recent results revealed that human YVH1 (hYVH1) protects cells from oxidative stress. This function requires phosphatase activity and the zinc binding domain. This current study provides evidence that the thiol-rich zinc-coordinating domain may act as a redox sensor to impede the active site cysteine from inactivating oxidation. Furthermore, using differential thiol labeling and mass spectrometry, it was determined that hYVH1 forms intramolecular disulfide bonds at the catalytic cleft as well as within the zinc binding domain to avoid irreversible inactivation during severe oxidative stress. Importantly, zinc ejection is readily reversible and required for hYVH1 activity upon returning to favorable conditions. This inimitable mechanism provides a means for hYVH1 to remain functionally responsive for protecting cells during oxidative stimuli.


Assuntos
Dissulfetos/química , Fosfatase 1 de Especificidade Dupla/química , Fosfatase 1 de Especificidade Dupla/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Fosfatase 1 de Especificidade Dupla/genética , Regulação Enzimológica da Expressão Gênica , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Ligação Proteica , Conformação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Zinco/metabolismo
9.
Am J Physiol Cell Physiol ; 296(2): C242-9, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19020052

RESUMO

Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is a nuclear, dual-specificity phosphatase that has been shown to dephosphorylate MAP kinases. We used a "substrate-trap" technique involving a mutation in MKP-1 of the catalytically critical cysteine to a serine residue ("CS" mutant) to capture novel MKP-1 substrates. We transfected the MKP-1 (CS) mutant and control (wild-type, WT) constructs into phorbol 12-myristate 13-acetate (PMA)-activated COS-1 cells. MKP-1-substrate complexes were immunoprecipitated, which yielded four bands of 17, 15, 14, and 10 kDa with the CS MKP-1 mutant but not the WT MKP-1. The bands were identified by mass spectrometry as histones H3, H2B, H2A, and H4, respectively. Histone H3 was phosphorylated, and purified MKP-1 dephosphorylated histone H3 (phospho-Ser-10) in vitro; whereas, histone H3 (phospho-Thr-3) was unaffected. We have previously shown that thrombin and vascular endothelial growth factor (VEGF) upregulated MKP-1 in human endothelial cells (EC). We now show that both thrombin and VEGF caused dephosphorylation of histone H3 (phospho-Ser-10) and histone H3 (phospho-Thr-3) in EC with kinetics consistent with MKP-1 induction. Furthermore, MKP-1-specific small interfering RNA (siRNA) prevented VEGF- and thrombin-induced H3 (phospho-Ser-10) dephosphorylation but had no effect on H3 (phospho-Thr-3 or Thr-11) dephosphorylation. In summary, histone H3 is a novel substrate of MKP-1, and VEGF- and thrombin-induced H3 (phospho-Ser-10) dephosphorylation requires MKP-1. We propose that MKP-1-mediated H3 (phospho-Ser-10) dephosphorylation is a key regulatory step in EC activation by VEGF and thrombin.


Assuntos
Fosfatase 1 de Especificidade Dupla/metabolismo , Células Endoteliais/enzimologia , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Células COS , Domínio Catalítico , Chlorocebus aethiops , Fosfatase 1 de Especificidade Dupla/química , Fosfatase 1 de Especificidade Dupla/genética , Células Endoteliais/efeitos dos fármacos , Epigênese Genética , Histonas/química , Humanos , Imunoprecipitação , Peso Molecular , Mutação , Fragmentos de Peptídeos/farmacologia , Fosforilação , Ligação Proteica , Interferência de RNA , Serina , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Trombina/metabolismo , Fatores de Tempo , Transfecção , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
Curr Med Chem ; 15(25): 2536-44, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18855677

RESUMO

Dual-specificity phosphatases (DSPs) are important, but poorly understood, cell signaling enzymes that remove phosphate groups from tyrosine and serine/threonine residues on their substrate. Deregulation of DSPs has been implicated in cancer, obesity, diabetes, inflammation, and Alzheimer's disease. Due to their biological and biomedical significance, DSPs have increasingly become the subject of drug discovery high-throughput screening (HTS) and focused compound library development efforts. Progress in identifying selective and potent DSP inhibitors has, however, been restricted by the lack of sufficient structural data on inhibitor-bound DSPs. The shallow, almost flat, substrate binding sites in DSPs have been a major factor in hampering the rational design and the experimental development of active site inhibitors. Recent experimental and virtual HTS studies, as well as advances in molecular modeling, provide new insights into the potential mechanisms for substrate recognition and binding by this important class of enzymes. We present herein an overview of the progress, along with a brief description of applications to two types of DSPs: Cdc25 and MAP kinase phosphatase (MKP) family members. In particular, we focus on combined computational and experimental efforts for designing Cdc25B and MKP-1 inhibitors and understanding their mechanisms of interactions with their target proteins. These studies emphasize the utility of developing computational models and methods that meet the two major challenges currently faced in structure-based in silico design of lead compounds: the conformational flexibility of the target protein and the entropic contribution to the selection and stabilization of particular bound conformers.


Assuntos
Técnicas de Química Combinatória/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Fosfatases de Especificidade Dupla/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Algoritmos , Sequência de Aminoácidos , Sítios de Ligação , Fosfatase 1 de Especificidade Dupla/antagonistas & inibidores , Fosfatase 1 de Especificidade Dupla/química , Fosfatase 1 de Especificidade Dupla/metabolismo , Fosfatases de Especificidade Dupla/química , Fosfatases de Especificidade Dupla/metabolismo , Inibidores Enzimáticos/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Fosfatases cdc25/antagonistas & inibidores , Fosfatases cdc25/química , Fosfatases cdc25/metabolismo
11.
J Biol Chem ; 283(35): 23581-8, 2008 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-18579522

RESUMO

The mitogen-activated protein kinases (MAPKs) are key signal transduction molecules, which respond to various external stimuli. The MAPK phosphatases (MKPs) are known to be negative regulators of MAPKs in eukaryotes. We screened an Arabidopsis cDNA library using horseradish peroxidase-conjugated calmodulin (CaM), and isolated AtMKP1 as a CaM-binding protein. Recently, tobacco NtMKP1 and rice OsMKP1, two orthologs of Arabidopsis AtMKP1, were reported to bind CaM via a single putative CaM binding domain (CaMBD). However, little is known about the regulation of phosphatase activity of plant MKP1s by CaM binding. In this study, we identified two Ca(2+)-dependent CaMBDs within AtMKP1. Specific binding of CaM to two different CaMBDs was verified using a gel mobility shift assay, a competition assay with a Ca(2+)/CaM-dependent enzyme, and a split-ubiquitin assay. The peptides for two CaMBDs, CaMBDI and CaMBDII, bound CaM in a Ca(2+)-dependent manner, and the binding affinity of CaMBDII was found to be higher than that of CaMBDI. CaM overlay assays using mutated CaMBDs showed that four amino acids, Trp(453) and Leu(456) in CaMBDI and Trp(678) and Ile(684) in CaMBDII, play a pivotal role in CaM binding. Moreover, the phosphatase activity of AtMKP1 was increased by CaM in a Ca(2+)-dependent manner. Our results suggest that two important signaling pathways, Ca(2+) signaling and the MAPK signaling cascade, are connected in plants via the regulation of AtMKP1 activity. To our knowledge, this is the first report to show that the biochemical activity of MKP1 in plants is regulated by CaM.


Assuntos
Arabidopsis/enzimologia , Sinalização do Cálcio/fisiologia , Calmodulina/metabolismo , Fosfatase 1 de Especificidade Dupla/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Arabidopsis/genética , Cálcio/química , Cálcio/metabolismo , Calmodulina/química , Calmodulina/genética , Clonagem Molecular , Fosfatase 1 de Especificidade Dupla/química , Fosfatase 1 de Especificidade Dupla/genética , MAP Quinases Reguladas por Sinal Extracelular/química , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Biblioteca Gênica , Mutação , Oryza/enzimologia , Oryza/genética , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Nicotiana/enzimologia , Nicotiana/genética
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