RESUMO
We developed cProSite, a website that provides online genomics, proteomics, and phosphoproteomics analysis for the data of The National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC). This tool focuses on comparisons and correlations between different proteins and mRNAs of tumors and normal tissues. Our website is designed with biologists and clinicians in mind, with a user-friendly environment and fast search engine. The search results of cProSite can be used for clinical data validation and provide useful strategic information to identify drug targets at proteomic, phosphoproteomic, or genomic levels. The site is available at http://cprosite.ccr.cancer.gov.
RESUMO
mRNA expression of the DLC1 tumor suppressor gene is downregulated in many lung cancers and their derived cell lines, with DLC1 protein levels being low or absent. Although the role of increased EZH2 methyltransferase in cancer is usually attributed to its histone methylation, we unexpectedly observed that post-translational destabilization of DLC1 protein is common and attributable to its methylation by cytoplasmic EZH2, leading to CUL-4A ubiquitin-dependent proteasomal degradation of DLC1. Furthermore, siRNA knockdown of KRAS in several lines increases DLC1 protein, associated with a drastic reduction in cytoplasmic EZH2. Pharmacologic inhibition of EZH2, CUL-4A, or the proteasome can increase the steady-state level of DLC1 protein, whose tumor suppressor activity is further increased by AKT and/or SRC kinase inhibitors, which reverse the direct phosphorylation of DLC1 by these kinases. These rational drug combinations induce potent tumor growth inhibition, with markers of apoptosis and senescence, that is highly dependent on DLC1 protein.
Assuntos
Antineoplásicos/farmacologia , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteínas Ativadoras de GTPase/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Supressoras de Tumor/metabolismo , Animais , Antineoplásicos/uso terapêutico , Benzodioxóis/farmacologia , Benzodioxóis/uso terapêutico , Compostos de Boro/farmacologia , Compostos de Boro/uso terapêutico , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteínas Ativadoras de GTPase/genética , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Glicina/análogos & derivados , Glicina/farmacologia , Glicina/uso terapêutico , Células HEK293 , Compostos Heterocíclicos com 3 Anéis/farmacologia , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Mutagênese Sítio-Dirigida , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Estabilidade Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In advanced cancer, the RHOA GTPase is often active together with reduced expression of genes encoding Rho-specific GTPase-accelerating proteins (Rho-GAP), which negatively regulate RHOA and related GTPases. Here we used the The Cancer Genome Atlas dataset to examine 12 tumor types (including colon, breast, prostate, pancreas, lung adenocarcinoma, and squamous cell carcinoma) for the frequency of codon mutations of 10 Rho-GAP and experimentally tested biochemical and biological consequences for cancer-associated mutants that arose in the DLC1 tumor suppressor gene. DLC1 was the Rho-GAP gene mutated most frequently, with 5%-8% of tumors in five of the tumor types evaluated having DLC1 missense mutations. Furthermore, 20%-26% of the tumors in four of these five tumor types harbored missense mutations in at least one of the 10 Rho-GAPs. Experimental analysis of the DLC1 mutants indicated 7 of 9 mutants whose lesions were located in the Rho-GAP domain were deficient for Rho-GAP activity and for suppressing cell migration and anchorage-independent growth. Analysis of a DLC1 linker region mutant and a START domain mutant showed each was deficient for suppressing migration and growth in agar, but their Rho-GAP activity was similar to that of wild-type DLC1. Compared with the wild-type, the linker region mutant bound 14-3-3 proteins less efficiently, while the START domain mutant displayed reduced binding to Caveolin-1. Thus, mutation of Rho-GAP genes occurs frequently in some cancer types and the majority of cancer-associated DLC1 mutants evaluated were deficient biologically, with various mechanisms contributing to their reduced activity. SIGNIFICANCE: These findings indicate that point mutation of Rho-GAP genes is unexpectedly frequent in several cancer types, with DLC1 mutants exhibiting reduced function by various mechanisms.
Assuntos
Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular Tumoral , Humanos , Mutação de Sentido Incorreto , Mutação PuntualRESUMO
SRC and ERK kinases control many cell biological processes that promote tumorigenesis by altering the activity of oncogenic and tumor suppressor proteins. We identify here a physiological interaction between DLC1, a focal adhesion protein and tumor suppressor, with SRC and ERK. The tumor suppressor function of DLC1 is attenuated by phosphorylation of tyrosines Y451 and Y701 by SRC, which down-regulates DLC1's tensin-binding and Rho-GAP activities. ERK1/2 phosphorylate DLC1 on serine S129, which increases both the binding of SRC to DLC1 and SRC-dependent phosphorylation of DLC1. SRC inhibitors exhibit potent antitumor activity in a DLC1-positive transgenic cancer model and a DLC1-positive tumor xenograft model, due to reactivation of the tumor suppressor activities of DLC1. Combined treatment of DLC1-positive tumors with SRC plus AKT inhibitors has even greater antitumor activity. Together, these findings indicate cooperation between the SRC, ERK1/2, and AKT kinases to reduce DLC1 Rho-GAP and tumor suppressor activities in cancer cells, which can be reactivated by the kinase inhibitors.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias Experimentais/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Quinases da Família src/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Transplante de Neoplasias , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Supressoras de Tumor/genética , Quinases da Família src/genéticaRESUMO
No efficient treatment exists for nephrotic syndrome (NS), a frequent cause of chronic kidney disease. Here we show mutations in six different genes (MAGI2, TNS2, DLC1, CDK20, ITSN1, ITSN2) as causing NS in 17 families with partially treatment-sensitive NS (pTSNS). These proteins interact and we delineate their roles in Rho-like small GTPase (RLSG) activity, and demonstrate deficiency for mutants of pTSNS patients. We find that CDK20 regulates DLC1. Knockdown of MAGI2, DLC1, or CDK20 in cultured podocytes reduces migration rate. Treatment with dexamethasone abolishes RhoA activation by knockdown of DLC1 or CDK20 indicating that steroid treatment in patients with pTSNS and mutations in these genes is mediated by this RLSG module. Furthermore, we discover ITSN1 and ITSN2 as podocytic guanine nucleotide exchange factors for Cdc42. We generate Itsn2-L knockout mice that recapitulate the mild NS phenotype. We, thus, define a functional network of RhoA regulation, thereby revealing potential therapeutic targets.
Assuntos
Resistência a Medicamentos/genética , Glucocorticoides/farmacologia , Síndrome Nefrótica/tratamento farmacológico , Mapas de Interação de Proteínas/genética , Proteína rhoA de Ligação ao GTP/genética , Adulto , Animais , Criança , Pré-Escolar , Análise Mutacional de DNA , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Glucocorticoides/uso terapêutico , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Mutação , Síndrome Nefrótica/genética , Linhagem , Podócitos , RNA Interferente Pequeno/metabolismo , Resultado do Tratamento , Sequenciamento do Exoma , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
We report several receptor tyrosine kinase (RTK) ligands increase RhoA-guanosine triphosphate (GTP) in untransformed and transformed cell lines and determine this phenomenon depends on the RTKs activating the AKT serine/threonine kinase. The increased RhoA-GTP results from AKT phosphorylating three serines (S298, S329, and S567) in the DLC1 tumor suppressor, a Rho GTPase-activating protein (RhoGAP) associated with focal adhesions. Phosphorylation of the serines, located N-terminal to the DLC1 RhoGAP domain, induces strong binding of that N-terminal region to the RhoGAP domain, converting DLC1 from an open, active dimer to a closed, inactive monomer. That binding, which interferes with the interaction of RhoA-GTP with the RhoGAP domain, reduces the hydrolysis of RhoA-GTP, the binding of other DLC1 ligands, and the colocalization of DLC1 with focal adhesions and attenuates tumor suppressor activity. DLC1 is a critical AKT target in DLC1-positive cancer because AKT inhibition has potent antitumor activity in the DLC1-positive transgenic cancer model and in a DLC1-positive cancer cell line but not in an isogenic DLC1-negative cell line.
Assuntos
Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Adesões Focais/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Sítios de Ligação , Linhagem Celular , Movimento Celular , Células Epiteliais/ultraestrutura , Fibroblastos/ultraestrutura , Adesões Focais/ultraestrutura , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica , Guanosina Trifosfato/metabolismo , Células HEK293 , Células HeLa , Humanos , Hidrólise , Cristalino , Fosforilação , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Maintaining genomic integrity is of paramount importance to embryonic stem cells (ESCs), as mutations are readily propagated to daughter cells. ESCs display hypersensitivity to DNA damage-induced apoptosis (DIA) to prevent such propagation, although the molecular mechanisms underlying this apoptotic response are unclear. Here, we report that the regulatory RNA Apela positively regulates p53-mediated DIA. Apela is highly expressed in mouse ESCs and is repressed by p53 activation, and Apela depletion compromises p53-dependent DIA. Although Apela contains a coding region, this coding ability is dispensable for Apela's role in p53-mediated DIA. Instead, Apela functions as a regulatory RNA and interacts with hnRNPL, which prevents the mitochondrial localization and activation of p53. Together, these results describe a tri-element negative feedback loop composed of p53, Apela, and hnRNPL that regulates p53-mediated DIA, and they further demonstrate that regulatory RNAs add a layer of complexity to the apoptotic response of ESCs after DNA damage.
Assuntos
Apoptose , Retroalimentação Fisiológica , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , RNA/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Apoptose/genética , Sequência de Bases , Dano ao DNA , Elementos Facilitadores Genéticos/genética , Loci Gênicos , Ribonucleoproteínas Nucleares Heterogêneas/química , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Camundongos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Ligação Proteica/genéticaRESUMO
Cyclin dependent kinase 5 (Cdk5), a proline-directed serine/threonine kinase, requires p39 for its enzymatic activity, and is implicated in cytoskeletal organization and contraction in numerous cell types. The C-terminus of p39 binds muskelin, a multi-domain scaffolding protein known to affect cytoskeletal organization, but the mechanisms by which muskelin affects cytoskeletal organization remain unclear. The present study sought to determine whether p39 might serve as an adaptor protein that links muskelin to stress fibers and to investigate the possible biological relevance of such an interaction. Double immunoprecipitation showed that muskelin, p39, and myosin II are components of a single intracellular complex, and suppressing p39 abrogated the interaction between muskelin and the myosin subunits, demonstrating that p39 is required to link muskelin to myosin II. Muskelin is colocalized with myosin regulatory light chain (MRLC) and on stress fibers. The suppression of muskelin reduced Rho-GTP, MRLC phosphorylation, disrupted stress fiber organization, and promoted cell migration, all of which closely mimic the effect of Cdk5 inhibition. Moreover, suppressing muskelin and inhibiting Cdk5 together have no additional effect, indicating that muskelin plays an important role in Cdk5-dependent signaling. p39 is necessary and sufficient for Cdk5-dependent regulation of MRLC phosphorylation, as suppression of p39, but not p35, reduces MRLC phosphorylation. Together, these results demonstrate that p39 specifically links muskelin to myosin II and consequently, to stress fibers and reveal a novel role for muskelin in regulating myosin phosphorylation and cytoskeletal organization.
Assuntos
Actinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cristalino/metabolismo , Miosina Tipo II/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fibras de Estresse/metabolismo , Sítios de Ligação , Linhagem Celular , Humanos , Cadeias Leves de Miosina/metabolismo , Proteínas do Tecido Nervoso/química , Fosforilação , Ligação Proteica , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
DLC1 is a tumor suppressor protein whose full activity depends on its presence at focal adhesions, its Rho-GTPase activating protein (Rho-GAP) function, and its ability to bind several ligands, including tensin and talin. However, the mechanisms that regulate and coordinate these activities remain poorly understood. Here we identify CDK5, a predominantly cytoplasmic serine/threonine kinase, as an important regulator of DLC1 functions. The CDK5 kinase phosphorylates four serines in DLC1 located N-terminal to the Rho-GAP domain. When not phosphorylated, this N-terminal region functions as an autoinhibitory domain that places DLC1 in a closed, inactive conformation by efficiently binding to the Rho-GAP domain. CDK5 phosphorylation reduces this binding and orchestrates the coordinate activation DLC1, including its localization to focal adhesions, its Rho-GAP activity, and its ability to bind tensin and talin. In cancer, these anti-oncogenic effects of CDK5 can provide selective pressure for the down-regulation of DLC1, which occurs frequently in tumors, and can contribute to the pro-oncogenic activity of CDK5 in lung adenocarcinoma.
Assuntos
Quinase 5 Dependente de Ciclina/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Adesões Focais/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Humanos , Camundongos , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
PURPOSE: Although cyclin-dependent kinase 5 (Cdk5) inhibits the formation of junctions containing N-cadherin, the effect of Cdk5 on junctions containing E-cadherin is less clear. The present study investigates the functional significance of Cdk5 in forming and maintaining cell-cell stability in corneal epithelial cells. METHODS: A Cdk5-deficient human corneal limbal epithelial cell line was generated by lentiviral transduction of small hairpin RNA specific for Cdk5 (shCdk5-HCLE cells). A blasticidin-inducible vector for expression of Cdk5-specific short hairpin RNA (ShCdk5) was generated by recombination and packaged into non-replicative lentiviral particles for transduction of human corneal limbal epithelial (HCLE) cells. Blasticidin-resistant cells were isolated for analysis. Cell aggregations were performed using HCLE, Cdk5 inhibitor olomoucine, ShCdk5, and MDA-MB 231 cells in the presence and absence of calcium, and particle size was measured using image analysis software. Relative protein concentrations were measured with immunoblotting and quantitative densitometry. Total internal reflection fluorescence (TIRF) microscopy was performed on cells transfected with green fluorescent protein (GFP)-E-cadherin or GFP-p120, and internalization of boundary-localized proteins was analyzed with particle tracking software. The stability of surface-exposed proteins was determined by measuring the recovery of biotin-labeled proteins with affinity chromatography. Rho and Rac activity was measured with affinity chromatography and immunoblotting. RESULTS: Examining the effect of Cdk5 on E-cadherin containing epithelial cell-cell adhesions using a corneal epithelial cell line (HCLE), we found that Cdk5 and Cdk5 (pY15) coimmunoprecipitate with E-cadherin and Cdk5 (pY15) colocalizes with E-cadherin at cell-cell junctions. Inhibiting Cdk5 activity in HCLE or suppressing Cdk5 expression in a stable HCLE-derived cell line (ShHCLE) decreased calcium-dependent cell adhesion, promoted the cytoplasmic localization of E-cadherin, and accelerated the loss of surface-biotinylated E-cadherin. TIRF microscopy of GFP-E-cadherin in transfected HCLE cells showed an actively internalized sub-population of E-cadherin, which was not bound to p120 as it was trafficked away from the cell-cell boundary. This population increased in the absence of Cdk5 activity, suggesting that Cdk5 inhibition promotes dissociation of p120/E-cadherin junctional complexes. These effects of Cdk5 inhibition or suppression were accompanied by decreased Rac activity, increased Rho activity, and enhanced binding of E-cadherin to the Rac effector Ras GTPase-activating-like protein (IQGAP1). Cdk5 inhibition also reduced adhesion in a cadherin-deficient cell line (MDA-MB-231) expressing exogenous E-cadherin, although Cdk5 inhibition promoted adhesion when these cells were transfected with N-cadherin, as previous studies of Cdk5 and N-cadherin predicted. Moreover, Cdk5 inhibition induced N-cadherin expression and formation of N-cadherin/p120 complexes in HCLE cells. CONCLUSIONS: These results indicate that loss of Cdk5 activity destabilizes junctional complexes containing E-cadherin, leading to internalization of E-cadherin and upregulation of N-cadherin. Thus, Cdk5 activity promotes stability of E-cadherin-based cell-cell junctions and inhibits the E-cadherin-to-N-cadherin switch typical of epithelial-mesenchymal transitions.
Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Epitélio Corneano/citologia , Epitélio Corneano/metabolismo , Junções Intercelulares/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Adesão Celular , Linhagem Celular , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Quinase 5 Dependente de Ciclina/fisiologia , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Transição Epitelial-Mesenquimal , Humanos , Limbo da Córnea/citologia , Limbo da Córnea/metabolismo , Microscopia de Fluorescência , RNA Interferente Pequeno/genéticaRESUMO
BACKGROUND: Lens degeneration in Fpr1(-/-) mice prompted us to search for functional FPR1 expression directly on lens epithelial cells. RESULTS: FPR1 is functionally expressed on human lens epithelial cells but has atypical properties compared with hematopoietic cell FPR1. CONCLUSION: Lens epithelial cell FPR1 may be involved in development and maintenance of the lens. SIGNIFICANCE: This is the first link between non-hematopoietic expression of FPR1 and an ophthalmologic phenotype. Formyl peptide receptor 1 (FPR1) is a G protein-coupled chemoattractant receptor expressed mainly on leukocytes. Surprisingly, aging Fpr1(-/-) mice develop spontaneous lens degeneration without inflammation or infection (J.-L. Gao et al., manuscript in preparation). Therefore, we hypothesized that FPR1 is functionally expressed directly on lens epithelial cells, the only cell type in the lens. Consistent with this, the human fetal lens epithelial cell line FHL 124 expressed FPR1 mRNA and was strongly FPR1 protein-positive by Western blot and FACS. Competition binding using FPR1 ligands N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys (Nle = Norleucine), formylmethionylleucylphenylalanine, and peptide W revealed the same profile for FHL 124 cells, neutrophils, and FPR1-transfected HEK 293 cells. Saturation binding with fluorescein-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys revealed ~2500 specific binding sites on FHL-124 cells (K(D) ~ 0.5 nm) versus ~40,000 sites on neutrophils (K(D) = 3.2 nm). Moreover, formylmethionylleucylphenylalanine induced pertussis toxin-sensitive Ca(2+) flux in FHL 124 cells, consistent with classic G(i)-mediated FPR1 signaling. FHL 124 cell FPR1 was atypical in that it resisted agonist-induced internalization. Expression of FPR1 was additionally supported by detection of the intact full-length open reading frame in sequenced cDNA from FHL 124 cells. Thus, FHL-124 cells express functional FPR1, which is consistent with a direct functional role for FPR1 in the lens, as suggested by the phenotype of Fpr1 knock-out mice.
Assuntos
Células Epiteliais/metabolismo , Cristalino/metabolismo , Receptores de Formil Peptídeo/genética , Animais , Linhagem Celular , Humanos , Camundongos , Camundongos Knockout , Receptores de Formil Peptídeo/metabolismoRESUMO
The tumor suppressor gene deleted in liver cancer-1 (DLC1), which encodes a protein with strong RhoGAP (GTPase activating protein) activity and weak Cdc42GAP activity, is inactivated in various human malignancies. Following Dlc1 inactivation, mouse embryo fibroblasts (MEF) with a conditional Dlc1 knockout allele reproducibly underwent neoplastic transformation. In addition to inactivation of Dlc1 and increased activity of Rho and Cdc42, transformation depended on the subsequent decreased expression of the Cdk4/6 inhibitors p15(Ink4b) and p16(Ink4a) together with increased expression and activation of Cdk4/6. The level of expression of these cell-cycle regulatory genes was relevant to human tumors with low DLC1 expression. Analysis of publicly available annotated datasets of lung and colon cancer with gene expression microarray profiles indicated that, in pairwise comparisons, low DLC1 expression occurred frequently together (P < 0.01) with downregulation of p15(Ink4b) or p16(Ink4a) or upregulation of CDK4 or CDK6. In addition, an unfavorable prognosis (P < 0.05) was associated with low DLC1 and low p15(Ink4b) in lung cancer and colon cancer, low DLC1 and low p16(Ink4a) in lung cancer, low DLC1 and high CDK4 in lung cancer, and low DLC1 and high CDK6 in colon cancer. Thus, several genes and biochemical activities collaborate with the inactivation of DLC1 to give rise to cell transformation in MEFs, and the identified genes are relevant to human tumors with low DLC1 expression.
Assuntos
Transformação Celular Neoplásica/genética , Inibidor de Quinase Dependente de Ciclina p15/genética , Proteínas Ativadoras de GTPase/genética , Neoplasias/genética , Proteínas Supressoras de Tumor/genética , Animais , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Regulação para Baixo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes p16 , Genes ras , Humanos , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Neoplasias/patologia , Prognóstico , Quinases Associadas a rho/metabolismoRESUMO
Cell adhesion is a fundamental property of epithelial cells required for anchoring, migration and survival. During cell migration, the formation and disruption of adhesion sites is stringently regulated by integration of multiple, sequential signals acting in distinct regions of the cell. Recent findings implicate cyclin dependent kinase 5 (Cdk5) in the signaling pathways that regulate cell adhesion and migration of a variety of cell types. Experiments with epithelial cell lines indicate that Cdk5 activity exerts its effects by limiting Src activity in regions where Rho activity is required for stress fiber contraction and by phosphorylating the talin head to stabilize nascent focal adhesions. Both pathways regulate cell migration by increasing adhesive strength.
Assuntos
Movimento Celular , Quinase 5 Dependente de Ciclina/metabolismo , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Animais , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Células Epiteliais/efeitos dos fármacos , Humanos , Modelos Biológicos , Cadeias Leves de Miosina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Talina/metabolismoRESUMO
Cdk5 regulates adhesion and migration in a variety of cell types. We previously showed that Cdk5 is strongly activated during stress fiber formation and contraction in spreading cells. Here we determine the mechanism linking Cdk5 to stress fiber contractility and its relevance to cell migration. Immunofluorescence showed that Cdk5 colocalized with phosphorylated myosin regulatory light chain (pMRLC) on contracting stress fibers. Inhibiting Cdk5 activity by various means significantly reduced pMRLC level and cytoskeletal contraction, with loss of central stress fibers. Blocking Cdk5 activity also reduced Rho-Rho kinase (ROCK) signaling, which is the principal pathway of myosin phosphorylation under these conditions. Next, we examined the effect of Cdk5 activity on Src, a known regulator of Rho. Inhibiting Cdk5 activity increased Src activation and phosphorylation of its substrate, p190RhoGAP, an upstream inhibitor of Rho. Inhibiting both Cdk5 and Src activity completely reversed the effect of Cdk5 inhibition on Rho and prevented the loss of central stress fibers, demonstrating that Cdk5 exerts its effects on Rho-ROCK signaling by suppressing Src activity. Moreover, inhibiting either Cdk5 or ROCK activity increased cell migration to an equal extent, while inhibiting both kinases produced no additional effect, demonstrating that Cdk5-dependent regulation of ROCK activity is a physiological determinant of migration rate.
Assuntos
Movimento Celular/fisiologia , Quinase 5 Dependente de Ciclina/metabolismo , Citoesqueleto/metabolismo , Células Epiteliais/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Repressoras/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Linhagem Celular , Quinase 5 Dependente de Ciclina/genética , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Células Epiteliais/citologia , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Cinetina/metabolismo , Cadeias Leves de Miosina/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/genética , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo , Quinases da Família src/genéticaRESUMO
Previous studies have shown that Cdk5 promotes lens epithelial cell adhesion. Here we use a cell spreading assay to investigate the mechanism of this effect. As cells spread, forming matrix adhesions and stress fibers, Cdk5(Y15) phosphorylation and Cdk5 kinase activity increased. Cdk5(Y15) phosphorylation was inhibited by PP1, a Src family kinase inhibitor. To identify the PP1-sensitive kinase, we transfected cells with siRNA oligonucleotides for cSrc and related kinases. Only cSrc siRNA oligonucleotides inhibited Cdk5(Y15) phosphorylation. Cdk5(pY15) and its activator, p35, colocalized with actin in stress fibers. To examine Cdk5 function, we inhibited Cdk5 activity under conditions that also prevent phosphorylation at Y15: expression of kinase inactive mutations Cdk5(Y15F) and Cdk5(K33T), and siRNA suppression of Cdk5. Stress fiber formation was severely inhibited. To distinguish between a requirement for Cdk5 kinase activity and a possible adaptor role for Cdk5(pY15), we used two methods that inhibit kinase activity without inhibiting phosphorylation at Y15: pharmacological inhibition with olomoucine and expression of the kinase inactive mutation, Cdk5(D144N). Stress fiber organization was altered, but stress fiber formation was not blocked. These findings indicate that Cdk5(Y15) phosphorylation and Cdk5 activity have distinct functions required for stress fiber formation and organization, respectively.
Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Fibras de Estresse/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Quinase 5 Dependente de Ciclina/análise , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Humanos , Mutação , Fosforilação , RNA Interferente Pequeno/metabolismo , Coelhos , Fibras de Estresse/ultraestrutura , Transfecção , Proteína Supressora de Tumor p53/análise , Proteína Supressora de Tumor p53/metabolismoRESUMO
PURPOSE: To investigate the effect of the Cdk5 inhibitor olomoucine on corneal debridement wound healing in vivo. METHODS: Corneal debridement wounds of 1.5 mm were made on the ocular surface of CD-1 mice. A 20 microl drop of 15 microM olomoucine in 1% DMSO was applied to the wound area immediately after wounding and again after 6 h. Control mice received identical applications of 1% DMSO. Mice were euthanized after 18 h, two weeks, and three weeks for evaluation of wound healing and restratification. Corneas were stained with Richardson's dye, photographed, and processed for histology and immunofluorescence as whole mounts or paraffin sections. The remaining wound area at 18 h was measured by image analysis. Scratch wounded cultures of human corneal-limbal epithelial cells (HCLE) were used to examine the effect of olomoucine on matrix metalloproteinase (MMP) expression in vitro. MMP-2 and MMP-9 were detected by immunofluorescence and immunoblotting. RESULTS: Olomoucine treatment significantly enhanced corneal wound closure without increasing inflammation or infiltration of polymorphonuclear leukocytes 18 h after wounding (p<0.05). The increased localization of MMP-9 within epithelial cells at the wound edge was further enhanced by olomoucine while the expression of MMP-2 was reduced. Olomoucine treatment of scratch wounded HCLE cells produced similar changes in MMP-9 and MMP-2 expression. The examination of treated corneas two and three weeks after wounding showed normal epithelial restratification with no evidence of inflammation or stromal disorganization. CONCLUSIONS: Topical application of olomoucine in 1% DMSO significantly enhances closure of small epithelial debridement wounds without increasing inflammation or impairing reepithelialization.
Assuntos
Córnea/fisiopatologia , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Desbridamento , Inibidores Enzimáticos/farmacologia , Cinetina/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Caderinas/metabolismo , Células Cultivadas , Córnea/metabolismo , Córnea/patologia , Córnea/cirurgia , Células Epiteliais/enzimologia , Humanos , Limbo da Córnea/efeitos dos fármacos , Limbo da Córnea/enzimologia , Limbo da Córnea/lesões , Limbo da Córnea/patologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos , Neutrófilos/patologia , Fatores de Tempo , Distribuição TecidualRESUMO
Cyclin-dependent kinase 5 (Cdk5) has been shown to regulate adhesion and migration of lens and corneal epithelial cells. To explore protein-protein interactions that may mediate these functions, we performed yeast two-hybrid screening on an embryonic rat lens library using Cdk5 and its regulators, p35 and p39 as baits. This screen identified an interaction between p39 and non-muscle myosin essential light chain (MLC(17)). GST pull-down experiments demonstrated that p39 binds directly to MLC(17) through a strong binding site in the N-terminal 109 amino acids of p39. Immunoprecipitation of proteins from Cos1 cells co-transfected with GFP-MLC(17) and HA-p39 confirmed that these proteins interact intracellularly. Immunofluorescence microscopy of co-transfected lens epithelial cells showed that GFP-MLC(17) and HA-p39 co-localize along cytoskeletal fibrils. Moreover, endogenous rat lens p39 co-immunoprecipitated with MLC(17) and myosin heavy chain II (MHC II), demonstrating that the interaction is physiological and serves to link p39 to the cytoskeleton.
Assuntos
Cadeias Leves de Miosina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Quinase 5 Dependente de Ciclina/metabolismo , Cristalino/citologia , Cristalino/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Coelhos , Ratos , Frações Subcelulares/química , Técnicas do Sistema de Duplo-HíbridoRESUMO
Protein tyrosine phosphatase 1B (PTP-1B) is an enzyme that plays a critical role in down-regulating insulin signaling through dephosphorylation of the insulin receptor. Studies have shown that PTP-1B knockout mice showed increased insulin sensitivity in muscle and liver as well as resistance to obesity. A series of hydroxy benzofuran methyl ketones and their naturally mimicking dimers and linear and angular furanochalcones and flavones have been evaluated as PTP-1B inhibitors. Screened compounds displayed good inhibitory activity.
Assuntos
Benzofuranos/síntese química , Benzofuranos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Cromatografia em Camada Fina , Ciclização , Avaliação Pré-Clínica de Medicamentos , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Vanadatos/farmacologiaRESUMO
Protein tyrosine phosphatase 1B (PTP1B) is an enzyme that plays a critical role in down-regulating insulin signaling through dephosphorylation of the insulin receptor. Studies have shown that PTP1B knock-out mice showed increased insulin sensitivity in muscle and liver as well as resistance to obesity. A series of functionalized acetophenones were synthesized and evaluated for their PTP1B inhibitory activity. Some of the screened compounds displayed good inhibitory activity.