RESUMO
Alpha/beta hydrolase domain-containing 5 (ABHD5), also termed CGI-58, is the key upstream activator of adipose triglyceride lipase (ATGL), which plays an essential role in lipid metabolism and energy storage. Mutations in ABHD5 disrupt lipolysis and are known to cause the Chanarin-Dorfman syndrome. Despite its importance, the structure of ABHD5 remains unknown. In this work, we combine computational and experimental methods to build a 3D structure of ABHD5. Multiple comparative and machine learning-based homology modeling methods are used to obtain possible models of ABHD5. The results from Gaussian accelerated molecular dynamics and experimental data of the apo models and their mutants are used to select the most likely model. Moreover, ensemble docking is performed on representative conformations of ABHD5 to reveal the binding mechanism of ABHD5 and a series of synthetic ligands. Our study suggests that the ABHD5 models created by deep learning-based methods are the best candidate structures for the ABHD5 protein. The mutations of E41, R116, and G328 disturb the hydrogen bonding network with nearby residues and suppress membrane targeting or ATGL activation. The simulations also reveal that the hydrophobic interactions are responsible for binding sulfonyl piperazine ligands to ABHD5. Our work provides fundamental insight into the structure of ABHD5 and its ligand-binding mode, which can be further applied to develop ABHD5 as a therapeutic target for metabolic disease and cancer.
RESUMO
Alpha/beta hydrolase domain-containing protein 5 (ABHD5) is a highly conserved protein that regulates various lipid metabolic pathways via interactions with members of the perilipin (PLIN) and Patatin-like phospholipase domain-containing protein (PNPLA) protein families. Loss of function mutations in ABHD5 result in Chanarin-Dorfman Syndrome (CDS), characterized by ectopic lipid accumulation in numerous cell types and severe ichthyosis. Recent data demonstrates that ABHD5 is the target of synthetic and endogenous ligands that might be therapeutic beneficial for treating metabolic diseases and cancers. However, the structural basis of ABHD5 functional activities, such as protein-protein interactions and ligand binding is presently unknown. To address this gap, we constructed theoretical structural models of ABHD5 by comparative modeling and topological shape analysis to assess the spatial patterns of ABHD5 conformations computed in protein dynamics. We identified functionally important residues on ABHD5 surface for lipolysis activation by PNPLA2, lipid droplet targeting and PLIN-binding. We validated the computational model by examining the effects of mutating key residues in ABHD5 on an array of functional assays. Our integrated computational and experimental findings provide new insights into the structural basis of the diverse functions of ABHD5 as well as pathological mutations that result in CDS.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/química , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Biologia Computacional/métodos , Lipase/metabolismo , Gotículas Lipídicas/metabolismo , Mutação , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Humanos , Ligantes , Gotículas Lipídicas/química , Conformação ProteicaRESUMO
Platinum drug-induced cross-link repair requires the concerted activities of translesion synthesis (TLS), Fanconi anemia (FA), and homologous recombination repair pathways. The E2 ubiquitin-conjugating enzyme RAD6 is essential for TLS. Here, we show that RAD6 plays a universal role in platinum-based drug tolerance. Using a novel RAD6-selective small-molecule inhibitor (SMI#9) targeting the RAD6 catalytic site, we demonstrate that SMI#9 potentiates the sensitivities of cancer cells with innate or acquired cisplatin or oxaliplatin resistance. 5-Iododeoxyuridine/5-chlorodeoxyuridine pulse-labeling experiments showed that RAD6 is necessary for overcoming cisplatin-induced replication fork stalling, as replication-restart was impaired in both SMI#9-pretreated and RAD6B-silenced cells. Consistent with the role of RAD6/TLS in late-S phase, SMI#9-induced DNA replication inhibition occurred preferentially in mid/late-S phase. The compromised DNA repair and chemosensitization induced by SMI#9 or RAD6B depletion were associated with decreased platinum drug-induced proliferating cell nuclear antigen (PCNA) and FANCD2 monoubiquitinations (surrogate markers of TLS and FA pathway activation, respectively) and with attenuated FANCD2, RAD6, γH2AX, and POL η foci formation and cisplatin-adduct removal. SMI#9 pretreatment synergistically increased cisplatin inhibition of MDA-MB-231 triple-negative breast cancer cell proliferation and tumor growth. Using an isogenic HCT116 colon cancer model of oxaliplatin resistance, we further show that γH2AX and monoubiquitinated PCNA and FANCD2 are constitutively up-regulated in oxaliplatin-resistant HCT116 (HCT116-OxR) cells and that γH2AX, PCNA, and FANCD2 monoubiquitinations are induced by oxaliplatin in parental HCT116 cells. SMI#9 pretreatment sensitized HCT116-OxR cells to oxaliplatin. These data deepen insights into the vital role of RAD6/TLS in platinum drug tolerance and reveal clinical benefits of targeting RAD6 with SMI#9 for managing chemoresistant cancers.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Reparo do DNA/efeitos dos fármacos , DNA de Neoplasias/biossíntese , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Compostos Organoplatínicos/farmacologia , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Replicação do DNA/efeitos dos fármacos , DNA de Neoplasias/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Oxaliplatina , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Alpha-beta hydrolase domain-containing 5 (ABHD5), the defective gene in human Chanarin-Dorfman syndrome, is a highly conserved regulator of adipose triglyceride lipase (ATGL)-mediated lipolysis that plays important roles in metabolism, tumor progression, viral replication, and skin barrier formation. The structural determinants of ABHD5 lipolysis activation, however, are unknown. We performed comparative evolutionary analysis and structural modeling of ABHD5 and ABHD4, a functionally distinct paralog that diverged from ABHD5 ~500 million years ago, to identify determinants of ABHD5 lipolysis activation. Two highly conserved ABHD5 amino acids (R299 and G328) enabled ABHD4 (ABHD4 N303R/S332G) to activate ATGL in Cos7 cells, brown adipocytes, and artificial lipid droplets. The corresponding ABHD5 mutations (ABHD5 R299N and ABHD5 G328S) selectively disrupted lipolysis without affecting ATGL lipid droplet translocation or ABHD5 interactions with perilipin proteins and ABHD5 ligands, demonstrating that ABHD5 lipase activation could be dissociated from its other functions. Structural modeling placed ABHD5 R299/G328 and R303/G332 from gain-of-function ABHD4 in close proximity on the ABHD protein surface, indicating they form part of a novel functional surface required for lipase activation. These data demonstrate distinct ABHD5 functional properties and provide new insights into the functional evolution of ABHD family members and the structural basis of lipase regulation.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Lipólise/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferase/química , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Adipócitos Marrons/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Linhagem Celular , Chlorocebus aethiops , Expressão Gênica , Técnicas de Silenciamento de Genes , Lipase/metabolismo , Gotículas Lipídicas , Lisofosfolipase/química , Lisofosfolipase/genética , Lisofosfolipase/metabolismo , Camundongos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Relação Estrutura-AtividadeRESUMO
Fat and muscle lipolysis involves functional interactions of adipose triglyceride lipase (ATGL), α-ß hydrolase domain-containing protein 5 (ABHD5), and tissue-specific perilipins 1 and 5 (PLIN1 and PLIN5). ABHD5 potently activates ATGL, but this lipase-promoting activity is suppressed when ABHD5 is bound to PLIN proteins on lipid droplets. In adipocytes, protein kinase A (PKA) phosphorylation of PLIN1 rapidly releases ABHD5 to activate ATGL, but mechanisms for rapid regulation of PLIN5-ABHD5 interaction in muscle are unknown. Here, we identify synthetic ligands that release ABHD5 from PLIN1 or PLIN5 without PKA activation and rapidly activate adipocyte and muscle lipolysis. Molecular imaging and affinity probe labeling demonstrated that ABHD5 is directly targeted by these synthetic ligands and additionally revealed that ABHD5-PLIN interactions are regulated by endogenous ligands, including long-chain acyl-CoA. Our results reveal a new locus of lipolysis control and suggest ABHD5 ligands might be developed into novel therapeutics that directly promote fat catabolism.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Proteínas de Transporte/metabolismo , Lipólise/genética , Fosfoproteínas/metabolismo , Proteínas/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Células 3T3-L1 , Acil Coenzima A/metabolismo , Adipócitos/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Humanos , Ligantes , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Perilipina-1 , Perilipina-5 , Fosfoproteínas/genética , Proteínas/genéticaRESUMO
Protein ubiquitination is important for cell signaling, DNA repair, and proteasomal degradation, and it is not surprising that alterations in ubiquitination occur frequently in cancer. Ubiquitin-conjugating enzymes (E2) mediate ubiquitination by selective interactions with ubiquitin-activating (E1) and ubiquitin ligase (E3) enzymes, and thus selective E2 small molecule inhibitor (SMI) will provide specificity unattainable with proteasome inhibitors. Here we describe synthesis and functional characterization of the first SMIs of human E2 Rad6B, a fundamental component of translesion synthesis DNA repair. A pharmacophore model for consensus E2 ubiquitin-binding sites was generated for virtual screening to identify E2 inhibitor candidates. Twelve triazine (TZ) analogs screened in silico by molecular docking to the Rad6B X-ray structure were verified by their effect on Rad6B ubiquitination of histone H2A. TZs #8 and 9 docked to the Rad6B catalytic site with highest complementarity. TZs #1, 2, 8, and 9 inhibited Rad6B-ubiquitin thioester formation and subsequent ubiquitin transfer to histone H2A. SMI #9 inhibition of Rad6 was selective as BCA2 ubiquitination by E2 UbcH5 was unaffected by SMI #9. SMI #9 more potently inhibited proliferation, colony formation, and migration than SMI #8, and induced MDA-MB-231 breast cancer cell G2-M arrest and apoptosis. Ubiquitination assays using Rad6 immunoprecipitated from SMI #8- or 9-treated cells confirmed inhibition of endogenous Rad6 activity. Consistent with our previous data showing Rad6B-mediated polyubiquitination stabilizes ß-catenin, MDA-MB-231 treatment with SMIs #8 or 9 decreased ß-catenin protein levels. Together these results describe identification of the first Rad6 SMIs.
Assuntos
Inibidores Enzimáticos/farmacologia , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Sequência de Bases , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sequência Conservada , Desenho de Fármacos , Inibidores Enzimáticos/química , Humanos , Conformação Molecular , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Triazinas/química , Triazinas/farmacologia , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , beta Catenina/metabolismoRESUMO
The ubiquitin conjugating enzyme Rad6B is overexpressed in breast cancer and induces ß-catenin transcriptional activation and stabilization via K63-linked polyubiquitination. Here we identify ß-catenin and Rad6B interacting regions, identify potential Rad6B ubiquitination sites in ß-catenin, and characterize their breast cancer tissue expression. ß-catenin and Rad6B colocalize in breast carcinoma and coimmunoprecipitate from MDA-MB-231 cells. Pull-down assays using GST-ß-catenin and His-Rad6B deletion mutants identified amino acids 131-181 and 50-116, respectively, as necessary for their interaction. Ubiquitination assays using ß-catenin deletion mutants mapped Rad6B-induced ubiquitination within ß-catenin 181-422 encompassing Armadillo repeats 2-7. Lysine to arginine mutations within repeats 5-7 identified K394 as the major Rad6B ubiquitination site in vitro and in vivo, and confirmed by Rad6B ubiquitination of a ß-catenin peptide encompassing K394. Ubiquitination of wild type- but not K394R-ß-catenin was decreased by Rad6B silencing. Compared to wild type-, K312R-, K335R-, K345R-, or K354R-ß-catenin, K394R mutation caused ~50% drop in TOP/Flash activity in Wnt-silent MCF-7 cells. Consistent with these data, expression of Rad6B, itself a ß-catenin/TCF transcriptional target, was also reduced in K394R-ß-catenin transfected cells. Steady-state K394R-ß-catenin levels are decreased compared to wild type-ß-catenin. The decreased expression is not due to proteasomal degradation as treatment with MG132 failed to rescue its levels. Lymph node-positive breast carcinomas express higher levels of Rad6 protein and Rad6 activity, and K63-linked ubiquitinated ß-catenin than reduction mammoplasties. These data suggest that K394 is a novel site of ß-catenin ubiquitination that may be important for the stability and activity of ß-catenin in breast cancer.
Assuntos
Lisina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação , beta Catenina/metabolismo , Proteínas do Domínio Armadillo/química , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Domínio Catalítico , Linhagem Celular Tumoral , Feminino , Inativação Gênica , Humanos , Mutação/genética , Invasividade Neoplásica , Ligação Proteica , Transcrição Gênica , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Regulação para Cima/genética , beta Catenina/química , beta Catenina/genéticaRESUMO
We have previously demonstrated that expression of the novel gene schlafen-3 (Slfn-3) correlates with intestinal epithelial cell differentiation (Patel VB, Yu Y, Das JK, Patel BB, Majumdar AP. Biochem Biophys Res Commun 388: 752-756, 2009). The present investigation was undertaken to examine whether Slfn-3 plays a role in regulating differentiation of FOLFOX-resistant (5-fluorouracil + oxaliplatin) colon cancer cells that are highly enriched in cancer stem cells (CSCs). Transfection of Slfn-3 in FOLFOX-resistant colon cancer HCT-116 cells resulted in increase of alkaline phosphatase activity, a marker of intestinal differentiation. Additionally, Slfn-3 transfection resulted in reduction of mRNA and protein levels of the CSC markers CD44, CD133, CD166, and aldehyde dehydrogenase 1 in both FOLFOX-resistant HCT-116 and HT-29 cells. This was accompanied by decreased formation of tumorosphere/colonosphere (an in vitro model of tumor growth) in stem cell medium and inhibition of expression of the chemotherapeutic drug transporter protein ABCG2. Additionally, Slfn-3 transfection of FOLFOX-resistant HCT-116 and HT-29 cells reduced Hoechst 33342 dye exclusion. Finally, Slfn-3 transfection inhibited the expression of transforming growth factor-α in both FOLFOX-resistant colon cancer cells, but stimulated apoptosis in response to additional FOLFOX treatment. In summary, our data demonstrate that Slfn-3 expression inhibits multiple characteristics of CSC-enriched, FOLFOX-resistant colon cancer cells, including induction of differentiation and reduction in tumorosphere/colonosphere formation, drug transporter activity, and autocrine stimulation of proliferation. Thus Slfn-3 expression may render colon CSCs more susceptible to cancer chemotherapeutics.
Assuntos
Comunicação Autócrina/genética , Proteínas de Ciclo Celular/genética , Neoplasias do Colo/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Células-Tronco Neoplásicas/metabolismo , Proteínas/genética , Proteínas/fisiologia , RNA Mensageiro/metabolismo , Antígeno AC133 , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Família Aldeído Desidrogenase 1 , Antígenos CD/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas de Ciclo Celular/fisiologia , Diferenciação Celular/genética , Neoplasias do Colo/genética , Receptores ErbB/metabolismo , Proteínas Fetais/metabolismo , Fluoruracila , Glicoproteínas/metabolismo , Células HCT116 , Células HT29 , Humanos , Receptores de Hialuronatos/metabolismo , Isoenzimas/metabolismo , Leucovorina , Proteínas de Neoplasias/metabolismo , Compostos Organoplatínicos , Peptídeos/metabolismo , Retinal Desidrogenase/metabolismo , Transdução de Sinais/genética , Transfecção , Fator de Crescimento Transformador alfa/metabolismoRESUMO
The cancer stem cell model was described for hematologic malignancies in 1997 and since then evidence has emerged to support it for many solid tumors as well, including colon cancer. This model proposes that certain cells within the tumor mass are pluripotent and capable of self-renewal and have an enhanced ability to initiate distant metastasis. The cancer stem cell model has important implications for cancer treatment, since most current therapies target actively proliferating cells and may not be effective against the cancer stem cells that are responsible for recurrence. In recent years great progress has been made in identifying markers of both normal and malignant colon stem cells. Proteins proposed as colon cancer stem cell markers include CD133, CD44, CD166, ALDH1A1, Lgr5, and several others. In this review we consider the evidence for these proteins as colon cancer stem cell markers and as prognostic indicators of colon cancer survival. Additionally, we discuss potential functions of these proteins and the implications this may have for development of therapies that target colon cancer stem cells.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias do Colo/etiologia , Células-Tronco Neoplásicas/patologia , Antígeno AC133 , Transportadores de Cassetes de Ligação de ATP/metabolismo , Aldeído Desidrogenase/metabolismo , Família Aldeído Desidrogenase 1 , Animais , Antígenos CD/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Neoplasias do Colo/patologia , Proteínas Fetais/metabolismo , Glicoproteínas/metabolismo , Receptores de Hialuronatos/metabolismo , Camundongos , Peptídeos/metabolismo , Prognóstico , Receptores Acoplados a Proteínas G/metabolismo , Retinal Desidrogenase , Células-Tronco/patologiaRESUMO
The intestinal epithelium is subjected to repetitive deformation during normal gut function by peristalsis and villous motility. Such repetitive strain promotes intestinal epithelial migration across fibronectin in vitro, but signaling mediators for this are poorly understood. We hypothesized that integrin-linked kinase (ILK) mediates strain-stimulated migration in intestinal epithelial cells cultured on fibronectin. ILK kinase activity increased rapidly 5 min after strain induction in both Caco-2 and intestinal epithelial cell-6 (IEC-6) cells. Wound closure in response to strain was reduced in ILK small interfering RNA (siRNA)-transfected Caco-2 cell monolayers when compared with control siRNA-transfected Caco-2 cells. Pharmacological blockade of phosphatidylinositol-3 kinase (PI3K) or Src or reducing Src by siRNA prevented strain activation of ILK. ILK coimmunoprecipitated with focal adhesion kinase (FAK), and this association was decreased by mutation of FAK Tyr925 but not FAK Tyr397. Strain induction of FAK Tyr925 phosphorylation but not FAK Tyr397 or FAK Tyr576 phosphorylation was blocked in ILK siRNA-transfected cells. ILK-Src association was stimulated by strain and was blocked by the Src inhibitor PP2. Finally, ILK reduction by siRNA inhibited strain-induced phosphorylation of myosin light chain and Akt. These results suggest a strain-dependent signaling pathway in which ILK association with FAK and Src mediates the subsequent downstream strain-induced motogenic response and suggest that ILK induction by repetitive deformation may contribute to recovery from mucosal injury and restoration of the mucosal barrier in patients with prolonged ileus. ILK may therefore be an important target for intervention to maintain the mucosa in such patients.
Assuntos
Mucosa Intestinal/enzimologia , Proteínas Serina-Treonina Quinases/fisiologia , Cicatrização , Animais , Linhagem Celular , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Mutação , Cadeias Leves de Miosina/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/administração & dosagem , RNA Interferente Pequeno/fisiologia , Ratos , Quinases da Família src/antagonistas & inibidoresRESUMO
The intestinal epithelium is subjected to repetitive deformation during normal gut function by peristalsis and villous motility. In vitro, cyclic strain promotes intestinal epithelial proliferation and induces an absorptive phenotype characterized by increased dipeptidyl dipeptidase (DPPIV) expression. Schlafen 3 is a novel gene recently associated with cellular differentiation. We sought to evaluate whether Schlafen 3 mediates the effects of strain on the differentiation of intestinal epithelial cell (IEC)-6 in the absence or presence of cyclic strain. Strain increased Schlafen 3 mRNA and protein. In cells transfected with a control-nontargeting siRNA, strain increased DPPIV-specific activity. However, Schlafen 3 reduction by siRNA decreased basal DPPIV and prevented any stimulation of DPPIV activity by strain. Schlafen 3 reduction also prevented DPPIV induction by sodium butyrate (1 mM) or transforming growth factor (TGF)-beta (0.1 ng/ml), two unrelated differentiating stimuli. However, Schlafen-3 reduction by siRNA did not prevent the mitogenic effect of strain or that of EGF. Blocking Src and phosphatidyl inositol (PI3)-kinase prevented strain induction of Schlafen 3, but Schlafen 3 induction required activation of p38 but not ERK. These results suggest that cyclic strain induces an absorptive phenotype characterized by increased DPPIV activity via Src-, p38-, and PI3-kinase-dependent induction of Schlafen 3 in rat IEC-6 cells on collagen, whereas Schlafen 3 may also be a key factor in the induction of intestinal epithelial differentiation by other stimuli such as sodium butyrate or TGF-beta. The induction of Schlafen 3 or its human homologs may modulate intestinal epithelial differentiation and preserve the gut mucosa during normal gut function.
Assuntos
Células Epiteliais/citologia , Células Epiteliais/fisiologia , Mucosa Intestinal/citologia , Proteínas/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Indução Enzimática , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibronectinas , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas/genética , RNA Interferente Pequeno , Ratos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
We observed previously that combined small interfering RNAs (siRNAs) targeting CrkII and CrkL, known activators of guanine nucleotide exchange factor DOCK1, strongly inhibit Caco-2 intestinal epithelial cell spreading and migration on collagen IV. DOCK1 siRNA reduced its expression >95% in Caco-2 cells but inhibited spreading much less than combined CrkII/CrkL siRNAs, suggesting that CrkII/CrkL interact with additional DOCK proteins. siRNA targeting DOCK5, a closely related DOCK1 family member, inhibited Caco-2 spreading similarly to DOCK1 siRNA, and the combined siRNAs synergistically inhibited spreading. Similar results were observed in human umbilical vein endothelial cells, and reverse transcriptase PCR demonstrated DOCK5 siRNA reduction of DOCK5 expression in both cell types. Combined DOCK1/DOCK5 siRNAs also inhibited Caco-2 migration and lamellipodial extension. Expression of DOCK5 cDNA, with silent mutations in the siRNA target region allowing expression simultaneously with DOCK5 siRNA, required CrkII/CrkL to restore cell spreading and DOCK5 coimmunoprecipitated with CrkII and CrkL. DOCK5 association with CrkII and CrkL was greatly reduced by mutations in their NH2-terminal SH3 domains. Expression of the DOCK5 COOH-terminal region (Met1738-Gln1870), containing potential Src homology 3 domain-binding proline-rich sites but lacking other functional regions, inhibited Caco-2 spreading and coimmunoprecipitated with CrkL. Coimmunoprecipitation of full-length DOCK5 with CrkL was strongly reduced by deletion of DOCK5 COOH-terminal amino acids 1832-1870. Green fluorescent protein-tagged DOCK5 localized to the membrane of Caco-2 cells spreading on collagen IV. In these studies, we describe human DOCK5 cloning and expression, our results indicating that, along with DOCK1, DOCK5 is an important mediator of CrkII/CrkL regulation of Caco-2 spreading and migration on collagen IV.
Assuntos
Movimento Celular/fisiologia , Colágeno Tipo IV , Regulação da Expressão Gênica/fisiologia , Mucosa Intestinal/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células CACO-2 , Humanos , Mucosa Intestinal/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteínas Proto-Oncogênicas c-crk/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-crk/genética , Proteínas Proto-Oncogênicas c-crk/metabolismo , RNA Interferente Pequeno/genética , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/genéticaRESUMO
We previously observed that collagen IV regulates Caco-2 intestinal epithelial cell spreading and migration via Src-dependent p130(Cas) phosphorylation and stimulates focal adhesion kinase (FAK). However, the role of FAK and the related kinase, Pyk2, in Caco-2 spreading and migration is unclear. FAK- or Pyk2-specific siRNAs reduced protein levels by 90%. However, when detached cells were replated on collagen IV neither individual nor combined FAK and Pyk2 siRNAs affected the cell spreading rate. As combined FAK and Pyk2 siRNAs increased p130(Cas) protein levels, we cotransfected cells with 1 nm p130(Cas) siRNA to partially reduce p130(Cas) protein to control levels. Although p130(Cas) Tyr(P)(249) phosphorylation was reduced by 60%, cell spreading was unaffected. Combined siRNA reduction of FAK, Pyk2 and p130(Cas) increased cell spreading by 20% compared to p130(Cas) siRNA alone, suggesting that FAK and Pyk2 negatively regulate spreading in addition to stimulating spreading via p130(Cas). FAK-binding mutant SH3 domain-deleted rat p130(Cas) was not phosphorylated after adhesion and, unlike full-length p130(Cas), did not restore spreading after human-specific p130(Cas) siRNA knockdown of endogenous p130(Cas). Together, these data suggest that FAK positively regulates Caco-2 spreading on collagen IV via p130(Cas) phosphorylation, but also suggests that FAK may negatively regulate spreading through other mechanisms and the presence of additional FAK-independent pathways regulating p130(Cas).
Assuntos
Colágeno Tipo IV/farmacologia , Proteína Substrato Associada a Crk/metabolismo , Quinase 2 de Adesão Focal/fisiologia , Transdução de Sinais/fisiologia , Células CACO-2 , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultura , Quinase 2 de Adesão Focal/antagonistas & inibidores , Quinase 2 de Adesão Focal/biossíntese , Humanos , Fosforilação , RNA Interferente Pequeno/farmacologia , TransfecçãoRESUMO
Mucosal healing requires migration and proliferation. Most studies of focal adhesion kinase (FAK), a protein that regulates motility, proliferation, and apoptosis, have focused on rapid phosphorylation. We reported lower FAK protein levels in motile Caco-2 colon cancer cells and postulated that this reduction in FAK available for activation might impact cell migration and mucosal healing. Therefore, total and active FAK (FAK(397)) immunoreactivity was assessed at the migrating fronts of human Caco-2 and rat IEC-6 intestinal epithelial cells. Caco-2 and IEC-6 motility, quantitated as migration into linear or circular wounds, was examined following FAK protein inhibition by small interfering RNA (siRNA). FAK protein stability and mRNA expression were ascertained by cycloheximide decay, RT-PCR, and in situ hybridization in static and migrating Caco-2 cells. Cells at the migrating front of Caco-2 and IEC-6 monolayers exhibited lower immunostaining for both total and activated FAK than cells immediately behind the front. Western blot analysis also demonstrated diminished FAK protein levels in motile cells by >/=30% in both the differential density seeding and multiple scrape models. siRNA FAK protein inhibition enhanced motility in both the linear scrape (20% in Caco-2) and circular wound (16% in Caco-2 and 19% in IEC-6 cells) models. FAK protein degradation did not differ in motile and static Caco-2 cells and was unaffected by FAK(397) phosphorylation, but FAK mRNA was lower in migrating Caco-2 cells. Thus FAK protein abundance appears regulated at the mRNA level during gut epithelial cell motility and may influence epithelial cell migration coordinately with signals that modify FAK phosphorylation.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Motilidade Gastrointestinal/fisiologia , Mucosa Intestinal/citologia , Mucosa Intestinal/fisiologia , Animais , Células CACO-2 , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Células HT29 , Humanos , RatosRESUMO
We have previously observed that collagen IV regulates Caco-2 intestinal epithelial cell spreading and migration via Src kinase and stimulates Src-dependent tyrosine phosphorylation of p130cas. We observed that collagen IV also stimulated Src-dependent phosphorylation of both paxillin Tyr31 and paxillin Tyr118. Caco-2 transfection with paxillin or p130cas siRNAs inhibited expression of these proteins by more than 90% for at least 5 days after transfection. Although p130cas siRNA inhibited cell spreading on collagen IV by 33%, three different paxillin siRNAs did not inhibit cell spreading. p130cas siRNA did not affect Src Tyr416 or Src Tyr527 phosphorylation, FAK Tyr397 phosphorylation, or Src-dependent phosphorylation of FAK Tyr925, suggesting that p130cas did not inhibit cell spreading by altering FAK or Src activity. Rat p130cas expression after siRNA knock-out of endogenous human p130cas in Caco-2 cells reduced cell spreading inhibition by 71%. In contrast, expression of rat p130cas from which the Src-phosphorylated substrate domain was deleted did not rescue siRNA inhibition of cell spreading. Combined treatment with siRNAs to Crk and CrkL, which bind to the p130cas substrate domain, inhibited cell spreading by 54%. Both p130cas siRNA and the combined Crk/CrkL siRNAs strongly inhibited (52 and 46% inhibition, respectively) Caco-2 sheet migration on collagen IV and noticeably inhibited lamellipodial extension, whereas paxillin siRNA only inhibited migration by 18% and did not noticeably affect lamellipodial extension. These results suggest that Src may regulate Caco-2 migration on collagen IV via both p130cas and paxillin but that Src phosphorylation of p130cas is more important for this process.
Assuntos
Movimento Celular/fisiologia , Colágeno Tipo IV/fisiologia , Proteínas do Citoesqueleto/fisiologia , Mucosa Intestinal/citologia , Fosfoproteínas/fisiologia , Proteínas/fisiologia , Animais , Células CACO-2 , Proteína Substrato Associada a Crk , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Camundongos , Paxilina , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Proteína p130 Retinoblastoma-Like , Tirosina/metabolismoRESUMO
Repetitive mechanical deformation may stimulate intestinal epithelial proliferation. Because the extracellular matrix modulates static intestinal epithelial biology, we examined whether matrix proteins influence intestinal epithelial responses to deformation. Human Caco-2BBE cells and nontransformed human enterocytes (HIPEC) were subjected to 10% average cyclic strain at 10 cycles/min on flexible membranes precoated with matrix proteins without or with plasma fibronectin or functional anti-integrin antibodies in the medium. Strain stimulated proliferation, focal adhesion kinase, extracellular signal-regulated protein kinase (ERK), p38, and Jun N-terminal kinase similarly on collagen I or IV, and more weakly on laminin, but had no effect on fibronectin. MEK blockade (PD98059) prevented strain-stimulated proliferation on collagen but did not affect proliferation on fibronectin. Adding tissue fibronectin to a collagen substrate or plasma fibronectin to the media suppressed strain s mitogenic and signal effects, but not those of epidermal growth factor. Functional antibodies to the alpha5 or alpha(v) integrin subunit blocked strain's effects on Caco-2 proliferation and ERK activation, although ligation of the alpha2 or alpha6 subunit did not. Repetitive strain also stimulated, and fibronectin inhibited, human intestinal primary epithelial cell proliferation. Repetitive deformation stimulates transformed and nontransformed human intestinal epithelial proliferation in a matrix-dependent manner. Tissue or plasma fibronectin may regulate the intestinal epithelial response to strain via integrins containing alpha5 or alpha(v).