RESUMO
AIMS/HYPOTHESIS: Forkhead box protein O1 (FOXO1) is a transcription factor essential for beta cell fate. Protein kinase B-dependent phosphorylation of FOXO1 at S256 (P-FOXO1) enables its binding to 14-3-3 dimers and nuclear export. Dephosphorylated FOXO1 enters nuclei and activates pro-apoptotic genes. Since our previous observations suggest that protein kinase C delta (PKCδ) induces nuclear accumulation of FOXO1, the underlying mechanism was examined. METHODS: In human islets, genetically modified mice and INS-1E cells apoptosis was assessed by TUNEL staining. Subcellular translocation of proteins was examined by confocal microscopy and signalling pathways were analysed by western blotting and overlay assay. RESULTS: In PKCδ-overexpressing (PKCδ-tg) mouse islet cells and INS-1E cells FOXO1 accumulated in nuclei, surprisingly, as P-FOXO1. PKCδ-tg decelerated IGF-1-dependent stimulation of nuclear export, indicating that changes in export caused nuclear retention of P-FOXO1. Nuclear accumulation of P-FOXO1 was accompanied by increased phosphorylation of 14-3-3ζ at S58 and reduced dimerisation of 14-3-3ζ. Palmitic acid further augmented phosphorylation of 14-3-3ζ and triggered nuclear accumulation of FOXO1 in both INS-1E and human islet cells. Furthermore, the overexpression of a phosphomimicking mutant of 14-3-3ζ (S58D) enhanced nuclear FOXO1. In accordance with the nuclear accumulation of P-FOXO1, PKCδ overexpression alone did not increase apoptotic cell death. Additionally, insulin secretion and glucose homeostasis in PKCδ-overexpressing mice remained unaffected. CONCLUSIONS/INTERPRETATION: These results suggest that PKCδ-mediated phosphorylation of 14-3-3ζ contributes to the nuclear retention of FOXO1, even when FOXO1 is phosphorylated as under non-stress conditions. P-FOXO1 does not induce pro-apoptotic genes, but may rather exert beneficial effects on beta cells.
Assuntos
Proteínas 14-3-3/genética , Fatores de Transcrição Forkhead/metabolismo , Proteína Quinase C-delta/metabolismo , Transporte Ativo do Núcleo Celular/genética , Animais , Núcleo Celular/metabolismo , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Humanos , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação/genética , Cultura Primária de Células , Proteína Quinase C-delta/genética , Transdução de Sinais/genéticaRESUMO
CUB-domain-containing-protein-1 (CDCP1) is an integral membrane protein whose expression is up-regulated in various cancer types. Although high CDCP1 expression has been correlated with poor prognosis in lung, breast, pancreas, and renal cancer, its functional role in tumor formation or progression is incompletely understood. So far it has remained unclear, whether CDCP1 is a useful target for antibody therapy of cancer and what could be a desired mode of action for a therapeutically useful antibody. To shed light on these questions, we have investigated the cellular effects of a therapeutic antibody candidate (RG7287). In focus formation assays, prolonged RG7287 treatment prevented the loss of contact inhibition caused by co-transformation of NIH3T3 cells with CDCP1 and Src. In a xenograft study, MCF7 cells stably overexpressing CDCP1 reached the predefined tumor volume faster than the parental MCF7 cells lacking endogenous CDCP1. This tumor growth advantage was abolished by RG7287 treatment. In vitro, RG7287 induced rapid tyrosine phosphorylation of CDCP1 by Src, which was accompanied by translocation of CDCP1 to a Triton X-100 insoluble fraction of the plasma membrane. Triggering these effects required bivalency of the antibody suggesting that it involves CDCP1 dimerization or clustering. However, this initial activation of CDCP1 was only transient and prolonged RG7287 treatment induced internalization and down-regulation of CDCP1 in different cancer cell lines. Antibody stimulated CDCP1 degradation required Src activity and was proteasome dependent. Also in three different xenograft models with endogenous CDCP1 expression RG7287 treatment resulted in significant tumor growth inhibition concomitant with substantially reduced CDCP1 levels as judged by immunohistochemistry and Western blotting. Thus, despite transiently activating CDCP1 signaling, the RG7287 antibody has a therapeutically useful mode of action.
Assuntos
Anticorpos Antineoplásicos/farmacologia , Antígenos CD , Antígenos de Neoplasias , Moléculas de Adesão Celular , Membrana Celular/metabolismo , Glicoproteínas de Membrana , Proteínas de Neoplasias , Neoplasias Experimentais , Proteólise/efeitos dos fármacos , Animais , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Membrana Celular/patologia , Humanos , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Camundongos , Células NIH 3T3 , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Akt kinases are important mediators of the insulin signal, and some Akt substrates are directly involved in glucose homeostasis. Recently, Girdin has been described as an Akt substrate that is expressed ubiquitously in mammals. Cells overexpressing Girdin show an enhanced Akt activity. However, not much is known about Girdin's role in insulin signaling. We therefore analyzed the role of Girdin in primary human myotubes and found a correlation between Girdin expression and insulin sensitivity of the muscle biopsy donors, as measured by a hyperinsulinemic-euglycemic clamp. To understand this finding on a cellular level, we then investigated the function of Girdin in C2C12 mouse myoblasts. Girdin knock-down reduced Akt and insulin receptor substrate-1 phosphorylation. In contrast, stable overexpression of Girdin in C2C12 cells strikingly increased insulin sensitivity through a massive upregulation of the insulin receptor and enhanced tyrosine phosphorylation of insulin receptor substrate-1. Furthermore, Akt and c-Abl kinases were constitutively activated. To investigate medium-term insulin responses we measured glucose incorporation into glycogen. The Girdin overexpressing cells showed a high basal glycogen synthesis that peaked already at 1nM insulin. Taken together, we characterized Girdin as a new and major regulator of the insulin signal in myoblasts and skeletal muscle.
Assuntos
Insulina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Mioblastos/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/metabolismo , Adulto , Animais , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Glicogênio/biossíntese , Humanos , Insulina/farmacologia , Masculino , Camundongos , Mioblastos/efeitos dos fármacos , Mioblastos/enzimologia , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacosRESUMO
The receptor protein tyrosine phosphatase alpha (PTPα) is involved in the regulation of tyrosine kinases like the Src kinase and the insulin receptor. As with other PTPs, its function is determined by alternative splicing, dimerisation, phosphorylation and proteolytical processing. PTPα is cleaved by calpain in its intracellular domain, which decreases its potential to dephosphorylate Src kinase. Here, we demonstrate that PTPα is also processed in the extracellular domain. Extracellular processing was exclusively found for a splice variant containing an extra nine amino acid insert three residues amino-terminal from the transmembrane domain. Processing was sensitive to the metalloprotease-inhibitor Batimastat, and CHO-M2 cells lacking a disintegrin and metalloproteinase 17 (ADAM17; tumor-necrosis-factor α converting enzyme) activity were not able to cleave PTPα. After transient overexpression of ADAM17 and PTPα in these cells, processing was restored, proving that ADAM17 is involved in this process. Further characterization of the consequences of processing revealed that dephosphorylation of the insulin receptor or activation of Src was not affected but focus formation was reduced. We conclude that extracellular proteolytic processing is a novel mechanism for PTPα regulation.
Assuntos
Proteínas ADAM/metabolismo , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Proteólise , Proteínas ADAM/deficiência , Proteína ADAM17 , Animais , Células CHO , Linhagem Celular , Cricetinae , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Estrutura Terciária de ProteínaRESUMO
Cub domain containing protein 1 (CDCP1) is strongly expressed in tumors derived from lung, colon, ovary, or kidney. It is a membrane protein that is phosphorylated and then bound by Src family kinases. Although expression and phosphorylation of CDCP1 have been investigated in many tumor cell lines, the CDCP1 features responsible for transformation have not been fully evaluated. This is in part due to the lack of an experimental system in which cellular transformation depends on expression of exogenous CDCP1 and Src. Here we use retrovirus mediated co-overexpression of c-Src and CDCP1 to induce focus formation of NIH3T3 cells. Employing different mutants of CDCP1 we show that for a full transformation capacity, the intact amino- and carboxy-termini of CDCP1 are essential. Mutation of any of the core intracellular tyrosine residues (Y734, Y743, or Y762) abolished transformation, and mutation of a palmitoylation motif (C689,690G) strongly reduced it. Src kinase binding to CDCP1 was not required since Src with a defective SH2 domain generated even more CDCP1 dependent foci whereas Src myristoylation was necessary. Taken together, the focus formation assay allowed us to define structural requirements of CDCP1/Src dependent transformation and to characterize the interaction of CDCP1 and Src.
Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Transformação Celular Neoplásica/metabolismo , Proteínas de Neoplasias/metabolismo , Quinases da Família src/metabolismo , Animais , Antígenos CD/genética , Antígenos de Neoplasias , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Humanos , Lipoilação , Camundongos , Mutação , Células NIH 3T3 , Proteínas de Neoplasias/genética , Fosforilação , Quinases da Família src/genéticaRESUMO
Among the multitude of dysregulated signalling mechanisms that comprise insulin resistance in divergent organs, the primary events in the development of type 2 diabetes are not well established. As protein kinase C (PKC) activation is consistently present in skeletal muscle of obese and insulin resistant subjects, we generated a transgenic mouse model that overexpresses constitutively active PKC-beta(2) in skeletal muscle to test whether activation of PKC is sufficient to cause an aversive whole-body phenotype. Upon this genetic modification, increased serine phosphorylation in Irs1 was observed and followed by impaired (3)H-deoxy-glucose uptake and muscle glycogen content, and transgenic mice exhibited insulin and glucose intolerance as they age. Muscle histochemistry revealed an increase in lipid deposition (intramyocellular lipids), and transgenic mice displayed impaired expression of transcriptional regulators of genes involved in fatty acid oxidation (peroxisome proliferator-activated receptor-gamma, PGC-1beta, acyl-CoA oxidase) and lipolysis (hormone-sensitive lipase). In this regard, muscle of transgenic mice exhibited a reduced capacity to oxidize palmitate and contained less mitochondria as determined by citrate synthase activity. Moreover, the phenotype included a profound decrease in the daily running distance, intra-abdominal and hepatic fat accumulation and impaired insulin action in the brain. Together, our data suggest that activation of a classical PKC in skeletal muscle as present in the pre-diabetic state is sufficient to cause disturbances in whole-body glucose and lipid metabolism followed by profound alterations in oxidative capacity, ectopic fat deposition and physical activity.
Assuntos
Encéfalo/patologia , Fígado Gorduroso/enzimologia , Resistência à Insulina , Atividade Motora , Músculo Esquelético/enzimologia , Proteína Quinase C/metabolismo , Animais , Encéfalo/metabolismo , Fígado Gorduroso/patologia , Insulina/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/patologia , Especificidade de Órgãos , Oxirredução , Condicionamento Físico Animal , Proteína Quinase C beta , Transdução de Sinais , Regulação para Cima/genéticaRESUMO
OBJECTIVE: In vitro models suggest that free fatty acid-induced apoptotic beta-cell death is mediated through protein kinase C (PKC)delta. To examine the role of PKCdelta signaling in vivo, transgenic mice overexpressing a kinase-negative PKCdelta (PKCdeltaKN) selectively in beta-cells were generated and analyzed for glucose homeostasis and beta-cell survival. RESEARCH DESIGN AND METHODS: Mice were fed a standard or high-fat diet (HFD). Blood glucose and insulin levels were determined after glucose loads. Islet size, cleaved caspase-3, and PKCdelta expression were estimated by immunohistochemistry. In isolated islet cells apoptosis was assessed with TUNEL/TO-PRO3 DNA staining and the mitochondrial potential by rhodamine-123 staining. Changes in phosphorylation and subcellular distribution of forkhead box class O1 (FOXO1) were analyzed by Western blotting and immunohistochemistry. RESULTS: PKCdeltaKN mice were protected from HFD-induced glucose intolerance. This was accompanied by increased insulin levels in vivo, by an increased islet size, and by a reduced staining of beta-cells for cleaved caspase-3 compared with wild-type littermates. In accordance, long-term treatment with palmitate increased apoptotic cell death of isolated islet cells from wild-type but not from PKCdeltaKN mice. PKCdeltaKN overexpression protected islet cells from palmitate-induced mitochondrial dysfunction and inhibited nuclear accumulation of FOXO1 in mouse islet and INS-1E cells. The inhibition of nuclear accumulation of FOXO1 by PKCdeltaKN was accompanied by an increased phosphorylation of FOXO1 at Ser256 and a significant reduction of FOXO1 protein. CONCLUSIONS: Overexpression of PKCdeltaKN in beta-cells protects from HFD-induced beta-cell failure in vivo by a mechanism that involves inhibition of fatty acid-mediated apoptosis, inhibition of mitochondrial dysfunction, and inhibition of FOXO1 activation.
Assuntos
Intolerância à Glucose/prevenção & controle , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/fisiologia , Proteína Quinase C-delta/genética , Animais , Apoptose , Glicemia/metabolismo , Técnicas de Cultura de Células , Morte Celular , Dieta , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Insulina/análise , Insulina/sangue , Insulina/genética , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Proteína Quinase C-delta/deficiência , Rodamina 123/farmacologiaRESUMO
Aberrant PI3K/Akt signaling has been implicated in many human cancers, including prostate carcinomas. Currently different therapeutic strategies target the inhibition of this survival pathway. The nucleoside analog triciribine (TCN), which was initially described as a DNA synthesis inhibitor, has recently been shown to function as an inhibitor of Akt. Here, we demonstrate that TCN inhibits Akt phosphorylation at Thr308 and Ser473 and Akt activity in the human prostate cancer cell line PC-3. In addition, TCN sensitized PC-3 cells to TRAIL- and anti-CD95-induced apoptosis, whereas the cells remained resistant to DNA damaging chemotherapeutics. The observed sensitization essentially depended on the phosphorylation status of Akt. Thus, prostate cancer cell lines displaying constitutively active Akt, e.g. PC-3 or LNCaP, were sensitized to death receptor-induced apoptosis. Most importantly with respect to therapeutic application, derivatives of both TCN and TRAIL are already tested in current clinical trials. Therefore, this combinatorial treatment might open a promising therapeutic approach for the elimination of hormone-refractory prostate cancers, which are largely resistant to conventional DNA damaging anticancer drugs or irradiation.
Assuntos
Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Ribonucleosídeos/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspases/metabolismo , Humanos , Immunoblotting , Masculino , Mitocôndrias/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , RNA Interferente Pequeno/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Teste de Sequência Alfanumérica , Células Tumorais CultivadasRESUMO
The Ca(2+)-dependent membrane-spanning classical cadherins bind directly to cytosolic catenins. This cadherin-catenin interaction is known to be critical for the fundamental role of cadherins in cell-cell adhesion. The small subfamily of the 7D-cadherins, however, cannot interact with catenins due to their highly truncated cytoplasmic tail. Thus far, no cytoplasmic interaction partner for the 7D-cadherins has been described. With the use of the cytoplasmic domain of the Ksp (kidney-specific)-cadherin, which belongs to the family of 7D-cadherins, as bait in affinity chromatography with human kidney lysates, the small heat-shock protein alpha B-crystallin was identified by matrix-assisted laser desorption/ionization-time-of-flight analysis as a cytosolic binding partner of Ksp-cadherin. This interaction was verified by co-immunoprecipitation analysis. With the use of overlapping peptides representing the entire alpha B-crystallin molecule, the N-terminal part of alpha B-crystallin, which does not possess chaperone activity, was identified as responsible for the binding to Ksp-cadherin. This interaction was found to be specific since only the cytoplasmic domain of Ksp-cadherin, but not LI (liver-intestine)-cadherin (another member of the 7D-cadherin family), interacted with alpha B-crystallin. In the human kidney, both alpha B-crystallin and Ksp-cadherin co-localize to cells of the collecting duct. They also co-localize with the actin cytoskeleton and co-precipitate with the latter. These findings suggest that the interaction of Ksp-cadherin with alpha B-crystallin is important for the connection of Ksp-cadherin to the cytoskeleton and thus for maintaining tissue integrity in the kidney.
Assuntos
Caderinas/metabolismo , Rim/metabolismo , Cadeia B de alfa-Cristalina/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Citoplasma/química , Citoplasma/metabolismo , Humanos , Rim/química , Dados de Sequência Molecular , Mapeamento de Interação de Proteínas , Cadeia B de alfa-Cristalina/análiseRESUMO
The extracellular domains of receptor-type protein-tyrosine phosphatases (PTPs) contain a diverse range of protein modules like fibronectin- or immunoglobulin-like structures. These are frequently expressed in a tissue- and development specific manner as splice variants. The extracellular domain of PTPalpha is rather short and heavily glycosylated. Two splice variants are known, which it differs by an exon encoding nine amino acids within the extracellular domain. We have analyzed the expression pattern of both variants and found that the smaller form is ubiquitously expressed while the larger form was found at an increased level only in brain, some skeletal muscle and differentiating cells like granule neurons, adipocytes and myotubes. The phosphatase activity of both forms was similar when tested in vitro using para-nitrophenylphosphate as a substrate and in a transient expression system with the substrates c-Fyn or c-Src. In a quantitative focus formation assay the capability of the larger form to activate Src-dependent focus formation in intact cells was increased more than twofold whereas the capability to dephosphorylate the insulin receptor in a BHK cell system was similar. We conclude that the two splice variants of PTPalpha are expressed differentially and regulate c-Src activity in different ways.
Assuntos
Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Células 3T3-L1 , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Células NIH 3T3 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Ratos , Receptor de Insulina/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores , TransfecçãoRESUMO
Microtubules are important for the turnover of podosomes, dynamic, actin-rich adhesions implicated in migration and invasion of monocytic cells. The molecular basis for this functional dependency, however, remained unclear. Here, we show that contact by microtubule plus ends critically influences the cellular fate of podosomes in primary human macrophages. In particular, we identify the kinesin KIF1C, a member of the Kinesin-3 family, as a plus-end-enriched motor that targets regions of podosome turnover. Expression of mutation constructs or small interfering RNA-/short hairpin RNA-based depletion of KIF1C resulted in decreased podosome dynamics and ultimately in podosome deficiency. Importantly, protein interaction studies showed that KIF1C binds to nonmuscle myosin IIA via its PTPD-binding domain, thus providing an interface between the actin and tubulin cytoskeletons, which may facilitate the subcellular targeting of podosomes by microtubules. This is the first report to implicate a kinesin in podosome regulation and also the first to describe a function for KIF1C in human cells.
Assuntos
Estruturas da Membrana Celular/fisiologia , Cinesinas/fisiologia , Macrófagos/fisiologia , Microtúbulos/fisiologia , Diferenciação Celular , Estruturas da Membrana Celular/ultraestrutura , Células Cultivadas , Clonagem Molecular , Escherichia coli , Humanos , Cinesinas/deficiência , Cinesinas/genética , Macrófagos/citologia , Microinjeções , Mutagênese , Plasmídeos , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
BACKGROUND: Members of the Kinesin-3 family of kinesin-like proteins mediate transport of axonal vesicles (KIF1A, KIF1Bbeta), distribution of mitochondria (KIF1Balpha) and anterograde Golgi to ER vesicle transport (KIF1C). Until now, little is known about the regulation of kinesin-like proteins. Several proteins interact with members of this protein family. Here we report on a novel, KIF1 binding protein (KBP) that was identified in yeast two-hybrid screens. RESULTS: KBP was identified by using the yeast-two-hybrid system with an amino-terminal fragment of KIF1C as a bait that is strongly homologous to KIF1B. Here we investigated the interaction of KBP and KIF1B. The full length proteins coimmunoprecipitated after overexpression and in untransfected 293 cells. Immunofluorescence experiments revealed that KBP was mainly localized to mitochondria, as has been described for KIF1Balpha. Overexpression of a deletion mutant or reduction of the KBP protein level using an anti-sense construct led to an aggregation of mitochondria. Such an effect is probably due to the lower activity of KIF1Balpha in the absence of KBP, as was revealed in motility assays. CONCLUSION: KBP is a new binding partner for KIF1Balpha that is a regulator of its transport function and thus represents a new type of kinesin interacting protein.
Assuntos
Cinesinas/metabolismo , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Linhagem Celular , Humanos , Cinesinas/genética , Cinesinas/fisiologia , Camundongos , Mitocôndrias/química , Proteínas Mitocondriais/fisiologia , Proteínas Motores Moleculares , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Serpinas/genética , Serpinas/metabolismo , Serpinas/fisiologia , Distribuição Tecidual , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
In recent years, recombinant DNA technology has been used to design insulin molecules that overcome the limitations of regular insulin in mealtime supplementation. However, safety issues have been raised with these alternatives, as the alteration of the three-dimensional structure may alter the interaction with the insulin and/or IGF-I receptors and therefore lead to the activation of alternate metabolic as well as mitogenic signaling pathways. It is therefore essential to carefully study acute and long-term effects in a preclinical state, as insulin therapy is meant to be a lifelong treatment. In this study, we determined in vivo the insulin receptor signaling characteristics activated by insulin glulisine (Lys(B3), Glu(B29)) at the level of insulin receptor phosphorylation, insulin receptor substrate phosphorylation, and downstream signaling elements such as phosphatidylinositol (PI) 3-kinase, AKT, and mitogen-activated protein kinase. C57BL/6 mice were injected with insulin glulisine or regular insulin and Western blot analysis was performed for liver and muscle tissue. The extent and time course of insulin receptor phosphorylation and activation of downstream signaling elements after insulin glulisine treatment was similar to that of human regular insulin in vivo. Moreover, insulin signaling in hypothalamic tissue determined by PI 3-kinase activity was comparable. Therefore, insulin glulisine may be a useful tool for diabetes treatment.
Assuntos
Insulina/análogos & derivados , Insulina/farmacologia , Receptor de Insulina/fisiologia , Animais , Humanos , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Receptor de Insulina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Cytoplasmic protein tyrosine kinases of the Src family play a pivotal role in the regulation of cellular processes including proliferation and differentiation. Among other functions, Src kinases are involved in regulating the cell architecture. In an approach to identify protein tyrosine kinases from the medically important parasite Schistosoma mansoni, we isolated the TK3 gene by degenerate primer PCR and cDNA library screening. Sequencing of the complete cDNA and data-base analyses indicated that TK3 is a Src family kinase. Its predicted size of 71 kDa was confirmed by Western blot analysis. Southern blot analysis showed that TK3 is a single-copy gene, and Northern blot and RT-PCR experiments indicated its expression in both sexes and throughout development. Localization studies by in situ hybridization and immunolocalization revealed that TK3 is predominantly expressed in the reproductive organs such as the testes of the male and the ovary as well as the vitellarium of the female. Its enzymatic activity was confirmed by functional analyses. In transient transfection experiments with HEK293 cells, TK3 phosphorylated the well-known Src-kinase substrate p130 Cas, an intracellular scaffolding protein. Yeast two-hybrid screenings in a heterologous invertebrate system identified dAbi, vinculin and tubulin as binding partners, representing molecules that fulfill functions in the cell architecture of many organisms. These findings suggest that TK3 may play a role in signal transduction pathways organizing the cytoskeleton in the gonads of schistosomes.
Assuntos
Regulação da Expressão Gênica , Schistosoma mansoni/enzimologia , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Citoesqueleto/fisiologia , DNA de Helmintos/química , DNA de Helmintos/isolamento & purificação , Gônadas/enzimologia , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Proteínas de Helminto/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Filogenia , Ligação Proteica , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Tubulina (Proteína)/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Vinculina/metabolismo , Quinases da Família src/química , Quinases da Família src/genética , Quinases da Família src/isolamento & purificaçãoRESUMO
CUB-domain-containing protein 1 (CDCP1) is a novel transmembrane molecule that is expressed in metastatic colon and breast tumors as well as on the surface of hematopoietic stem cells. In this study, we used multiparameter flow cytometry and antibodies against CDCP1 to analyze the expression of CDCP1 on defined hematopoietic cell subsets of different sources. In addition, CDCP1 expression on leukemic blasts and on cells with nonhematopoietic stem/progenitor cell phenotypes was determined. Here we demonstrate that a subset of bone marrow (BM), cord blood (CB), and mobilized peripheral blood (PB) CD34+ cells expressed this marker and that CDCP1 was detected on CD34(+)CD38- BM stem/progenitor cells but not on mature PB cells. Analysis of leukemic blasts from patients with acute lymphoblastic leukemia, acute myeloid leukemia, and chronic myeloid leukemia in blast crisis revealed that CDCP1 is predominantly expressed on CD34(+)CD133+ myeloid leukemic blasts. However, CDCP1 was not strictly correlated with CD34 and/or CD133 expression, suggesting that CDCP1 is a novel marker for leukemia diagnosis. Stimulation of CD34+ BM cells with CDCP1-reactive monoclonal antibody CUB1 resulted in an increased (approximately twofold) formation of erythroid colony-forming units, indicating that CDCP1 plays an important role in early hematopoiesis. Finally, we show that CDCP1 is also expressed on cells phenotypically identical to mesenchymal stem/progenitor cells (MSCs) and neural progenitor cells (NPCs). In conclusion, CDCP1 is not only a novel marker for immature hematopoietic progenitor cell subsets but also unique in its property to recognize cells with phenotypes reminiscent of MSC and NPC.
Assuntos
Medula Óssea/metabolismo , Moléculas de Adesão Celular/metabolismo , Sangue Fetal/metabolismo , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Neoplasias/metabolismo , Antígeno AC133 , ADP-Ribosil Ciclase/metabolismo , ADP-Ribosil Ciclase 1 , Animais , Antígenos CD/metabolismo , Antígenos CD34/metabolismo , Antígenos de Neoplasias , Biomarcadores , Neoplasias da Mama/metabolismo , Moléculas de Adesão Celular/genética , Clonagem Molecular , Neoplasias do Colo/metabolismo , Sangue Fetal/citologia , Citometria de Fluxo , Glicoproteínas/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , Leucemia Mieloide Aguda/metabolismo , Glicoproteínas de Membrana , Camundongos , Células NIH 3T3 , Proteínas de Neoplasias/genética , Peptídeos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismoRESUMO
Insulin receptor substrate-1 (IRS-1) was recently identified as a novel upstream substrate for the insulin-activated protein kinase C (PKC)-zeta. This interaction down-regulates insulin signal transduction under hyper-insulinemic conditions. To clarify the molecular mechanism of this feedback loop, we sought to identify the PKC-zeta phosphorylation sites of IRS-1 and to investigate their biological significance. Upon incubation of recombinant IRS-1 fragments with PKC-zeta, we identified Ser(318) of rat IRS-1 (Ser(323) in human IRS-1) as the major in vitro phosphorylation site (confirmed by mutation of Ser(318) to alanine). To monitor phosphorylation of Ser(318) in cellular extracts, we prepared a polyclonal phosphosite-specific antibody. The biological significance was studied in baby hamster kidney cells stably expressing the insulin receptor (BHK(IR)). Using the phospho-Ser(318)-specific antibody we observed that insulin stimulates phosphorylation of Ser(318) in IRS-1, which is mediated, at least partially, by PKC-zeta. Moreover, we found that the previously described insulin-stimulated, PKC-zeta-mediated inhibition of the interaction of IRS-1 with the insulin receptor and the reduced tyrosine phosphorylation of IRS-1 was abrogated by mutation of IRS-1 Ser(318) to alanine. These results, generated in BHK(IR) cells, suggest that phosphorylation of Ser(318) by PKC-zeta might contribute to the inhibitory effect of prolonged hyperinsulinemia on IRS-1 function.
Assuntos
Chaperonas Moleculares/fisiologia , Fosfoproteínas/metabolismo , Receptor de Insulina/metabolismo , Tirosina/metabolismo , Proteínas 14-3-3 , Sequência de Aminoácidos , Animais , Linhagem Celular , Cricetinae , Humanos , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Dados de Sequência Molecular , Fosforilação , Ratos , Serina/metabolismoRESUMO
The atypical isoforms of protein kinase C (aPKCs) play an important role in insulin signaling and are involved in insulin-stimulated glucose uptake in different cell systems. On the other hand, aPKCs also are able to negatively regulate important proteins for insulin signaling, like phosphatidylinositol 3-kinase and protein kinase B/Akt. To find aPKC-interacting proteins that may promote positive or negative activities of aPKCs, a yeast two-hybrid screen was performed. Partitioning-defective protein 6 (Par6) was detected in human cDNA libraries of different adult insulin-sensitive tissues. Although Par6 is known as an aPKC-interacting protein during development, no role for Par6 in insulin signaling has been reported so far. We therefore studied the effects of Par6 overexpression in C2C12 murine myoblasts. In these cells, Par6 associated constitutively with endogenous aPKCs, and the expression level as well as the activity of aPKCs were increased. Insulin-dependent association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate 1 was hampered and the phosphorylation of Akt/glycogen synthase kinase-3alpha/beta was significantly impaired after stimulation with insulin or with platelet-derived growth factor. Consequently, insulin-dependent glycogen synthesis was down-regulated (1.44 vs. 2.24 fold, P < 0.01). We therefore suggest that Par6 acts as a negative regulator of the insulin signal.
Assuntos
Glicogênio/biossíntese , Insulina/metabolismo , Mioblastos/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Regulação para Baixo/fisiologia , Ativação Enzimática/fisiologia , Humanos , Isoenzimas/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Técnicas do Sistema de Duplo-HíbridoRESUMO
Increasing evidence indicates a role of insulin signalling for insulin secretion from the pancreatic beta-cells. Therefore, regulators of insulin signalling, like protein tyrosine phosphatases, could also have an impact on insulin secretion. Here, we investigated a possible role of the negative regulator protein tyrosine phosphatase alpha (PTP alpha) for insulin secretion. RT-PCR analysis confirmed that both splice variants of the extracellular domain of PTP alpha that vary by an insert of 9 amino acids are expressed in human islets and insulinoma cells (INS-1E, RIN1046-38). Overexpression of the wild type PTP alpha splice variant containing the 9 amino acids reduced insulin secretion, as did a mutant form unable to bind Grb2 (Tyr798Phe). By contrast, overexpression of a phosphatase inactive mutant improved insulin secretion. These data reveal a functional relevance of PTP alpha for insulin secretion.
Assuntos
Glucose/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais/fisiologia , Linhagem Celular , Espaço Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Secreção de Insulina , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores , Proteínas Recombinantes/metabolismoRESUMO
CDCP1 is a transmembrane protein that contains three CUB domains within the extracellular region and a hexalysine stretch within the cytoplasmic region. CDCP1 mRNA is highly expressed in lung and colon tumors and in the erythroleukemic cell line K562. To analyze CDCP1 protein expression, monoclonal antibodies against the extracellular domain of CDCP1 were raised. For this purpose, CDCP1 was overexpressed in NIH-3T3 cells. Balb/c mice were then immunized with the resultant cell line NIH-3T3/huCDCP1. After fusion of SP2/0 cells with immune spleen cells, hybridoma clones were selected that secreted antibodies reacting with NIH-3T3/huCDCP1 cells but not with parental cells. Four antibodies (CUB1-CUB4) were obtained that fulfilled these criteria. Screening of peripheral blood cells revealed that the antibodies did not recognize mature lymphocytes, monocytes, granulocytes, erythrocytes, or platelets. In contrast, multi-color analyses revealed that CDCP1 protein is almost exclusively expressed on a subset of CD34(+) stem/progenitor cells in bone marrow. Transplantation of purified CDCP1(+) cells into NOD/SCID mice resulted in engraftment of human cells with multi-lineage differentiation potential, suggesting that CDCP1 is a novel marker for hematopoietic stem cells.
Assuntos
Moléculas de Adesão Celular/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Neoplasias/metabolismo , Células 3T3 , Animais , Antígenos CD , Antígenos de Neoplasias , Biomarcadores , Moléculas de Adesão Celular/genética , Diferenciação Celular , Citometria de Fluxo , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Camundongos SCID , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
Previously we have shown that protein kinase C (PKC)-mediated reorganization of the actin cytoskeleton in smooth muscle cells is transmitted by the non-receptor tyrosine kinase, Src. Several authors have described how 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation of cells results in an increase of Src activity, but the mechanism of the PKC-mediated Src activation is unknown. Using PKC isozymes purified from Spodoptera frugiperda insect cells, we show here that PKC is not able to activate Src directly. Our data reveal that the PKC-dependent Src activation occurs via the activation of the protein tyrosine phosphatase (PTP) PTP alpha. PTP alpha becomes activated in vivo after TPA stimulation. Further, we show that PKC delta phosphorylates and activates only PTP alpha in vitro but not any other of the TPA-responsive PKC isozymes that are expressed in A7r5 rat aortic smooth muscle cells. To further substantiate our data, we show that cells lacking PKC delta have a markedly reduced PTP alpha and Src activity after 12-O-tetradecanoylphorbol-13-acetate stimulation. These data support a model in which the main mechanism of 12-O-tetradecanoylphorbol-13-acetate-induced Src activation is the direct phosphorylation and activation of PTP alpha by PKC delta, which in turn dephosphorylates and activates Src.