RESUMO
IGF-I and its receptor IGF-IR are seen as critical effectors of muscle hypertrophy, a notion recently questioned. Using MKR transgenic mice that express a dominant negative IGF-IR only in skeletal muscle, we have examined the role of the IGF-IR signaling in differentiation and repair of muscle fibers after damage-induced muscle regeneration. This process is impaired in MKR muscle, with incomplete regeneration, persistence of infiltrating cells and sustained expression of differentiation markers. Analysis of MKR and WT muscle-derived progenitor stem cells and myoblasts showed twice as many such cells in MKR muscle and an incomplete in vitro differentiation, that is, despite similar levels of myogenin expression, the level of fusion of MKR myoblasts was significantly reduced in comparison to WT myoblasts. These data show IGF-IR signaling is not only required at early hyperplasia stages of muscle differentiation, but also for late stages of myofiber maturation and hypertrophy.
Assuntos
Diferenciação Celular/fisiologia , Músculo Esquelético/fisiologia , Mioblastos/fisiologia , Receptor IGF Tipo 1/metabolismo , Regeneração/fisiologia , Animais , Células Cultivadas , Técnicas de Inativação de Genes , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Músculo Esquelético/citologia , Músculo Esquelético/patologia , Mioblastos/citologia , Receptor IGF Tipo 1/genética , Transdução de Sinais/fisiologia , Células-Tronco/fisiologiaRESUMO
Proper chromosome segregation is required to maintain the appropriate number of chromosomes from one cell generation to another and to prevent aneuploidy, which is mainly found in solid cancers. A correct mitotic spindle is necessary to accomplish such a process. Aurora kinases play critical roles in chromosome segregation and cell division; their deregulation impairs spindle assembly, checkpoint function and cell division causing chromosome mis-segregation. These kinases have been implicated in tumorigenesis. Aurora-A (AurA), in particular has been identified as a cancer-susceptibility gene, is overexpressed in a number of tumors and is required for G2/M transition and spindle assembly. ASAP is a novel spindle-associated protein, the deregulation of which induces severe mitotic defects. We show here that ASAP is a novel substrate of AurA kinase. We have identified serine 625 as the major phosphorylation site for AurA in vivo and localized the phosphorylated form of ASAP to centrosomes from late G2 to telophase, and around the midbody during cytokinesis. AurA depletion induces a proteasome-dependent degradation of ASAP. ASAP depletion induces spindle defects rescued by the expression of the phosphorylation-mimetic mutant ASAP-S625E and not by the non-phosphorylatable mutant ASAP-S625A. Microinjection of mono-specific S625 phospho-antibodies also impaired spindle formation and mitosis. These results strongly indicate that the phosphorylation of ASAP on S625 by AurA is required for bipolar spindle assembly and is essential for a correct mitotic progression. All together, these results suggest that we have identified a novel AurA substrate, pointing out ASAP as a new potential target for antitumoral drugs.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/metabolismo , Aurora Quinases , Linhagem Celular Tumoral , Centrossomo , Citocinese , Humanos , Fosforilação , Serina/metabolismoRESUMO
MyoD is a critical myogenic factor induced rapidly upon activation of quiescent satellite cells, and required for their differentiation during muscle regeneration. One of the two enhancers of MyoD, the distal regulatory region, is essential for MyoD expression in postnatal muscle. This enhancer contains a functional divergent serum response factor (SRF)-binding CArG element required for MyoD expression during myoblast growth and muscle regeneration in vivo. Electrophoretic mobility shift assay, chromatin immunoprecipitation, and microinjection analyses show this element is a hybrid SRF- and MEF2 Binding (SMB) sequence where myocyte enhancer factor 2 (MEF2) complexes can compete out binding of SRF at the onset of differentiation. As cells differentiate into postmitotic myotubes, MyoD expression no longer requires SRF but instead MEF2 binding to this dual-specificity element. As such, the MyoD enhancer SMB element is the site for a molecular relay where MyoD expression is first initiated in activated satellite cells in an SRF-dependent manner and then increased and maintained by MEF2 binding in differentiated myotubes. Therefore, SMB is a DNA element with dual and stage-specific binding activity, which modulates the effects of regulatory proteins critical in controlling the balance between proliferation and differentiation.
Assuntos
Diferenciação Celular , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento Muscular/fisiologia , Proteína MyoD , Fatores de Regulação Miogênica/metabolismo , Fator de Resposta Sérica/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Genes Reporter , Fatores de Transcrição MEF2 , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Proteína MyoD/genética , Proteína MyoD/metabolismo , Fatores de Regulação Miogênica/genética , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ligação Proteica , Regeneração/fisiologia , Fator de Resposta Sérica/genéticaRESUMO
BACKGROUND: Immune activities of monocytes (MOs) can be altered within the microenvironment of solid malignancies, including breast cancer. Metformin (1,1-dimethylbiguanide hydrochloride, MET), has been shown to decrease tumor cell proliferation, but its effects have yet to be explored with respect to MOs (monocytes) activity during their crosstalk with breast cancer cells. Here, we investigated the effects of MET on overall phenotypic functional activities, including cellular immunometabolism and protective redox signaling based-biomarkers, intracellular free calcium ions (ifCa2+), phagocytosis and co-operative cytokines (IFN-γ and IL-10) of autologous MOs before and during their interplay with primary ER-/PR-/HER2+ breast cancer cells. METHODS: Human primary breast cancer cells were either cultured alone or co-cultured with autologous MOs before treatment with MET. RESULTS: MET downregulated breast cancer cell proliferation and phagocytosis, while having no significant effect on the ratio of phosphorylated Akt (p-Akt) to total Akt. Additionally, we observed that, in the absence of MET treatment, the levels of lactate dehydrogenase (LDH)-based cytotoxicity, catalase, ifCa2+, IL-10 and arginase activity were significantly reduced in co-cultures compared to levels in MOs cultured alone whereas levels of inducible nitric oxide synthase (iNOS) activity were significantly increased. In contrast, MET treatment reduced the effects measured in co-culture on the levels of LDH-based cytotoxicity, arginase activity, catalase, ifCa2+, and IFN-γ. MET also induced upregulation of both iNOS and arginase in MO cells, although the increase did not reach significant difference for iNOS activity. Moreover, MET induced a robust increase of superoxide dismutase (SOD) activity in MOs, but not in MOs co-cultured with breast cancer cells. Furthermore, MET markedly upregulated the levels of IFN-γ production and downregulated those of IL-10 in isolated MOs, while inducing a slight opposing up-regulation of IL-10 production in co-cultures. CONCLUSIONS: Our results show that the biomarkers of phenotypic functional activities of MOs are modified after co-culturing with primary human breast cancer cells. Treatment of co-cultures with MET resulted in increased release of antitumor cytokine IFN-γ and ifCa2+, and increased cell necrosis during breast cancer cells-MOs crosstalk.
Assuntos
Biomarcadores/metabolismo , Neoplasias da Mama/metabolismo , Metformina/farmacologia , Monócitos/citologia , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/metabolismo , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismoRESUMO
Protein kinase B (PKB/Akt) is an important modulator of insulin signaling, cell proliferation, and survival. Using small interfering RNA duplexes in nontransformed mammalian cells, we show that only Akt1 is essential for cell proliferation, while Akt2 promotes cell cycle exit. Silencing Akt1 resulted in decreased cyclin A levels and inhibition of S-phase entry, effects not seen with Akt2 knockdown and specifically rescued by microinjection of Akt1, not Akt2. In differentiating myoblasts, Akt2 knockout prevented myoblasts from exiting the cell cycle and showed sustained cyclin A expression. In contrast, overexpression of Akt2 reduced cyclin A and hindered cell cycle progression in M-G1 with increased nuclear p21. p21 is a major target in the differential effects of Akt isoforms, with endogenous Akt2 and not Akt1 binding p21 in the nucleus and increasing its level. Accordingly, Akt2 knockdown cells, and not Akt1 knockdown cells, showed reduced levels of p21. A specific Akt2/p21 interaction can be reproduced in vitro, and the Akt2 binding site on p21 is similar to that in cyclin A spanning T145 to T155, since (i) prior incubation with cyclin A prevents Akt2 binding, (ii) T145 phosphorylation on p21 by Akt1 prevents Akt2 binding, and (iii) binding Akt2 prevents phosphorylation of p21 by Akt1. These data show that specific interaction of the Akt2 isoform with p21 is key to its negative effect on normal cell cycle progression.
Assuntos
Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Sítios de Ligação , Ciclo Celular , Linhagem Celular , Proliferação de Células , Humanos , Camundongos , Modelos Biológicos , Células NIH 3T3 , Ligação Proteica , Isoformas de Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , TransfecçãoRESUMO
Akt1 and Akt2 are the major isoforms of Akt expressed in muscle cells and muscle tissue. We have performed siRNA silencing of Akt1 and Akt2 in C2 myoblasts to characterize their specific implication in muscle differentiation. Whereas silencing Akt2, and not Akt1, inhibited cell cycle exit and myoblast differentiation, Akt2 overexpression led to an increased proportion of differentiated myoblasts. In addition, we demonstrate that Akt2 is required for myogenic conversion induced by MyoD overexpression in fibroblasts. We show Akt2, but not Akt1, binds Prohibitin2/Repressor of Estrogen Activator, PHB2/REA, a protein recently implicated in transcriptionnal repression of myogenesis. Co-immunoprecipitation experiments on endogenous proteins showed the Akt2-REA complex does not contain Prohibitin1. We have analyzed expression and localization of PHB2/REA during proliferation and differentiation of mouse and human myoblasts. PHB2/REA shows punctated nuclear staining which partially co-localizes with Akt2 in differentiated myotubes and PHB2 levels decrease at the onset of myogenic differentiation concomitant with an increase in Akt2. There appears to be an inverse correlation between Akt2 and PHB2 protein levels where cells silenced for Akt2 expression show increased level of PHB2/REA and overexpression of Akt2 resulted in decreased Prohibitin2/REA. Taken together, these results, along with our previous observations, clearly show that Akt2 and not Akt1 plays a major and early role in cell cycle exit and myogenic differentiation and this function involves its specific interaction with PHB2/REA.
Assuntos
Diferenciação Celular/fisiologia , Músculo Esquelético/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/fisiologia , Proteínas Repressoras/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular , Núcleo Celular/metabolismo , Meios de Cultura , Citoplasma/metabolismo , Fibroblastos/metabolismo , Técnica Direta de Fluorescência para Anticorpo , Perfilação da Expressão Gênica , Camundongos , Microinjeções , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/enzimologia , Miogenina/metabolismo , Testes de Precipitina , Proibitinas , Ligação Proteica , Isoformas de Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/genética , TransfecçãoRESUMO
The process through which macromolecules penetrate the plasma membrane of mammalian cells remains poorly defined. We have examined whether natural cellular events modulate the capacity of cells to take up agents applied extraneously. Herein, we report that during mitosis and in a cell type-independent manner, cells exhibit a natural ability to absorb agents present in the extracellular environment up to 150 kDa as assessed using fluorescein isothiocyanate-dextrans. This event is exclusive to the mitotic period and not observed during G0, G1, S, or G2 phase. During mitosis, starting in advanced prophase, oligonucleotides, active enzymes, and polypeptides are efficiently taken into mitotic cells. This uptake of macromolecules during mitosis still takes place in the presence of cytochalasin D or nocodazole, showing no requirement for intact microtubules or actin filaments in this process. However, cell rounding up, which still takes place in the presence of either of these drugs in mitotic cells, appears to be a key event in this process. Indeed, limited trypsinization of adherent cells mimics both the cell retraction and macromolecule uptake observed as cells enter mitosis. A plasmid DNA encoding green fluorescent protein (3.3Mda) coated with an 18 amino acid peptide is efficiently expressed when applied onto synchronized G2/M fibroblasts, whereas little or no expression is observed when the coated plasmid is applied onto asynchronous cell cultures. This shows that such coating peptides are only efficient for their encapsulating and protective effect on the plasmid DNA to be "vectorized" rather than acting as true vectors.
Assuntos
Fibroblastos/fisiologia , Vetores Genéticos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Mitose/fisiologia , Animais , Transporte Biológico , Proteínas de Transporte/metabolismo , Células Cultivadas , Fibroblastos/citologia , Peroxidase do Rábano Silvestre , Fragmentos de Peptídeos/metabolismo , Plasmídeos/metabolismo , Ratos , TransfecçãoRESUMO
In view of the common regulatory mechanism that induces transcription of the mitotic phosphatase cdc25C and cyclin A at the beginning of S-phase, we investigated whether cdc25C was required for S-phase transit. Here, we show that in both nontransformed human fibroblasts and HeLa cells, cdc25C protein levels significantly increased concomitant with S-phase onset and cyclin A synthesis. Activity measurements on immunoprecipitates from synchronized HeLa cells revealed a sharp rise in cdc25C-associated phosphatase activity that coincided with S-phase. Microinjection of various antisense-cdc25C molecules led to inhibition of DNA synthesis in both HeLa cells and human fibroblasts. Furthermore, transfection of small interfering RNA directed against cdc25C specifically depleted cdc25C in HeLa cells without affecting cdc25A or cdc25B levels. Cdc25C RNA interference was also accompanied by S-phase inhibition. In cells depleted of cdc25C by antisense or siRNA, normal cell cycle progression could be re-established through microinjection of wild-type cdc25C protein but not inactive C377S mutant protein. Taken together, these results show that cdc25C not only plays a role at the G2/M transition but also in the modulation of DNA replication where its function is distinct from that of cdc25A.
Assuntos
Ciclina A/biossíntese , Fase S/fisiologia , Fosfatases cdc25/metabolismo , Ciclo Celular/fisiologia , Células Cultivadas , Células HeLa , Humanos , Mutação , Inibidores da Síntese de Ácido Nucleico/metabolismo , Fosforilação , Interferência de RNA/fisiologia , RNA Antissenso/fisiologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fosfatases cdc25/biossínteseRESUMO
We show here that the distal regulatory region (DRR) of the mouse and human MyoD gene contains a conserved SRF binding CArG-like element. In electrophoretic mobility shift assays with myoblast nuclear extracts, this CArG sequence, although slightly divergent, bound two complexes containing, respectively, the transcription factor YY1 and SRF associated with the acetyltransferase CBP and members of C/EBP family. A single nucleotide mutation in the MyoD-CArG element suppressed binding of both SRF and YY1 complexes and abolished DRR enhancer activity in stably transfected myoblasts. This MyoD-CArG sequence is active in modulating endogeneous MyoD gene expression because microinjection of oligonucleotides corresponding to the MyoD-CArG sequence specifically and rapidly suppressed MyoD expression in myoblasts. In vivo, the expression of a transgenic construct comprising a minimal MyoD promoter fused to the DRR and beta-galactosidase was induced with the same kinetics as MyoD during mouse muscle regeneration. In contrast induction of this reporter was no longer seen in regenerating muscle from transgenic mice carrying a mutated DRR-CArG. These results show that an SRF binding CArG element present in MyoD gene DRR is involved in the control of MyoD gene expression in skeletal myoblasts and in mature muscle satellite cell activation during muscle regeneration.
Assuntos
Genes Reguladores , Proteína MyoD/metabolismo , Mioblastos/metabolismo , Fator de Resposta Sérica/metabolismo , Animais , Sequência de Bases , Humanos , Camundongos , Dados de Sequência MolecularRESUMO
During ex vivo myoblast differentiation, a pool of quiescent mononucleated myoblasts, reserve cells, arise alongside myotubes. Insulin/insulin-like growth factor (IGF) and PKB/Akt-dependent phosphorylation activates skeletal muscle differentiation and hypertrophy. We have investigated the role of glycogen synthase kinase 3 (GSK-3) inhibition by protein kinase B (PKB)/Akt and Wnt/beta-catenin pathways in reserve cell activation during myoblast differentiation and myotube hypertrophy. Inhibition of GSK-3 by LiCl or SB216763, restored insulin-dependent differentiation of C2ind myoblasts in low serum, and cooperated with insulin in serum-free medium to induce MyoD and myogenin expression in C2ind myoblasts, quiescent C2 or primary human reserve cells. We show that LiCl treatment induced nuclear accumulation of beta-catenin in C2 myoblasts, thus mimicking activation of canonical Wnt signaling. Similarly to the effect of GSK-3 inhibitors with insulin, coculturing C2 reserve cells with Wnt1-expressing fibroblasts enhanced insulin-stimulated induction of MyoD and myogenin in reserve cells. A similar cooperative effect of LiCl or Wnt1 with insulin was observed during late ex vivo differentiation and promoted increased size and fusion of myotubes. We show that this synergistic effect on myotube hypertrophy involved an increased fusion of reserve cells into preexisting myotubes. These data reveal insulin and Wnt/beta-catenin pathways cooperate in muscle cell differentiation through activation and recruitment of satellite cell-like reserve myoblasts.
Assuntos
Diferenciação Celular/fisiologia , Tamanho Celular , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fibras Musculares Esqueléticas/citologia , Transdução de Sinais/fisiologia , Animais , Fusão Celular , Linhagem Celular , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Hipertrofia , Indóis/metabolismo , Cloreto de Lítio/metabolismo , Maleimidas/metabolismo , Camundongos , Células Musculares/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Fosforilação , Proteínas Wnt , Proteína Wnt1RESUMO
BACKGROUND: Pancreatic beta cells are unique effectors in the control of glucose homeostasis and their deficiency results in impaired insulin production leading to severe diabetic diseases. Here, we investigated the potential of a population of nonadherent muscle-derived stem cells (MDSC) from adult mouse muscle to differentiate in vitro into beta cells when transplanted as undifferentiated stem cells in vivo to compensate for beta-cell deficiency. RESULTS: In vitro, cultured MDSC spontaneously differentiated into insulin-expressing islet-like cell clusters as revealed using MDSC from transgenic mice expressing GFP or mCherry under the control of an insulin promoter. Differentiated clusters of beta-like cells co-expressed insulin with the transcription factors Pdx1, Nkx2.2, Nkx6.1, and MafA, and secreted significant levels of insulin in response to glucose challenges. In vivo, undifferentiated MDSC injected into streptozotocin (STZ)-treated mice engrafted within 48 h specifically to damaged pancreatic islets and were shown to differentiate and express insulin 10-12 days after injection. In addition, injection of MDSC into hyperglycemic diabetic mice reduced their blood glucose levels for 2-4 weeks. CONCLUSION: These data show that MDSC are capable of differentiating into mature pancreatic beta islet-like cells, not only upon culture in vitro, but also in vivo after systemic injection in STZ-induced diabetic mouse models. Being nonteratogenic, MDSC can be used directly by systemic injection, and this potential reveals a promising alternative avenue in stem cell-based treatment of beta-cell deficiencies.
Assuntos
Células-Tronco Adultas/citologia , Diferenciação Celular , Diabetes Mellitus Experimental/terapia , Células Secretoras de Insulina/citologia , Fatores de Transcrição Maf Maior , Fibras Musculares Esqueléticas/citologia , Transplante de Células-Tronco , Células-Tronco Adultas/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Gerbillinae , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição Maf Maior/genética , Fatores de Transcrição Maf Maior/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Ratos , Ratos Sprague-Dawley , Transativadores/genética , Transativadores/metabolismoRESUMO
Specificity of the cAMP-dependent protein kinase (PKA) pathway relies on an extremely sophisticated compartmentalization mechanism of the kinase within a given cell, based on high-affinity binding of PKA tetramer pools to different A-Kinase Anchoring Proteins (AKAPs). We and others have previously shown that AKAPs-dependent PKA subcellular targeting is a requisite for optimal cAMP-dependent potentiation of insulin exocytosis. We thus hypothesized that a PKA pool may directly anchor to the secretory compartment to potentiate insulin exocytosis. Here, using immunofluorescence analyses combined to subcellular fractionations and purification of insulin secretory granules (ISGs), we identified discrete subpools of type II PKAs, RIIα and RIIß PKAs, along with the catalytic subunit, physically associated with ISGs within pancreatic insulin-secreting ß-cells. Ultrastructural analysis of native rodent ß-cells confirmed in vivo the occurrence of PKA on dense-core ISGs. Isoform-selective disruption of binding of PKAs to AKAPs reinforced the requirement of type II PKA isoforms for cAMP potentiation of insulin exocytosis. This granular localization of PKA was of critical importance since siRNA-mediated depletion of either RIIα or RIIß PKAs resulted in a significant reduction of cAMP-dependent potentiation of insulin release. The present work provides evidence for a previously unrecognized pool of type II PKAs physically anchored to the ß-cell ISGs compartment and supports a non-redundant function for type II PKAs during cAMP potentiation of exocytosis.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Exocitose , Células Secretoras de Insulina/metabolismo , Vesículas Secretórias/metabolismo , Linhagem Celular , Humanos , Isoenzimas/metabolismoRESUMO
We describe a reliable and efficient method for the purification of catalytically active and mutant inactive full-length forms of the human dual specificity phosphatase cdc25C from bacteria. The protocol involves isolating insoluble cdc25C protein in inclusion bodies, solubilization in guanidine HCL, and renaturation through rapid dilution into low salt buffer. After binding renatured proteins to an ion exchange resin, cdc25C elutes in two peaks at 350 and 450 mM NaCl. Analysis by gel exclusion chromatography and enzymatic assays reveals the highest phosphatase activity is associated with the 350 mM NaCl with little or no activity present in the 450 mM peak. Furthermore, active cdc25C has a native molecular mass of 220 kDa consistent with a potential tetrameric complex of the 55-kDa cdc25C protein. Assaying phosphatase activity against artificial substrates pNPP and 3-OMFP reveals a 220 kDa form of the phosphatase is active in a non-phosphorylated state. The protein effectively activates cdk1/cyclin B prokinase complexes in vitro in the absence of cdk1 kinase activity in an orthovanadate sensitive manner but is inactivated by A-kinase phosphorylation. In vitro phosphorylation of purified cdc25C by cdk1/cyclin B1, cdk2/cyclin A2 and cdk2/cyclin E shows that distinct TP/SP mitotic phosphorylation sites on cdc25C are differentially phosphorylated by these 3 cdk/cyclin complexes associated with different levels of cdc25C activation. Finally, we show that endogenous native cdc25C from human cells is present in high molecular weight complexes with other proteins and resolves mostly above 200-kDa. These data show that untagged cdc25C can be purified with a simple protocol as an active dual specificity phosphatase with a native molecular mass consistent with a homo-tetrameric configuration.
Assuntos
Fosfatases cdc25/isolamento & purificação , Fosfatases cdc25/metabolismo , Compostos de Anilina/química , Compostos de Anilina/metabolismo , Proteína Quinase CDC2/metabolismo , Catálise , Ciclina B1 , Quinase 2 Dependente de Ciclina/metabolismo , Humanos , Peso Molecular , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , Fosfatases cdc25/químicaRESUMO
The binding of the cdk inhibitor p21cip1 to Akt2 in the nucleus is an essential component in determining the specific role of Akt2 in the cell cycle arrest that precedes myogenic differentiation. Here, through a combination of biochemical and cell biology approaches, we have addressed the molecular basis of this binding. Using amino-terminal truncation of Akt2, we show that p21cip1 binds at the carboxy terminal of Akt2 since deletion of the first 400 amino acids did not affect the interaction between Akt2 and p21cip1. Pull down using carboxy terminal-truncated Akt2 protein revealed the importance of the region between amino acids 400 and 445 for the binding to p21cip1. Since Akt2_400-445 and Akt2_420-445 peptides could both bind p21cip1, this refines the binding domain on Akt2 between amino acids 420 and 445. In order to confirm these data in living cells, we developed a protocol to synchronize myoblasts at the cell cycle exit point when p21cip1 expression is induced by MyoD before myogenic differentiation. When a synthetic Akt2 peptide spanning the region (410-437) was microinjected in p21-expressing myoblasts, p21cip1 no longer localized exclusively in the nucleus, instead being redistributed throughout the cell, thus showing that injected peptide 410-437 acts to compete with the binding of endogenous Akt2 to p21cip1. Taken together, our data suggest that this 27 amino acid sequence on Akt2 is necessary and sufficient to bind p21cip1 both in vitro and in living cells.
Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Desenvolvimento Muscular/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Western Blotting , Pontos de Checagem do Ciclo Celular/genética , Diferenciação Celular/genética , Clonagem Molecular , Imunofluorescência , Humanos , Camundongos , Dados de Sequência Molecular , Desenvolvimento Muscular/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Homologia de SequênciaRESUMO
Kinases of the Akt family are integral and essential components in growth factor signaling pathways activated downstream of the membrane bound phospho-inositol-3 kinase. In light of strong homologies in the primary amino acid sequence, the three Akt kinases were long surmised to play redundant and overlapping roles in insulin signaling across the spectra of cell and tissue types. Over the last 10 years, work using mouse knockout models, cell specific inactivation, and more recently targeted gene inactivation, has brought into question the redundancy within Akt kinase isoforms and instead pointed to isoform specific functions in different cellular events and diseases. Here we concentrate on the differential roles played by Akt1 and Akt2 in a variety of cellular processes and in particular during cancer biogenesis. In this overview, we illustrate that while Akt1 and 2 are often implicated in many aspects of cellular transformation, the two isoforms frequently act in a complementary opposing manner. Furthermore, Akt1 and Akt2 kinases interact differentially with modulating proteins and are necessary in relaying roles during the evolution of cancers from deregulated growth into malignant metastatic killers. These different actions of the two isoforms point to the importance of treatments targeting isoform specific events in the development of effective approaches involving Akt kinases in human disease.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , Proliferação de Células , Humanos , Insulina/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Metástase Neoplásica , Isoformas de Proteínas , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
BACKGROUND: The dual specificity phosphatase cdc25C was the first human cdc25 family member found to be essential in the activation of cdk1/cyclin B1 that takes place at the entry into mitosis. Human cdc25C is phosphorylated on Proline-dependent SP and TP sites when it becomes active at mitosis and the prevalent model is that this phosphorylation/activation of cdc25C would be part of an amplification loop with cdk1/cyclin B1. METHODOLOGY/PRINCIPAL FINDINGS: Using highly specific antibodies directed against cdc25C phospho-epitopes, pT67 and pT130, we show here that these two phospho-forms of cdc25C represent distinct pools with differential localization during human mitosis. Phosphorylation on T67 occurs from prophase and the cdc25C-pT67 phospho-isoform closely localizes with condensed chromosomes throughout mitosis. The phospho-T130 form of cdc25C arises in late G2 and associates predominantly with centrosomes from prophase to anaphase B where it colocalizes with Plk1. As shown by immunoprecipitation of each isoform, these two phospho-forms are not simultaneously phosphorylated on the other mitotic TP sites or associated with one another. Phospho-T67 cdc25C co-precipitates with MPM2-reactive proteins while pT130-cdc25C is associated with Plk1. Interaction and colocalization of phosphoT130-cdc25C with Plk1 demonstrate in living cells, that the sequence around pT130 acts as a true Polo Box Domain (PBD) binding site as previously identified from in vitro peptide screening studies. Overexpression of non-phosphorylatable alanine mutant forms for each isoform, but not wild type cdc25C, strongly impairs mitotic progression showing the functional requirement for each site-specific phosphorylation of cdc25C at mitosis. CONCLUSIONS/SIGNIFICANCE: These results show for the first time that in human mitosis, distinct phospho-isoforms of cdc25C exist with different localizations and interacting partners, thus implying that the long-standing model of a cdc25C/cdk1 multi-site auto amplification loop is implausible.
Assuntos
Mitose/fisiologia , Fosfatases cdc25/metabolismo , Células Cultivadas , Centrossomo/metabolismo , Imunofluorescência , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Mitose/genética , Mutagênese Sítio-Dirigida , Mutação , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Treonina/metabolismo , Fosfatases cdc25/genéticaRESUMO
Stem cells with the ability to differentiate in specialized cell types can be extracted from a wide array of adult tissues including skeletal muscle. Here we have analyzed a population of cells isolated from skeletal muscle on the basis of their poor adherence on uncoated or collagen-coated dishes that show multi-lineage differentiation in vitro. When analysed under proliferative conditions, these cells express stem cell surface markers Sca-1 (65%) and Bcrp-1 (80%) but also MyoD (15%), Neuronal beta III-tubulin (25%), GFAP (30%) or Nkx2.5 (1%). Although capable of growing as non-attached spheres for months, when given an appropriate matrix, these cells adhere giving rise to skeletal muscle, neuronal and cardiac muscle cell lineages. A similar cell population could not be isolated from either bone marrow or cardiac tissue suggesting their specificity to skeletal muscle. When injected into damaged muscle, these non-adherent muscle-derived cells are retrieved expressing Pax7, in a sublaminar position characterizing satellite cells and participate in forming new myofibers. These data show that a non-adherent stem cell population can be specifically isolated and expanded from skeletal muscle and upon attachment to a matrix spontaneously differentiate into muscle, cardiac and neuronal lineages in vitro. Although competing with resident satellite cells, these cells are shown to significantly contribute to repair of injured muscle in vivo supporting that a similar muscle-derived non-adherent cell population from human muscle may be useful in treatment of neuromuscular disorders.
Assuntos
Linhagem da Célula , Músculo Esquelético/citologia , Miocárdio/citologia , Neurônios/citologia , Células-Tronco/citologia , Animais , Western Blotting , Adesão Celular , Diferenciação Celular , Separação Celular , Células Cultivadas , Técnicas de Cocultura , Imunofluorescência , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas , Cadeias Pesadas de Miosina/metabolismo , Fenótipo , Regeneração , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante de Células-TroncoRESUMO
Recent studies in Xenopus have identified a new checkpoint protein called Claspin that is believed to transduce the checkpoint DNA damage signals to Chk1 kinase. Here we show that the human Claspin homolog is a chromatin bound protein either in the absence or in the presence of damaged DNA, independent of its association with ATR. Furthermore, we show that human Claspin is found in complex with PCNA, an essential component of the DNA replication machinery, and is released upon DNA replication arrest. Interfering with PCNA function by overexpression of p21 mutant, impaired in its interaction with Cdks but not with PCNA, leads to ATR-dependent Chk1 activation. These findings suggest that the dissociation of Claspin-PCNA could be part of the signal leading to Chk1 activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Ciclo Celular/fisiologia , Cromatina/fisiologia , Replicação do DNA/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Quinase 1 do Ponto de Checagem , Dano ao DNA , Células HeLa , Humanos , Proteínas Quinases/fisiologia , Proteínas de XenopusRESUMO
Mutations in genes encoding presenilins (PS1 and PS2) are responsible for the majority of early onset familial Alzheimer's disease. PS, a critical component of gamma-secretase, is responsible for the intramembranous cleavage of amyloid precursor protein and Notch. Other physiological functions have been assigned to PS without any clear identification of the mechanisms underlying these multiple biological roles. The early embryonic lethality of PS1 and PS2 double knock-out (PS1/2 null) mice prevents the evaluation of physiological roles of PS. To investigate new functions for presenilins, we performed a proteomic approach by using cells derived from PS1/2 null blastocysts and wild type controls. We identified a presenilin-dependent cell-surface binding of albumin. Binding of albumin depends on intact caveolae on the cellular surface. Abnormal caveolin 1 localization in PS1/2 null cells was associated with a loss of caveolae and an absence of caveolin 1 expression within lipid rafts. Expressing PS1 or PS2 but not the intracellular form of Notch1 in PS1/2 null cells restored normal caveolin 1 localization, demonstrating that presenilins are required for the subcellular trafficking of caveolin 1 independently from Notch activity. Despite an expression of both caveolin 1 and PS1 within lipid raft-enriched fractions after sucrose density centrifugation in wild type cells, no direct interaction between these two proteins was detected, implying that presenilins affect caveolin 1 trafficking in an indirect manner. We conclude that presenilins are required for caveolae formation by controlling transport of intracellular caveolin 1 to the plasma membrane.