RESUMO
PTP1B, a non-transmembrane protein tyrosine phosphatase that has long been studied as a negative regulator of insulin and leptin signaling, has received renewed attention as an unexpected positive factor in tumorigenesis. Here, we highlight how views of this enzyme have evolved from regarding it as a simple metabolic off-switch to a more complex view of PTP1B as an enzyme that can play both negative and positive roles in diverse signaling pathways. These dual characteristics make PTP1B a particularly attractive therapeutic target for diabetes, obesity, and perhaps breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica , Diabetes Mellitus/metabolismo , Terapia de Alvo Molecular , Obesidade/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/patologia , Humanos , Obesidade/tratamento farmacológico , Obesidade/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Protein tyrosine phosphatase (PTP)1B is an abundant non-transmembrane enzyme that plays a major role in regulating insulin and leptin signaling. Recently, we reported that PTP1B is inhibited by sumoylation, and that sumoylated PTP1B accumulates in a perinuclear distribution, consistent with its known localization in the endoplasmic reticulum (ER) and the contiguous outer nuclear membrane. Here, we report that, in addition to its localization at the ER, PTP1B also is found at the inner nuclear membrane, where it is heavily sumoylated. We also find that PTP1B interacts with emerin, an inner nuclear membrane protein that is known to be tyrosine phosphorylated, and that PTP1B expression levels are inversely correlated with tyrosine phosphorylation levels of emerin. PTP1B sumoylation greatly increases as cells approach mitosis, corresponding to the stage where tyrosine phosphorylation of emerin is maximal. In addition, expression of a non-sumoylatable mutant of PTP1B greatly reduced levels of emerin tyrosine phosphorylation. These results suggest that PTP1B regulates the tyrosine phosphorylation of a key inner nuclear membrane protein in a sumoylation- and cell-cycle-dependent manner.
Assuntos
Proteínas de Membrana/metabolismo , Membrana Nuclear/enzimologia , Proteínas Nucleares/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Sumoilação , Tirosina/metabolismo , Animais , Ciclo Celular , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 1/análise , Proteína Tirosina Fosfatase não Receptora Tipo 1/genéticaRESUMO
Protein-tyrosine phosphatase 1B (PTP1B) is an ubiquitously expressed enzyme that negatively regulates growth-factor signalling and cell proliferation by binding to and dephosphorylating key receptor tyrosine kinases, such as the insulin receptor. It is unclear how the activity of PTP1B is regulated. Using a yeast two-hybrid assay, a protein inhibitor of activated STAT1 (PIAS1) was isolated as a PTP1B-interacting protein. Here, we show that PIAS1, which functions as a small ubiquitin-like modifier (SUMO) E3 ligase, associates with PTP1B in mammalian fibroblasts and catalyses sumoylation of PTP1B. Sumoylation of PTP1B reduces its catalytic activity and inhibits the negative effect of PTP1B on insulin receptor signalling and on transformation by the oncogene v-crk. Insulin-stimulated sumoylation of endogenous PTP1B results in a transient downregulation of the enzyme; this event does not occur when the endogenous enzyme is replaced with a sumoylation-resistant mutant of PTP1B. These results suggest that sumoylation, which has been implicated primarily in processes in the nucleus and nuclear pore, also modulates a key enzyme-substrate signalling complex that regulates metabolism and cell proliferation.
Assuntos
Regulação Enzimológica da Expressão Gênica , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Tirosina Fosfatases/genética , Proteína SUMO-1/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Proteínas Inibidoras de STAT Ativados/isolamento & purificação , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
Differing spatial scales of signaling cascades are critical for cell orientation during chemotactic responses. We used biotin EGF bound to streptavidin-coupled magnetic beads to locally stimulate cells overexpressing the EGF receptor. We have found that EGF-induced actin polymerization remains localized even under conditions of receptor overexpression. Conversely, EGF-induced ERK activation spreads throughout the cell body after EGF bead stimulation. The localized actin polymerization is independent of PI3-kinase and rho protein activity and requires Arp2/3 complex and cofilin function. Thus, we find differing spatial scales of signaling from the EGF receptor, supporting models of chemotaxis that integrate short- and long-range signaling.
Assuntos
Receptores ErbB/metabolismo , Proteínas Serina-Treonina Quinases , Transdução de Sinais/fisiologia , Fatores de Despolimerização de Actina , Actinas/metabolismo , Androstadienos/metabolismo , Animais , Linhagem Celular Tumoral , Inibidores Enzimáticos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfotirosina/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , RNA Interferente Pequeno/metabolismo , Ratos , WortmaninaRESUMO
The epidermal growth factor (EGF)-induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF.
Assuntos
Carcinoma/metabolismo , Quimiotaxia/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Proteínas dos Microfilamentos/fisiologia , Metástase Neoplásica/fisiopatologia , Fosfolipases Tipo C/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina , Actinas/biossíntese , Actinas/efeitos dos fármacos , Animais , Anticorpos/farmacologia , Carcinoma/patologia , Linhagem Celular Tumoral , Quimiotaxia/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Proteínas dos Microfilamentos/efeitos dos fármacos , Proteínas dos Microfilamentos/genética , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Interferência de RNA , Ratos , Fosfolipases Tipo C/efeitos dos fármacosRESUMO
Class IA PI3Ks (phosphoinositide 3-kinases) generate the secondary messenger PtdIns(3,4,5)P(3), which plays an important role in many cellular responses. The accumulation of PtdIns(3,4,5)P(3) in cell membranes is routinely measured using GFP (green fluorescent protein)-labelled PH (pleckstrin homology) domains. However, the kinetics of membrane PtdIns(3,4,5)P(3) synthesis and turnover as detected by PH domains have not been validated using an independent method. In the present study, we measured EGF (epidermal growth factor)-stimulated membrane PtdIns(3,4,5)P(3) production using a specific monoclonal anti-PtdIns(3,4,5)P(3) antibody, and compared the results with those obtained using PH-domain-dependent methods. Anti-PtdIns(3,4,5)P(3) staining rapidly accumulated at the leading edge of EGF-stimulated carcinoma cells. PtdIns(3,4,5)P(3) levels were maximal at 1 min, and returned to basal levels by 5 min. In contrast, membrane PtdIns(3,4,5)P(3) production, measured by the membrane translocation of an epitope-tagged (BTK)PH (PH domain of Bruton's tyrosine kinase), remained approx. 2-fold above basal level throughout 4-5 min of EGF stimulation. To determine the reason for this disparity, we measured the rate of PtdIns(3,4,5)P(3) hydrolysis by measuring the decay of the PtdIns(3,4,5)P(3) signal after LY294002 treatment of EGF-stimulated cells. LY294002 abolished anti-PtdIns(3,4,5)P(3) membrane staining within 10 s of treatment, suggesting that PtdIns(3,4,5)P(3) turnover occurs within seconds of synthesis. In contrast, (BTK)PH membrane recruitment, once initiated by EGF, was relatively insensitive to LY294002. These data suggest that sequestration of PtdIns(3,4,5)P(3) by PH domains may affect the apparent kinetics of PtdIns(3,4,5)P(3) accumulation and turnover; consistent with this hypothesis, we found that GRP-1 (general receptor for phosphoinositides 1) PH domains [which, like BTK, are specific for PtdIns(3,4,5)P(3)] inhibit PTEN (phosphatase and tensin homologue deleted on chromosome 10) dephosphorylation of PtdIns(3,4,5)P(3) in vitro. These data suggest that anti-PtdIns(3,4,5)P(3) antibodies are a useful tool to detect localized PtdIns(3,4,5)P(3), and illustrate the importance of using multiple approaches for the estimation of membrane phosphoinositides.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Imunoensaio/métodos , Neoplasias/imunologia , Neoplasias/metabolismo , Fosfatos de Fosfatidilinositol/análise , Fosfatos de Fosfatidilinositol/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular Tumoral , Epitopos/imunologia , Hormônios Gastrointestinais/metabolismo , Regulação da Expressão Gênica , Cinética , Neoplasias/patologia , PTEN Fosfo-Hidrolase/metabolismo , Ratos , Sensibilidade e EspecificidadeRESUMO
The base excision repair DNA N-glycosylase MBD4 (also known as MED1), an interactor of the DNA mismatch repair protein MLH1, plays a central role in the maintenance of genomic stability of CpG sites by removing thymine and uracil from G:T and G:U mismatches, respectively. MBD4 is also involved in DNA damage response and transcriptional regulation. The interaction with other proteins is likely critical for understanding MBD4 functions. To identify novel proteins that interact with MBD4, we used tandem affinity purification (TAP) from HEK-293 cells. The MBD4-TAP fusion and its co-associated proteins were purified sequentially on IgG and calmodulin affinity columns; the final eluate was shown to contain MLH1 by western blotting, and MBD4-associated proteins were identified by mass spectrometry. Bands with molecular weight higher than that expected for MBD4 (Ë66â¯kD) yielded peptides corresponding to MBD4 itself and the small ubiquitin-like molecule-1 (SUMO1), suggesting that MBD4 is sumoylated in vivo. MBD4 sumoylation was validated by co-immunoprecipitation in HEK-293 and MCF7 cells, and by an in vitrosumoylation assay. Sequence and mutation analysis identified three main sumoylation sites: MBD4 is sumoylated preferentially on K137, with additional sumoylation at K215 and K377. Patterns of MBD4 sumoylation were altered, in a DNA damage-specific way, by the anti-metabolite 5-fluorouracil, the alkylating agent N-Methyl-N-nitrosourea and the crosslinking agent cisplatin. MCF7 extract expressing sumoylated MBD4 displays higher thymine glycosylase activity than the unmodified species. Of the 67 MBD4 missense mutations reported in The Cancer Genome Atlas, 14 (20.9%) map near sumoylation sites. These results indicate that MBD4 is sumoylated in vivo in a DNA damage-specific manner, and suggest that sumoylation serves to regulate its repair activity and could be compromised in cancer. This study expands the role played by sumoylation in fine-tuning DNA damage response and repair.
Assuntos
Reparo do DNA , Endodesoxirribonucleases/metabolismo , Proteína SUMO-1/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Dano ao DNA , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Células HEK293 , Humanos , Células MCF-7 , Mutação , Neoplasias/genética , SumoilaçãoRESUMO
Protein tyrosine phosphatase (PTP) 1B plays a major role in inhibiting signaling from the insulin and leptin receptors. Recently, PTP1B was found to have an unexpected positive role in ErbB2 signaling in a mouse model of breast cancer, but the mechanism underlying this effect has been unclear. Using human breast epithelial cells grown in a three-dimensional matrix, we found that PTP1B, but not the closely related enzyme T-cell PTP, is required for ErbB2 transformation in vitro. Activation of ErbB2, but not ErbB1, increases PTP1B expression, and increased expression of PTP1B activates Src and induces a Src-dependent transformed phenotype. These findings identify a molecular mechanism by which PTP1B links an important oncogenic receptor tyrosine kinase to signaling pathways that promote aberrant cell division and survival in human breast epithelial cells.
Assuntos
Transformação Celular Neoplásica/metabolismo , Genes erbB-2/fisiologia , Glândulas Mamárias Humanas/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/fisiologia , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Divisão Celular/genética , Divisão Celular/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Transformação Celular Neoplásica/genética , Células Cultivadas , Ativação Enzimática , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/fisiologia , Humanos , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Transdução de Sinais/genética , TransfecçãoRESUMO
Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.
Assuntos
Carcinoma/enzimologia , Carcinoma/patologia , Pseudópodes/enzimologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Amidas/farmacologia , Animais , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Movimento Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Adesões Focais/efeitos dos fármacos , Adesões Focais/enzimologia , Humanos , Transporte Proteico/efeitos dos fármacos , Pseudópodes/efeitos dos fármacos , Piridinas/farmacologia , Ratos , Quinases Associadas a rho/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
Lamellipodial protrusion and directional migration of carcinoma cells towards chemoattractants, such as epidermal growth factor (EGF), depend upon the spatial and temporal regulation of actin cytoskeleton by actin-binding proteins (ABPs). It is generally hypothesized that the activity of many ABPs are temporally and spatially regulated by PIP(2); however, this is mainly based on in vitro-binding and structural studies, and generally in vivo evidence is lacking. Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP(2) in living cells. We show that EGF induces a rapid loss of PIP(2) through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin. Upon release, we find that cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation. Moreover, our data provide evidence for how PLC is involved in the formation of protrusions in breast carcinoma cells during chemotaxis and metastasis towards EGF.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fatores de Despolimerização de Actina/análise , Actinas/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Feminino , Hidrólise , Fosfatidilinositol 4,5-Difosfato/análise , Transporte Proteico , RatosRESUMO
Cell migration involves the localized extension of actin-rich protrusions, a process that requires Class I phosphoinositide 3-kinases (PI 3-kinases). Both Rac and Ras have been shown to regulate actin polymerization and activate PI 3-kinase. However, the coordination of Rac, Ras and PI 3-kinase activation during epidermal growth factor (EGF)-stimulated protrusion has not been analyzed. We examined PI 3-kinase-dependent protrusion in MTLn3 rat adenocarcinoma cells. EGF-stimulated phosphatidyl-inositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] levels showed a rapid and persistent response, as PI 3-kinase activity remained elevated up to 3 minutes. The activation kinetics of Ras, but not Rac, coincided with those of leading-edge PtdIns(3,4,5)P(3) production. Small interfering RNA (siRNA) knockdown of K-Ras but not Rac1 abolished PtdIns(3,4,5)P(3) production at the leading edge and inhibited EGF-stimulated protrusion. However, Rac1 knockdown did inhibit cell migration, because of the inhibition of focal adhesion formation in Rac1 siRNA-treated cells. Our data show that in EGF-stimulated MTLn3 carcinoma cells, Ras is required for both PtdIns(3,4,5)P(3) production and lamellipod extension, whereas Rac1 is required for formation of adhesive structures. These data suggest an unappreciated role for Ras during protrusion, and a crucial role for Rac in the stabilization of protrusions required for cell motility.
Assuntos
Movimento Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Adenocarcinoma , Animais , Linhagem Celular Tumoral , Ativação Enzimática , Fosfatos de Fosfatidilinositol/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas rac de Ligação ao GTP/genética , Proteínas ras/genéticaRESUMO
p85/p110 phosphoinositide 3-kinases regulate multiple cell functions and are frequently mutated in human cancer. The p85 regulatory subunit stabilizes and inhibits the p110 catalytic subunit. The minimal fragment of p85 capable of regulating p110 is the N-terminal SH2 domain linked to the coiled-coil iSH2 domain (referred to as p85ni). We have previously proposed that the conformationally rigid iSH2 domain tethers p110 to p85, facilitating regulatory interactions between p110 and the p85 nSH2 domain. In an oncogenic mutant of murine p85, truncation at residue 571 leads to constitutively increased phosphoinositide 3-kinase activity, which has been proposed to result from either loss of an inhibitory Ser-608 autophosphorylation site or altered interactions with cellular regulatory factors. We have examined this mutant (referred to as p65) in vitro and find that p65 binds but does not inhibit p110, leading to constitutive p110 activity. This activated phenotype is observed with recombinant proteins in the absence of cellular factors. Importantly, this effect is also produced by truncating p85ni at residue 571. Thus, the phenotype is not because of loss of the Ser-608 inhibitory autophosphorylation site, which is not present in p85ni. To determine the structural basis for the phenotype of p65, we used a broadly applicable spin label/NMR approach to define the positioning of the nSH2 domain relative to the iSH2 domain. We found that one face of the nSH2 domain packs against the 581-593 region of the iSH2 domain. The loss of this interaction in the truncated p65 would remove the orienting constraints on the nSH2 domain, leading to a loss of p110 regulation by the nSH2. Based on these findings, we propose a general model for oncogenic mutants of p85 and p110 in which disruption of nSH2-p110 regulatory contacts leads to constitutive p110 activity.
Assuntos
Mutação , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Sítios de Ligação , Ativação Enzimática , Glutationa Transferase/metabolismo , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fenótipo , Fosforilação , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Serina/química , Domínios de Homologia de srcRESUMO
Phosphoinositide 3-kinase (PI 3-kinase) activity is required for growth factor-induced cytoskeletal regulation and cell migration. We previously found that in MTLn3 rat adenocarcinoma cells, EGF-stimulated induction of actin barbed ends and lamellipod extension specifically requires the p85/p110alpha isoform of PI 3-kinase. To further characterize signaling by distinct PI 3-kinase isoforms, we have developed MTLn3 cells that transiently or stably overexpress either p110alpha or p110beta. Transient overexpression of p110beta inhibited EGF-stimulated lamellipod extension, whereas p110alpha-transfected cells showed normal EGF-stimulated lamellipod extension. Similar results were obtained by overexpression of kinase-dead p110beta, suggesting that effects on cytoskeletal signaling were due to competition with p85/p110alpha complexes. Stable overexpression of p110alpha appeared to be toxic, based on the difficulty in obtaining stable overexpressing clones. In contrast, cells expressing a 2-fold increase in p110beta were readily obtainable. Interestingly, cells stably expressing p110beta showed a marked inhibition of EGF-stimulated lamellipod extension. Using computer-assisted analysis of time-lapse images, we found that overexpression of p110beta caused a nearly complete inhibition of motility. Cells overexpressing p110beta showed normal activation of Akt and Erk, suggesting that overall PI 3-kinase signaling was intact. A chimeric p110 molecule containing the p85-binding and Ras-binding domains of p110alpha and the C2, helical, and kinase domains of p110beta, was catalytically active yet also inhibited EGF-stimulated lamellipod extension. These data highlight the differential signaling by distinct p110 isoforms. Identification of effectors that are differently regulated by p110alpha versus p110beta will be important for understanding cell migration and its role in metastasis.