RESUMO
cAMP is an ubiquitous second messenger. Localized areas with high cAMP concentration, i.e. cAMP microdomains, provide an elegant mechanism to generate signaling specificity and transduction efficiency. However, the mechanisms underlying cAMP effector targeting into these compartments is still unclear. Here we report the identification of radixin as a scaffolding unit for both cAMP effectors, Epac and PKA. This complex localizes in a submembrane compartment where cAMP synthesis occurs. Compartment disruption by shRNA and dominant negative approaches negatively affects cAMP action. Inhibition can be rescued by expression of Rap1b, a substrate for both Epac1 and PKA, but only in its GTP-bound and phosphorylated state. We propose that radixin scaffolds both cAMP effectors in a functional cAMP-sensing compartment for efficient signal transduction, using Rap1 as a downstream signal integrator.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Ancoragem à Quinase A/metabolismo , Adenilil Ciclases/metabolismo , Ciclo Celular , Proliferação de Células , Proteínas do Citoesqueleto/química , Fatores de Troca do Nucleotídeo Guanina/química , Células HEK293 , Humanos , Proteínas de Membrana/química , Modelos Moleculares , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Rap1b has been implicated in the transduction of the cAMP mitogenic response. Agonists that increase intracellular cAMP rapidly activate (i.e. GTP binding) and phosphorylate Rap1b on Ser(179) at its C terminus. cAMP-dependent protein kinase (PKA)-mediated phosphorylation of Rap1b is required for cAMP-dependent mitogenesis, tumorigenesis, and inhibition of AKT activity. However, the role of phosphorylation still remains unknown. In this study, we utilized amide hydrogen/deuterium exchange mass spectroscopy (DXMS) to assess potential conformational changes and/or mobility induced by phosphorylation. We report here DXMS data comparing exchange rates for PKA-phosphorylated (Rap1-P) and S179D phosphomimetic (Rap1-D) Rap1b proteins. Rap1-P and Rap1-D behaved exactly the same, revealing an increased exchange rate in discrete regions along the protein; these regions include a domain around the phosphorylation site and unexpectedly the two switch loops. Thus, local effects induced by Ser(179) phosphorylation communicate allosterically with distal domains involved in effector interaction. These results provide a mechanistic explanation for the differential effects of Rap1 phosphorylation by PKA on effector protein interaction.
Assuntos
Proteínas rap de Ligação ao GTP/química , Proteínas rap de Ligação ao GTP/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Linhagem Celular , Medição da Troca de Deutério , Humanos , Espectrometria de Massas , Modelos Moleculares , Fosforilação , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
ZFP36L2 protein destabilizes AU-rich element-containing transcripts and has been implicated in female fertility. In the C57BL/6NTac mouse, a mutation in Zfp36l2 that results in the decreased expression of a form of ZFP36L2 in which the 29 N-terminal amino acid residues have been deleted, ΔN-ZFP36L2, leads to fertilized eggs that arrest at the two-cell stage. Interestingly, homozygous ΔN-Zfp36l2 females in the C57BL/6NTac strain release 40% fewer eggs than the WT littermates (Ramos et al., 2004), suggesting an additional defect in ovulation and/or oocyte maturation. Curiously, the same ΔN-Zfp36l2 mutation into the SV129 strain resulted in anovulation, prompting us to investigate a potential problem in ovulation and oocyte maturation. Remarkably, only 20% of ΔN-Zfp36l2 oocytes in the 129S6/SvEvTac strain matured ex vivo, suggesting a defect on the oocyte meiotic maturation process. Treatment of ΔN-Zfp36l2 oocytes with a PKA inhibitor partially rescued the meiotic arrested oocytes. Furthermore, cAMP levels were increased in ΔN-Zfp36l2 oocytes, linking the cAMP/PKA pathway and ΔN-Zfp36l2 with meiotic arrest. Since ovulation and oocyte maturation are both triggered by LHR signaling, the downstream pathway was investigated. Adenylyl cyclase activity was increased in ΔN-Zfp36l2 ovaries only upon LH stimulation. Moreover, we discovered that ZFP36L2 interacts with the 3'UTR of LHR mRNA and that decreased expression levels of Zfp36l2 correlates with higher levels of LHR mRNA in synchronized ovaries. Furthermore, overexpression of ZFP36L2 decreases the endogenous expression of LHR mRNA in a cell line. Therefore, we propose that lack of the physiological down regulation of LHR mRNA levels by ZFP36L2 in the ovaries is associated with anovulation and oocyte meiotic arrest.
Assuntos
Oócitos/citologia , Ovulação/fisiologia , Proteínas de Ligação a RNA/fisiologia , Tristetraprolina/fisiologia , Regiões 3' não Traduzidas , Adenilil Ciclases/metabolismo , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Deleção de Genes , Células HEK293 , Homozigoto , Humanos , Meiose , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Oócitos/metabolismo , Oogênese , Ovário/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Receptores do LH/metabolismo , Tristetraprolina/genéticaRESUMO
cAMP stimulates proliferation in many cell types. For many years, cAMP-dependent protein kinase (PKA) represented the only known cAMP effector. PKA, however, does not fully mimic the action of cAMP, indicating the existence of a PKA-independent component. Since cAMP-mediated activation of the G-protein Rap1 and its phosphorylation by PKA are strictly required for the effects of cAMP on mitogenesis, we hypothesized that the Rap1 activator Epac might represent the PKA-independent factor. Here we report that Epac acts synergistically with PKA in cAMP-mediated mitogenesis. We have generated a new dominant negative Epac mutant that revealed that activation of Epac is required for thyroid-stimulating hormone or cAMP stimulation of DNA synthesis. We demonstrate that Epac's action on cAMP-mediated activation of Rap1 and cAMP-mediated mitogenesis depends on the subcellular localization of Epac via its DEP domain. Disruption of the DEP-dependent subcellular targeting of Epac abolished cAMP-Epac-mediated Rap1 activation and thyroid-stimulating hormone-mediated cell proliferation, indicating that an Epac-Rap-PKA signaling unit is critical for the mitogenic action of cAMP.
Assuntos
Proliferação de Células , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Transdução de Sinais/fisiologia , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , DNA/biossíntese , DNA/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Fatores de Troca do Nucleotídeo Guanina/agonistas , Fatores de Troca do Nucleotídeo Guanina/genética , Mutação , Fosforilação/efeitos dos fármacos , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Ratos , Tireotropina/metabolismo , Tireotropina/farmacologia , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
Rap1b has been implicated in the transduction of the cAMP mitogenic signal. It is phosphorylated and activated by cAMP, and its expression in models where cAMP is mitogenic leads to proliferation and tumorigenesis. Akt is a likely downstream effector of cAMP-Rap1 action. cAMP elevation induced a rapid and transient Akt inhibition that required activated and phosphorylated Rap1b. However, the mechanism(s) by which cAMP-Rap regulates Akt remains unclear. Here we show that (i) upstream regulators, PIK and PDK1, are not the target(s) of the cAMP inhibitory action; (ii) constitutively active Akt and calyculin A-stimulated Akt are resistant to cAMP inhibition, suggesting the action of a phosphatase; (iii) cAMP increases the rate of Akt dephosphorylation, directly implicating an Akt-phosphatase; (iv) Epac- and protein kinase A (PKA)-specific analogs synergistically inhibit Akt, indicating the involvement of both cAMP-dependent effector pathways; (v) H89 and dominant negative Epac 279E block cAMP-inhibitory action; (vi) Epac associates in a complex with Akt and PP2A, and the associated-phosphatase activity is positively modulated by cAMP in a PKA- and Rap1-dependent manner; (vii) like its action on Akt inhibition, PKA- and Epac-specific analogs synergistically activate Epac-associated PP2A; and (viii) dominant negative PP2A blocks cAMP-inhibitory action. Thus, we uncovered a novel cAMP-Epac/PKA-Rap1b-PP2A signaling module involved in Akt regulation that may represent a physiological event in the process of cAMP stimulation of thyroid mitogenesis.
Assuntos
Regulação Enzimológica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Isoquinolinas/farmacologia , Ratos , Transdução de Sinais , Sulfonamidas/farmacologia , Glândula Tireoide/metabolismo , Fatores de TempoRESUMO
Rap1b has been implicated in the transduction of the cAMP mitogenic signal. Rap1b is phosphorylated and activated by cAMP, and its expression in cells where cAMP is mitogenic leads to an increase in G(1)/S phase entry and tumor formation. The PCCL3 thyroid follicular cells represent a differentiated and physiologically relevant system that requires thyrotropin (TSH), acting via cAMP, for a full mitogenic response. In this model system, cAMP stimulation of DNA synthesis requires activation and phosphorylation of Rap1b by the cAMP-dependent protein kinase A (PKA). This scenario presents the challenge of identifying biochemical processes involved in the phosphorylation-dependent Rap1b mitogenic action. In thyroid cells, Akt has been implicated in the stimulation of cell proliferation by TSH and cAMP. However, the mechanism(s) by which cAMP regulates Akt activity remains unclear. In this study we show that in PCCL3 cells 1) TSH inhibits Akt activity via cAMP and PKA; 2) Rap1b is required for cAMP inhibition of Akt; and 3) transduction of the cAMP signal into Akt requires activation as well as phosphorylation of Rap1b by PKA.
Assuntos
AMP Cíclico/metabolismo , Inibidores Enzimáticos/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Proteínas rap de Ligação ao GTP/metabolismo , Animais , Western Blotting , Divisão Celular , Linhagem Celular , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Mitógenos/farmacologia , Mutação , Fosforilação , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Transdução de Sinais , Fatores de Tempo , TransfecçãoRESUMO
Guanine nucleotide exchange factors (GEFs) and their associated GTP-binding proteins (G-proteins) are key regulatory elements in the signal transduction machinery that relays information from the extracellular environment into specific intracellular responses. Among them, the MAPK cascades represent ubiquitous downstream effector pathways. We have previously described that, analogous to the Ras-dependent activation of the Erk-1/2 pathway, members of the Rho family of small G-proteins activate the JNK cascade when GTP is loaded by their corresponding GEFs. Searching for novel regulators of JNK activity we have identified Epac (exchange protein activated by cAMP) as a strong activator of JNK-1. Epac is a member of a growing family of GEFs that specifically display exchange activity on the Rap subfamily of Ras small G-proteins. We report here that while Epac activates the JNK severalfold, a constitutively active (G12V) mutant of Rap1b does not, suggesting that Rap-GTP is not sufficient to transduce Epac-dependent JNK activation. Moreover, Epac signaling to the JNKs was not blocked by inactivation of endogenous Rap, suggesting that Rap activation is not necessary for this response. Consistent with these observations, domain deletion mutant analysis shows that the catalytic GEF domain is dispensable for Epac-mediated activation of JNK. These studies identified a region overlapping the Ras exchange motif domain as critical for JNK activation. Consistent with this, an isolated Ras exchange motif domain from Epac is sufficient to activate JNK. We conclude that Epac signals to the JNK cascade through a new mechanism that does not involve its canonical catalytic action, i.e. Rap-specific GDP/GTP exchange. This represents not only a novel way to activate the JNKs but also a yet undescribed mechanism of downstream signaling by Epac.
Assuntos
Produtos do Gene vpr/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Motivos de Aminoácidos , Western Blotting , Linhagem Celular , DNA/metabolismo , DNA Complementar/metabolismo , Ativação Enzimática , Deleção de Genes , Genes Dominantes , Glutationa Transferase/metabolismo , Humanos , MAP Quinase Quinase 4 , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
We have shown that the small GTPase Rap1b, a protein known to antagonize the mitogenic and transforming activity of Ras, is endowed with both mitogenic and tumorigenic properties. Rap1b can be activated by cAMP, an intracellular message known to either stimulate or inhibit cell proliferation. The oncogenic property of Rap1b was revealed in a model system in which cAMP stimulates cell proliferation and was linked to Rap's ability to promote S phase entry. We have now tested the significance of the mitogenic action of Rap1b in a physiologically relevant model, the differentiated thyroid follicular cells, a system that requires thyroid-stimulating hormone (TSH), acting via cAMP, to mediate a full mitogenic response. Here we report that cAMP-dependent hormonal stimulation of DNA synthesis requires Rap1b in a manner dependent on its phosphorylation by protein kinase A.
Assuntos
Fase G1 , Fase S , Proteínas rap de Ligação ao GTP/metabolismo , Células 3T3 , Animais , Western Blotting , Diferenciação Celular , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , DNA/biossíntese , Relação Dose-Resposta a Droga , Genes Dominantes , Glutationa Transferase/metabolismo , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Fosforilação , Plasmídeos/metabolismo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Timidina/metabolismo , Timidina/farmacologia , Tireotropina/farmacologia , Fatores de Tempo , Transfecção , Proteínas rap de Ligação ao GTP/químicaRESUMO
cAMP signaling leads to activation and phosphorylation of Rap1b. Using cellular models where cAMP stimulates cell proliferation, we have demonstrated that cAMP-mediated activation, as well as phosphorylation of Rap1b, is critical for cAMP stimulation of DNA synthesis. To determine whether Rap1b stimulates mitogenesis in vivo, we have constructed a transgenic mouse where a constitutively active G12V-Rap1b, flanked by Cre recombinase LoxP sites, is followed by the dominant negative S17N mutant. Employing this novel mouse model, we have switched, in a tissue-specific (thyroid) and temporally controlled manner, the expression of Rap1b from a stimulatory to an inhibitory form. These experiments provide conclusive evidence that Rap1b is oncogenic in the thyroid in ways linked to transduction of the cAMP mitogenic signal.
Assuntos
AMP Cíclico/química , Glândula Tireoide/metabolismo , Proteínas rap de Ligação ao GTP/biossíntese , Animais , Antitireóideos/farmacologia , Bromodesoxiuridina/farmacologia , Carcinoma/patologia , Bovinos , Diferenciação Celular , AMP Cíclico/metabolismo , DNA/metabolismo , Genes Dominantes , Imuno-Histoquímica , Antígeno Ki-67/biossíntese , Camundongos , Camundongos Transgênicos , Fenótipo , Fosforilação , Regiões Promotoras Genéticas , Recombinação Genética , Transdução de Sinais , Glândula Tireoide/patologia , Fatores de Tempo , Transgenes , Proteínas rap de Ligação ao GTP/químicaRESUMO
The CCCH tandem zinc finger protein, Zfp36l2, like its better-known relative tristetraprolin (TTP), can decrease the stability of AU-rich element-containing transcripts in cell transfection studies; however, its physiological importance is unknown. We disrupted Zfp36l2 in mice, resulting in decreased expression of a truncated protein in which the N-terminal 29 amino acids had been deleted (DeltaN-Zfp36l2). Mice derived from different clones of ES cells exhibited complete female infertility, despite evidence from embryo and ovary transplantation experiments that they could gestate and rear wild-type young. DeltaN-Zfp36l2 females apparently cycled and ovulated normally, and their ova could be fertilized; however, the embryos did not progress beyond the two-cell stage of development. These mice represent a specific model of disruption of the earliest stages of embryogenesis, implicating Zfp36l2, a probable mRNA-binding and destabilizing protein, in the physiological control of female fertility at the level of early embryonic development. This newly identified biological role for Zfp36l2 may have implications for maternal mRNA turnover in normal embryogenesis, and conceivably could be involved in some cases of unexplained human female infertility.