RESUMO
Cellular pathways relay information through dynamic protein interactions. We have assessed the kinetic properties of the murine double minute protein (MDM2) and von Hippel-Lindau (VHL) ubiquitin ligases in living cells under physiological conditions that alter the stability of their respective p53 and hypoxia-inducible factor substrates. Photobleaching experiments reveal that MDM2 and VHL are highly mobile proteins in settings where their substrates are efficiently degraded. The nucleolar architecture converts MDM2 and VHL to a static state in response to regulatory cues that are associated with substrate stability. After signal termination, the nucleolus is able to rapidly release these proteins from static detention, thereby restoring their high mobility profiles. A protein surface region of VHL's beta-sheet domain was identified as a discrete [H+]-responsive nucleolar detention signal that targets the VHL/Cullin-2 ubiquitin ligase complex to nucleoli in response to physiological fluctuations in environmental pH. Data shown here provide the first evidence that cells have evolved a mechanism to regulate molecular networks by reversibly switching proteins between a mobile and static state.
Assuntos
Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Nucléolo Celular/ultraestrutura , Recuperação de Fluorescência Após Fotodegradação , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Sinais Direcionadores de Proteínas , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2 , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Proteína Supressora de Tumor Von Hippel-LindauRESUMO
Glucocorticoids are synthesized locally in adipose tissue and contribute to metabolic disease through the facilitation of adipose tissue expansion. Here we report that exposure of human primary preadipocytes to glucocorticoids increases their sensitivity to insulin and enhances their subsequent response to stimuli that promote differentiation. This effect was observed in primary human preadipocytes but not in immortalized 3T3-L1 murine preadipocytes or in fully differentiated primary human adipocytes. Stimulation of insulin signaling was mediated through induction of insulin receptor (IR), IR substrate protein 1 (IRS1), IRS2, and the p85 regulatory subunit of phosphoinositide-3-3-kinase, which led to enhanced insulin-mediated activation of Akt. Although induction of IRS2 was direct, induction of IR and IRS1 by glucocorticoids occurred subsequent to primary induction of the forkhead family transcription factors FoxO1A and FoxO3A. These results reveal a new role for glucocorticoids in preparing preadipocytes for differentiation.
Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Insulina/farmacologia , Células 3T3-L1 , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Fatores de Transcrição Forkhead/genética , Perfilação da Expressão Gênica , Glucocorticoides/fisiologia , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Fatores de TempoRESUMO
The glucocorticoid receptor (GR) cycles between a naive chaperone-complexed form in the cytoplasm and a transcriptionally active steroid-bound nuclear form. Nuclear import of GR occurs rapidly and is mediated through the importin alpha/beta karyopherin import pathway. By contrast, nuclear export of GR occurs only slowly under most conditions, despite a dependence on active signaling. In this study we have defined a nuclear retention signal (NRS) in the hinge region of GR that actively opposes the nuclear export of GR as well as the nuclear export mediated through an ectopic CRM1-dependent nuclear export signal (NES). The GR NRS overlaps closely with the basic NL1 nuclear localization signal (NLS) but can be distinguished from NL1 by targeted mutagenesis. Substitution of the classical NLS from SV40 T antigen for the GR NL1 results in a receptor in which nuclear export is accelerated. Remarkably, although the SV40-modified GR remains predominantly nuclear in the presence of steroid and is recruited to transcriptional regulatory regions indistinguishably from wild-type GR, the substitution dramatically weakens the ability of GR to activate transcription of a mouse mammary tumor virus reporter gene. These results suggest that active nuclear retention of GR plays an integral role in glucocorticoid signaling.
Assuntos
Receptores de Glucocorticoides/metabolismo , Transdução de Sinais , Ativação Transcricional/genética , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Células COS , Chlorocebus aethiops , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Ratos , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genéticaRESUMO
The mineralocorticoid receptor (MR) is a tightly regulated nuclear hormone receptor that selectively transmits corticosteroid signals. Steroid treatment transforms MR from a transcriptionally inert state, in which it is distributed equally between the nucleus and cytoplasm, to an active completely nuclear transcription factor. We report here that MR is an atypical nuclear hormone receptor that moves unidirectionally from the cytoplasm to the nucleus. We show that nuclear import of MR is controlled through three nuclear localization signals (NLSs) of distinct types. Nuclear localization of naive MR was mediated primarily through a novel serine/threonine-rich NLS (NL0) in the receptor N terminus. Specific amino acid substitutions that mimicked phosphorylation selectively enhanced or repressed NL0 activity, highlighting the potential for active regulation of this new type of NLS. The second NLS (NL2) within the ligand-binding domain also lacks a recognizable basic motif. Nuclear transfer through this signal was strictly dependent on steroid agonist, but was independent of the interaction of MR with coactivator proteins. The third MR NLS (NL1) is a bipartite basic motif localized to the C terminus of the MR DNA-binding domain with properties distinct from those of NL1 of the closely related glucocorticoid receptor. NL1 acted in concert with NL0 and NL2 to stimulate nuclear uptake of the agonist-treated receptor, but also directed the complete nuclear localization of MR in response to treatment with steroid antagonist. These results present MR as a nuclear hormone receptor whose unidirectional transfer to the nucleus may be regulated through multiple pathways.