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1.
Analyst ; 144(12): 3756-3764, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31070195

RESUMO

Protein phosphorylation is a very important regulatory mechanism in a majority of biological processes, and the determination of protein kinase activity plays a key role in the pathological study and drug development of kinase-related diseases. However, it is very challenging to in situ study endogenous protein kinase activity in a single living cell due to the shortage of in vivo efficient methods. Here, we propose a new strategy for direct determination of protein kinase activity in a single living cell by combining single molecule fluorescence correlation spectroscopy (FCS) with activity-based probes (ABPs). Ribosomal S6 kinase-2 (RSK2) was used as a model, and the ABPs were synthesized on the basis of RSK2 inhibitor FMK to specially label active RSK2 in living cells. Conventional FCS and MEMFCS (maximum entropy method) single molecule techniques were used to in situ determine RSK2 activity in living cells based on the difference in molecular weight between free probes and probe-RSK2 complexes. Furthermore, wild-type and mutated RSK2 were fused with enhanced green fluorescent protein (EGFP) using lentivirus infection, and fluorescence cross-correlation spectroscopy (FCCS) was used to verify the selective binding of ABPs to RSK2-EGFP fusion protein in living cells. Finally, FCS with ABPs was applied for in situ monitoring of the activation of endogenous RSK2 in the stimulation of serum, epidermal growth factor, kinase inhibitors and ultraviolet irradiation; we observed that endogenous RSK2 showed different behaviors in the cytoplasm and the nucleus in some stimulation. Our results document that FCS with ABPs is a very promising method for studying endogenous protein kinases in living cells.


Assuntos
Ensaios Enzimáticos/métodos , Proteínas Quinases S6 Ribossômicas 90-kDa/análise , Análise de Célula Única/métodos , Espectrometria de Fluorescência/métodos , Compostos de Boro/síntese química , Compostos de Boro/química , Carbocianinas/síntese química , Carbocianinas/química , Linhagem Celular Tumoral , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Imagem Individual de Molécula/métodos
2.
Exp Mol Med ; 48: e250, 2016 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-27491410

RESUMO

RSK2, also known as RPS6KA3 (ribosomal protein S6 kinase, 90 kDa, polypeptide 3), is a downstream kinase of the mitogen-activated protein kinase (MAPK) pathway, which is important in regulating survival, transcription, growth and proliferation. However, its biological role in mitotic progression is not well understood. In this study, we examined the potential involvement of RSK2 in the regulation of mitotic progression. Interestingly, depletion of RSK2, but not RSK1, caused the accumulation of mitotic cells. Time-lapse analysis revealed that mitotic duration, particularly the duration for metaphase-to-anaphase transition was prolonged in RSK2-depleted cells, suggesting activation of spindle assembly checkpoint (SAC). Indeed, more BubR1 (Bub1-related kinase) was present on metaphase plate kinetochores in RSK2-depleted cells, and depletion of BubR1 abolished the mitotic accumulation caused by RSK2 depletion, confirming BubR1-dependent SAC activation. Along with the shortening of inter-kinetochore distance, these data suggested that weakening of the tension across sister kinetochores by RSK2 depletion led to the activation of SAC. To test this, we analyzed the RSK2 effects on the stability of kinetochore-microtubule interactions, and found that RSK2-depleted cells formed less kinetochore-microtubule fibers. Moreover, RSK2 depletion resulted in the decrease of basal level of microtubule as well as an irregular distribution of mitotic spindles, which might lead to observed several mitotic progression defects such as increase in unaligned chromosomes, defects in chromosome congression and a decrease in pole-to-pole distance in these cells. Taken together, our data reveal that RSK2 affects mitotic progression by regulating the distribution, basal level and the stability of mitotic spindles.


Assuntos
Mitose , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Fuso Acromático/metabolismo , Células HeLa , Humanos , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/análise , Fuso Acromático/ultraestrutura
3.
Int J Clin Exp Pathol ; 7(8): 4959-70, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25197367

RESUMO

BACKGROUND: Colorectal cancer (CRC) is one of the leading causes of cancer-related death all over the world. Ribosomal s6 kinase4 (RSK4), an X-linked gene, firstly was found as to be a potential tumor suppressive gene in a variety of cancers and is widely participated in signaling pathway. However its role in CRC is unclear. This study is to explore the correlation between the protein expression of RSK4 and clinical pathologic characteristics in colorectal tumors, which might serve as a prognostic determinant of colorectal cancers. METHODS: Biopsies of 103 colorectal cancer and 46 matched adjacent noncancerous tissues were collected for analysis of RSK4 protein by immunohistochemistry. The correlation between RSK4 protein expression and the clinical pathological features of colorectal cancers were evaluated by Chi-square test and Fisher's exact test. The survival rates were analyzed by the Kaplan-Meier method, and the relationship between prognostic factors and patient survival was analyzed by the Cox proportional hazard models. RESULTS: RSK4 was conversely correlated with some pathological classifications (P<0.05 for N, G and clinical staging), and there were no statistically significant differences in age, CEA expression in blood, CA199 and tumors t-staging (x(2) test, P>0.05 for all categories) respectively. Furthermore, patients with high protein level of RSK4 showed prolonged overall survivals (P<0.05). Moreover, multivariate analysis showed that low expression level of RSK is an independent risk factor for high mortality in colorectal cancer. CONCLUSIONS: Low RSK4 expression is correlated with advanced clinical pathologic classifications and is a poor overall survival in colorectal cancer patients. These findings suggest that RSK4 may serve as a useful marker in prognostic evaluation for patients with colorectal cancer.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias Colorretais/patologia , Proteínas Quinases S6 Ribossômicas 90-kDa/biossíntese , Idoso , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Proteínas Quinases S6 Ribossômicas 90-kDa/análise
4.
Carcinogenesis ; 33(12): 2529-37, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22918890

RESUMO

Our previous report demonstrated that RSK2 plays an important role in cell proliferation and transformation induced by tumor promoters such as epidermal growth factor mediated through the N-terminal kinase domain of RSK2 in JB6 Cl41 mouse skin epidermal cells in vitro. However, no direct evidence has been reported regarding the relationship of RSK2 activity and human skin cancer. To elucidate the relationship of RSK2 activity and human skin cancer, we examined the effect of knocking down RSK2 expression on epidermal growth factor-induced anchorage-independent transformation in the premalignant HaCaT human skin keratinocyte cell line and on soft agar colony growth of SK-MEL-28 malignant melanoma cells. We found that the phosphorylated protein levels of RSK2 were enhanced in cancer tissues compared with normal tissues in a human skin cancer tissue array. We found that UVB stimulation induced increased in not only the total and phosphorylated protein levels of ERKs and RSK2 but also the nuclear localization and gene expression of RSK2. RSK2 knockdown inhibited proliferation and anchorage-independent transformation of HaCaT cells and soft agar colony growth of malignant melanoma cells. Moreover, RSK2(-/-) mouse embryonic fibroblast (MEF) showed enhanced sub-G(1) accumulation induced by UVB stimulation compared with RSK2(+/+) MEFs, indicating that RSK2 might play an important role in tolerance against stress associated with ultraviolet. Importantly, activated RSK2 protein levels were highly abundant in human skin cancer tissues compared with matched skin normal tissues. Taken together, our results demonstrated that RSK2 plays a key role in neoplastic transformation of human skin cells and in skin cancer growth.


Assuntos
Proteínas Quinases S6 Ribossômicas 90-kDa/fisiologia , Neoplasias Cutâneas/etiologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Fator de Crescimento Epidérmico/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Humanos , Proteínas Quinases S6 Ribossômicas 90-kDa/análise , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Transdução de Sinais/efeitos da radiação , Análise Serial de Tecidos , Raios Ultravioleta
5.
J Clin Invest ; 120(4): 1165-77, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20234090

RESUMO

Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of human cancer and frequently metastasizes to LNs. Identifying metastasis-promoting factors is of immense clinical interest, as the prognosis for patients with even a single unilateral LN metastasis is extremely poor. Here, we report that p90 ribosomal S6 kinase 2 (RSK2) promotes human HNSCC cell invasion and metastasis. We determined that RSK2 was overexpressed and activated in highly invasive HNSCC cell lines compared with poorly invasive cell lines. Expression of RSK2 also correlated with metastatic progression in patients with HNSCC. Ectopic expression of RSK2 substantially enhanced the invasive capacity of HNSCC cells, while inhibition of RSK2 activity led to marked attenuation of invasion in vitro. Additionally, shRNA knockdown of RSK2 substantially reduced the invasive and metastatic potential of HNSCC cells in vitro and in vivo in a xenograft mouse model, respectively. Mechanistically, we determined that cAMP-responsive element-binding protein (CREB) and Hsp27 are phosphorylated and activated by RSK2 and are important for the RSK2-mediated invasive ability of HNSCC cells. Our findings suggest that RSK2 is involved in the prometastatic programming of HNSCC cells, through phosphorylation of proteins in a putative signaling network. Moreover, targeting RSK2 markedly attenuates in vitro invasion and in vivo metastasis of HNSCC cells, suggesting that RSK2 may represent a therapeutic target in the treatment of metastatic HNSCC.


Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/patologia , Proteínas Quinases S6 Ribossômicas 90-kDa/fisiologia , Actinas/química , Animais , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Progressão da Doença , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Imuno-Histoquímica , Metástase Linfática , Camundongos , Invasividade Neoplásica , Fosforilação , RNA Interferente Pequeno/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/análise , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores
6.
J Affect Disord ; 124(1-2): 164-9, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19913919

RESUMO

BACKGROUND: The extracellular-regulated protein kinase (ERK) pathway has been implicated in processes such as neuronal plasticity and resilience in psychiatric disorders including major depressive disorder (MDD), bipolar disorder (BPD), and schizophrenia. The extent of the possible involvement of this pathway in psychiatric disorders remains unknown, as does its potential utility as a pharmacological target for the future development of novel therapeutics. METHODS: Western blot analyses were used to measure levels of different proteins-Rap1, B-Raf, MEK1, MEK2, ERK1/2, RSK1, CREB, NSE, and beta-actin-in the postmortem frontal cortex of individuals with schizophrenia, MDD, and BPD, as well as healthy non-psychiatric controls. RESULTS: Levels of most studied protein members of the ERK cascade were lower in individuals with psychiatric disorders than controls; differences between psychiatric groups were not statistically significant. In general, protein levels were lower in individuals with schizophrenia than in those with BPD or MDD, but protein levels varied across groups. LIMITATIONS: The small number of individuals in each diagnostic group may limit our interpretation of the results. Factors such as postmortem interval, medication status at time of death, and mood state at time of death may also have influenced the findings. DISCUSSION: The results are consistent with the hypothesis that the ERK pathway is implicated in reduced neuronal plasticity associated with the course of these psychiatric illnesses. The results warrant an expanded investigation into the activity of other members of this pathway as well as other brain areas of interest.


Assuntos
Transtorno Bipolar/patologia , Transtorno Depressivo Maior/patologia , MAP Quinases Reguladas por Sinal Extracelular/análise , Lobo Frontal/patologia , Esquizofrenia/patologia , Actinas/análise , Adulto , Western Blotting , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/análise , Feminino , Humanos , MAP Quinase Quinase 1/análise , MAP Quinase Quinase 2/análise , Masculino , Pessoa de Meia-Idade , Proteína Quinase 3 Ativada por Mitógeno/análise , Plasticidade Neuronal/fisiologia , Fosfopiruvato Hidratase/análise , Proteínas Proto-Oncogênicas B-raf/análise , Valores de Referência , Proteínas Quinases S6 Ribossômicas 90-kDa/análise , Proteínas de Ligação a Telômeros/análise
7.
Exp Hematol ; 37(10): 1238-1249.e5, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19619605

RESUMO

OBJECTIVE: Megakaryopoiesis and platelet formation is a multistep process through which hematopoietic progenitor cells develop into mature megakaryocytes (MKs) and form proplatelets. The present study investigates the regulation of different steps of megakaryopoiesis (i.e., differentiation, migration, and proplatelet formation) by extracellar signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) in two models of primary murine MKs derived from bone marrow (BM) cells and fetal liver (FL) cells. MATERIALS AND METHODS: A preparation of MKs was generated from BM obtained from femora and tibiae of C57BL6 mice. FL-derived MKs were obtained from the liver of mouse fetuses aged 13 to 15 days. RESULTS: For both cell populations, activation of MEK-ERK1/2 pathway by thrombopoietin was found to have a critical role in MK differentiation, regulating polyploidy and surface expression of CD34, GPIIb, and GPIb. The MEK-ERK1/2 pathway plays a major role in migration of BM-derived MKs toward a stromal-cell-derived factor 1alpha (SDF1alpha) gradient, whereas unexpectedly, FL-derived cells fail to migrate in response to the chemokine due to negligible expression of its receptor, CXCR4. The MEK-ERK1/2 pathway also plays a critical role in the generation of proplatelets. In contrast, p38MAPK pathway was not involved in any of these processes. CONCLUSION: This report demonstrates a critical role of MEK-ERK1/2 pathway in MK differentiation, motility, and proplatelet formation. This study highlights several differences between BM- and FL-derived MKs, which are discussed.


Assuntos
Plaquetas/citologia , Células da Medula Óssea/enzimologia , Quimiotaxia/fisiologia , Fígado/citologia , Sistema de Sinalização das MAP Quinases/fisiologia , Megacariócitos/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Trombopoese/fisiologia , Compostos de Anilina/farmacologia , Animais , Benzamidas/farmacologia , Células da Medula Óssea/citologia , Células Cultivadas/citologia , Células Cultivadas/enzimologia , Quimiocina CXCL12/farmacologia , Quimiotaxia/efeitos dos fármacos , Fígado/embriologia , Fígado/enzimologia , Megacariócitos/citologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Complexo Glicoproteico GPIb-IX de Plaquetas/biossíntese , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/biossíntese , Glicoproteína IIb da Membrana de Plaquetas/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/análise , Trombopoetina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/análise
8.
J Virol ; 82(4): 1838-50, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18057234

RESUMO

The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway is essential for infection by a variety of viruses. The p90 ribosomal S6 kinases (RSKs) are direct substrates of ERK and functional mediators of ERK MAPK signaling, but their roles in viral infection have never been examined. We demonstrate that ORF45 of Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with RSK1 and RSK2 and strongly stimulates their kinase activities. The activation of RSK by ORF45 is correlated with ERK activation but does not require MEK. We further demonstrate that RSK1/RSK2 is activated during KSHV primary infection and reactivation from latency; a subset of RSK1/RSK2 is present in the viral replication compartment in the nucleus. Depletion of RSK1/RSK2 by small interfering RNA or the specific inhibitor BI-D1870 suppresses KSHV lytic gene expression and progeny virion production, suggesting an essential role of RSK1/RSK2 in KSHV lytic replication.


Assuntos
Herpesvirus Humano 8/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteínas Virais/metabolismo , Ativação Viral , Replicação Viral , Linhagem Celular , Núcleo Celular/enzimologia , Citoplasma/enzimologia , Herpesvirus Humano 8/genética , Humanos , Proteínas Imediatamente Precoces/análise , Proteínas Imediatamente Precoces/genética , Fosforilação , Interferência de RNA , Proteínas Quinases S6 Ribossômicas 90-kDa/análise , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Virais/análise , Proteínas Virais/genética
9.
J Invest Dermatol ; 127(8): 2012-9, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17429437

RESUMO

The activity of the p38 mitogen-activated protein kinases (MAPKs) is increased in lesional psoriatic skin, supporting a possible role of these kinases in the pathogenesis of psoriasis. Recently, increased focal activation of the downstream target mitogen- and stress-activated protein kinase 1 (MSK1) was demonstrated in psoriatic epidermis. The purpose of this study is to investigate MSK2 and the transcription factor cyclic adenosine monophosphate response element-binding protein (CREB) in psoriatic skin and in cultured normal human keratinocytes. In lesional psoriatic skin, significantly increased MSK2 (Ser196) and CREB (Ser133) activation was demonstrated by phospho blotting. Immunofluorescence staining of phosphorylated MSK2 (Ser196) revealed colocalization with phosphorylated MSK1 (Thr 581) in the epidermis. Keratinocyte cultures stimulated with anisomycin and IL-1beta showed increased MSK2 (Ser196) and CREB (Ser133) phosphorylation. Such activation was abolished during preincubation with a p38 inhibitor. Keratinocytes transfected with small interfering RNA showed a stronger decrease in CREB phosphorylation in MSK1/2 double-transfected cells than in MSK1 and MSK2 single-transfected cells. This study demonstrate for the first time the expression of MSK2 in keratinocytes and increased MSK2 and CREB activation in lesional psoriatic skin. Our results indicate that the p38-MAPK/MSK1/MSK2 and CREB signalling pathway may play a role in the pathogenesis of psoriasis.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Epiderme/metabolismo , Psoríase/etiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/fisiologia , Adulto , Células Cultivadas , Reações Cruzadas , Imunofluorescência , Humanos , Fosforilação , Psoríase/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/análise , Proteínas Quinases S6 Ribossômicas 90-kDa/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia
10.
Development ; 133(9): 1823-30, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16571626

RESUMO

The cell cycle in oocytes generally arrests at a particular meiotic stage to await fertilization. This arrest occurs at metaphase of meiosis II (meta-II) in frog and mouse, and at G1 phase after completion of meiosis II in starfish. Despite this difference in the arrest phase, both arrests depend on the same Mos-MAPK (mitogen-activated protein kinase) pathway, indicating that the difference relies on particular downstream effectors. Immediately downstream of MAPK, Rsk (p90 ribosomal S6 kinase, p90(Rsk)) is required for the frog meta-II arrest. However, the mouse meta-II arrest challenges this requirement, and no downstream effector has been identified in the starfish G1 arrest. To investigate the downstream effector of MAPK in the starfish G1 arrest, we used a neutralizing antibody against Rsk and a constitutively active form of Rsk. Rsk was activated downstream of the Mos-MAPK pathway during meiosis. In G1 eggs, inhibition of Rsk activity released the arrest and initiated DNA replication without fertilization. Conversely, maintenance of Rsk activity prevented DNA replication following fertilization. In early embryos, injection of Mos activated the MAPK-Rsk pathway, resulting in G1 arrest. Moreover, inhibition of Rsk activity during meiosis I led to parthenogenetic activation without meiosis II. We conclude that immediately downstream of MAPK, Rsk is necessary and sufficient for the starfish G1 arrest. Although CSF (cytostatic factor) was originally defined for meta-II arrest in frog eggs, we propose to distinguish ;G1-CSF' for starfish from ;meta-II-CSF' for frog and mouse. The present study thus reveals a novel role of Rsk for G1-CSF.


Assuntos
Asterina/fisiologia , Fase G1 , Óvulo/enzimologia , Óvulo/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Blastômeros/citologia , Blastômeros/enzimologia , Ativação Enzimática , Feminino , Glutationa Transferase/metabolismo , Histidina/química , Cinética , Meiose , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Óvulo/citologia , Partenogênese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/análise , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/genética
11.
Mol Biol Cell ; 17(5): 2223-35, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16481405

RESUMO

Biochemical and microscopic studies have indicated that FGFR1 is a transmembrane and soluble protein present in the cytosol and nucleus. How FGFR1 enters the cytosol and subsequently the nucleus to control cell development and associated gene activities has become a compelling question. Analyses of protein synthesis, cytoplasmic subcompartmental distribution and movement of FGFR1-EGFP and FGFR1 mutants showed that FGFR1 exists as three separate populations (a) a newly synthesized, highly mobile, nonglycosylated, cytosolic receptor that is depleted by brefeldin A and resides outside the ER-Golgi lumen, (b) a slowly diffusing membrane receptor population, and (c) an immobile membrane pool increased by brefeldin A. RSK1 increases the highly mobile cytosolic FGFR1 population and its overall diffusion rate leading to increased FGFR1 nuclear accumulation, which coaccumulates with RSK1. A model is proposed in which newly synthesized FGFR1 can enter the (a) "nuclear pathway," where the nonglycosylated receptor is extruded from the pre-Golgi producing highly mobile cytosolic receptor molecules that rapidly accumulate in the nucleus or (b) "membrane pathway," in which FGFR1 is processed through the Golgi, where its movement is spatially restricted to trans-Golgi membranes with limited lateral mobility. Entrance into the nuclear pathway is favored by FGFR1's interaction with kinase active RSK1.


Assuntos
Citoplasma/metabolismo , Biossíntese de Proteínas , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Brefeldina A/farmacologia , Bovinos , Núcleo Celular/química , Núcleo Celular/metabolismo , Células Cultivadas , Recuperação de Fluorescência Após Fotodegradação , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Humanos , Modelos Biológicos , Transporte Proteico/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/análise , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/análise
12.
Mol Cell Biol ; 24(24): 10573-83, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15572664

RESUMO

We investigated the role of Rsk proteins in the nerve growth factor (NGF) signaling pathway in PC12 cells. When rat Rsk1 or murine Rsk2 proteins were transiently expressed, NGF treatment (100 ng/ml for 3 days) caused three- and fivefold increases in Rsk1 and Rsk2 activities, respectively. Increased activation of both wild-type Rsk proteins could be achieved by coexpression of a constitutively active (CA) mitogen-activated protein kinase (MAPK) kinase, MEK1-DD, which is known to cause differentiation of PC12 cells even in the absence of NGF. Rsk1 and Rsk2 mutated in the PDK1-binding site were not activated by either NGF or MEK1-DD. Expression of constitutively active Rsk1 or Rsk2 in PC12 cells resulted in highly active proteins whose levels of activity did not change either with NGF treatment or after coexpression with MEK1-DD. Rsk2-CA expression had no detectable effect on the cells. However, expression of Rsk1-CA led to differentiation of PC12 cells even in the absence of NGF, as evidenced by neurite outgrowth. Differentiation was not observed with a nonactive Rsk1-CA that was mutated in the PDK1-binding site. Expression of Rsk1-CA did not lead to activation of the endogenous MAPK pathway, indicating that Rsk1 is sufficient to induce neurite outgrowth and is the only target of MAPK required for this effect. Collectively, our data demonstrate a key role for Rsk1 in the differentiation process of PC12 cells.


Assuntos
Diferenciação Celular , Neurônios/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Embrião não Mamífero , Ativação Enzimática , Feminino , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , MAP Quinase Quinase 1/metabolismo , Microinjeções , Microscopia de Fluorescência , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Cultura de Órgãos , Células PC12 , RNA Mensageiro/metabolismo , Ratos , Proteínas Quinases S6 Ribossômicas 90-kDa/análise , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transdução de Sinais , Fatores de Tempo , Xenopus
14.
Methods ; 32(4): 389-97, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15003601

RESUMO

Intracellular signaling by protein kinases controls many aspects of cellular biochemistry and physiology. Determining the direct substrates of protein kinases is important in understanding how these signaling enzymes exert their effect on cellular functions. One of the recent developments in this area takes advantage of the similarity in the ATP binding domains of protein kinases, where a few conserved amino acids containing large side chains come in close contact with the N-6 position of bound ATP. Mutation of one or more of these residues generates a "pocket" in the ATP binding site that allows the mutant kinase, but not other cellular kinases, to utilize analogs of ATP with bulky substituents synthesized onto the N-6 position. The use of such a mutated kinase and radiolabeled ATP analogs allows for the specific labeling of direct substrates of the kinase within a mixture of cellular proteins. We have recently reported the generation of "pocket" mutants of extracellular regulated kinase 2 (ERK2) and their use in the identification of two novel substrates of ERK2. In this report, we discuss the generation and characterization of ERK2 mutants that utilize analogs of ATP and describe the methodology used to identify ERK2-associated substrates. We also describe the direct labeling of ERK2 substrates in cell lysates. These methodologies can be adapted for use with other protein kinases to increase the understanding of intracellular signal transduction.


Assuntos
Trifosfato de Adenosina/metabolismo , Engenharia de Proteínas/métodos , Proteínas Quinases/metabolismo , Transdução de Sinais/fisiologia , Trifosfato de Adenosina/análogos & derivados , Animais , Sítios de Ligação/genética , Western Blotting , Células COS , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Cercopithecus aethiops , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Fator de Crescimento Epidérmico/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Marcação por Isótopo , Espectrometria de Massas , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Mutagênese Sítio-Dirigida/genética , Proteína Básica da Mielina/metabolismo , Fosforilação , Testes de Precipitina , Proteínas Quinases/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/análise , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Especificidade por Substrato , Fatores de Transcrição/metabolismo , Transformação Genética , Ubiquitina-Proteína Ligases/análise , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Elk-1 do Domínio ets
15.
Endocrinology ; 144(8): 3344-50, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12865312

RESUMO

In this study we investigated diurnal changes in the activation state of the 90-kDa ribosomal S6 kinase (p90RSK) in the rat pineal gland. In animals housed under a lighting regimen with 12 h of light, we found an increase in phosphorylated p90RSK during the dark phase, and this increase was abolished by treatment with propranolol or continuous exposure to light. To determine the intracellular mechanism involved, rat pinealocytes were treated with norepinephrine. Norepinephrine caused a parallel increase in phosphorylated p42/44 MAPK (p42/44(MAPK)) and p90RSK that was reduced by prazosin or propranolol, indicating involvement of both alpha(1)- and beta-adrenergic receptors. Treatment with dibutyryl cGMP, 4beta-phorbol 12-myristate 13-acetate, or ionomycin mimicked norepinephrine-stimulated p90RSK phosphorylation, whereas dibutyryl cAMP caused a decrease in p90RSK phosphorylation. Inhibition of p42/44(MAPK) activation by UO126 was effective in reducing norepinephrine-stimulated p90RSK phosphorylation. Moreover, UO126 had an inhibitory effect on norepinephrine-stimulated arylalkyl-N-acetyltransferase activity. These results indicate that the adrenergically regulated nocturnal increase in p90RSK phosphorylation is mainly mediated through a cGMP-->p42/44(MAPK)-dependent mechanism.


Assuntos
Homeostase , Glândula Pineal/enzimologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Arilamina N-Acetiltransferase/metabolismo , Ritmo Circadiano , GMP Cíclico/farmacologia , Inibidores Enzimáticos/farmacologia , Luz , MAP Quinase Quinase 1 , MAP Quinase Quinase 2 , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Norepinefrina/farmacologia , Fosforilação , Glândula Pineal/efeitos dos fármacos , Glândula Pineal/efeitos da radiação , Propranolol/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos alfa 1/fisiologia , Receptores Adrenérgicos beta/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/análise , Transdução de Sinais
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