RESUMO
The PI3K/PTEN/Akt/mTOR/p70S6K pathway is one of the most frequently deregulated signaling pathways in solid tumors and has a functional role in drug resistance. However, targeting this pathway leads to compensatory activation of several mediators of cell survival. Expression of the reactive oxygen species-controlling kinase Mirk/dyrk1B was increased severalfold by the mammalian target of rapamycin (mTOR) inhibitors RAD001, WYE354 and rapamycin, with less effect by the Akt inhibitors AZD5363 and MK-2206. Upregulation of Mirk messenger RNA (mRNA) expression was mediated by cyclic AMP response element binding protein (CREB) binding to two sites in the Mirk promoter upstream of the transcription start site and one site within exon 4. Depletion of CREB reduced Mirk expression, whereas depletion of mTOR increased it. Moreover, hydroxytamoxifen activation of an Akt-estrogen receptor construct blocked an increase in Mirk mRNA and protein. Addition of a Mirk/dyrk1B kinase inhibitor increased the sensitivity of Panc1 pancreatic cancer cells and three different ovarian cancer cell lines to the mTOR inhibitor RAD001. Targeting Mirk kinase could improve the utility of mTOR inhibitors and so presents an attractive drug target.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima/efeitos dos fármacos , Linhagem Celular Tumoral , Cromonas/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Sinergismo Farmacológico , Ativação Enzimática , Everolimo , Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Morfolinas/farmacologia , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Quinases DyrkRESUMO
A major problem in the treatment of cancer arises from quiescent cancer cells that are relatively insensitive to most chemotherapeutic drugs and radiation. Such residual cancer cells can cause tumor regrowth or recurrence when they reenter the cell cycle. Earlier studies showed that levels of the serine/theronine kinase Mirk/dyrk1B are elevated up to 10-fold in quiescent G(0) tumor cells. Mirk uses several mechanisms to block cell cycling, and Mirk increases expression of antioxidant genes that decrease reactive oxygen species (ROS) levels and increase quiescent cell viability. We now show that a novel small molecule Mirk kinase inhibitor blocked tumor cells from undergoing reversible arrest in a quiescent G(0) state and enabled some cells to exit quiescence. The inhibitor increased cycling in Panc1, AsPc1, and SW620 cells that expressed Mirk, but not in HCT116 cells that did not. Mirk kinase inhibition elevated ROS levels and DNA damage detected by increased phosphorylation of the histone protein H2AX and by S-phase checkpoints. The Mirk kinase inhibitor increased cleavage of the apoptotic proteins PARP and caspase 3, and increased tumor cell kill several-fold by gemcitabine and cisplatin. A phenocopy of these effects occurred following Mirk depletion, showing drug specificity. In previous studies Mirk knockout or depletion had no detectable effect on normal tissue, suggesting that the Mirk kinase inhibitor could have a selective effect on cancer cells expressing elevated levels of Mirk kinase.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Pancreáticas/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Apoptose , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Senescência Celular , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Sinergismo Farmacológico , Éxons , Humanos , Neoplasias Pancreáticas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Gencitabina , Quinases DyrkRESUMO
Mirk/Dyrk1B is a serine/threonine kinase widely expressed in colon cancers. Serum starvation induced HD6 colon carcinoma cells to enter a quiescent G0 state, characterized by a 2N DNA content and a lower RNA content than G1 cells. Compared with cycling cells, quiescent cells exhibited 16-fold higher levels of the retinoblastoma protein p130/Rb2, which sequesters E2F4 to block entry into G1, 10-fold elevated levels of the CDK inhibitor p27kip1, and 10-fold higher levels of Mirk. However, depletion of Mirk did not prevent entry into G0, but enabled quiescent HD6, SW480, and colo320 colon carcinoma cells to acquire some biochemical characteristics of G1 cells, including increased levels of cyclin D1 and cyclin D3 because of slower turnover, increased activity of their CDK4/cyclin D complexes, and increased phosphorylation and decreased E2F4 sequestering ability of the CDK4 target, p130/Rb2. As a result, depletion of Mirk allowed some cells to escape quiescence and enabled cells released from quiescence to traverse G1 more quickly. The kinase activity of Mirk was increased by the chemotherapeutic drug 5-fluorouracil (5-FU). Treatment of p53 mutant colon cancer cells with 5-FU led to an elongated G1 in a Mirk-dependent manner, as G1 was shortened by ectopic overexpression of cyclin D1 mutated at the Mirk phosphorylation site (T288A), but not by wild-type cyclin D1. Mirk, through regulating cyclin D turnover, and the CDK inhibitor p27, as shown by depletion studies, functioned independently and additively to regulate the exit of tumor cells from quiescence.
Assuntos
Neoplasias do Colo/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Antimetabólitos/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular , Neoplasias do Colo/patologia , Ciclina D , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/fisiologia , Ciclinas/metabolismo , Diploide , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Fluoruracila/farmacologia , Células HT29 , Humanos , Imunoprecipitação , Proteínas Serina-Treonina Quinases/genética , Estabilidade Proteica , Proteínas Tirosina Quinases/genética , Interferência de RNA , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologia , Quinases DyrkRESUMO
The kinase Mirk/dyrk1B mediated the clonogenic growth of pancreatic cancer cells in earlier studies. It is now shown that Mirk levels increased 7-fold in SU86.86 pancreatic cancer cells when over a third of the cells were accumulated in a quiescent G(0) state, defined by Hoechst/Pyronin Y staining. Depletion of Mirk by a doxycycline-inducible short hairpin RNA increased the G(0) fraction to approximately 50%, suggesting that Mirk provided some function in G(0). Mirk reduced the levels of reactive oxygen species (ROS) in quiescent cultures of SU86.86 cells and of Panc1 cells by increasing transcription of the antioxidant genes ferroxidase, superoxide dismutase (SOD)2, and SOD3. These genes were functional antioxidant genes in pancreatic cancer cells because ectopic expression of SOD2 and ferroxidase in Mirk-depleted cells lowered ROS levels. Quiescent pancreatic cancer cells quickly lost viability when depleted of Mirk because of elevated ROS levels, exhibiting up to 4-fold less colony-forming activity and 4-fold less capability for dye exclusion. As a result, reduction of ROS by N-acetyl cysteine led to more viable cells. Mirk also destabilizated cyclin D1 and D3 in quiescent cells. Thus, quiescent pancreatic cancer cells depleted of Mirk became less viable because they were damaged by ROS, and had increased levels of G(1) cyclins to prime cells to escape quiescence.
Assuntos
Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sobrevivência Celular/fisiologia , Ciclina D , Ciclinas/metabolismo , Fase G1/fisiologia , Humanos , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Interferência de RNA , Fase de Repouso do Ciclo Celular/fisiologia , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Transcrição Gênica , Transfecção , Regulação para Cima , Quinases DyrkRESUMO
The kinase Mirk is overexpressed in many resected pancreatic adenocarcinomas and is amplified in a subset of pancreatic cancer cell lines. Depletion of Mirk has been shown to lead to apoptosis in pancreatic cancer cell lines, and thus to inhibit their clonogenic growth. Mirk is activated by signaling from activated Rac1 to MKK3 in MDCK cells, but the mechanism of activation of Mirk in pancreatic cancers is unknown. In this report, Mirk is shown to be a novel effector of K-ras, a gene mutated in approximately 90% of pancreatic cancers. Activation of Mirk signaling from oncogenic K-ras through Rac1 was shown in transient expression systems and reporter assays. Mirk activation in pancreatic cancer cells was blocked by RNA interference using three different synthetic duplex RNAis to K-ras, or two RNAis to Rac1, by pharmacologic inhibition of Rac1, or by expression of dominant negative K-rasS17N. Rac1 was activated in four out of five pancreatic cancer cell lines, and was activated by signaling from oncogenic K-ras. Mirk knockout does not induce embryonic lethality, and depletion of Mirk had no effect on the survival of normal diploid fibroblasts. In contrast, the clonogenic ability of Panc1 and AsPc1 pancreatic cancer cell lines was reduced 8- to 12-fold by the depletion of Mirk, with a greater reduction seen following the depletion of K-ras or both genes. Mirk is a novel downstream effector of oncogenic K-ras and mediates some of the survival signals activated by ras signaling.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Genes ras/fisiologia , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Adesão Celular , Linhagem Celular , Ensaio de Unidades Formadoras de Colônias , Cães , Ativação Enzimática , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Imunoprecipitação , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , RNA Interferente Pequeno/genética , Transdução de Sinais , Transfecção , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases DyrkRESUMO
Rhabdomyosarcoma is the most common sarcoma in children and is difficult to treat if the primary tumor is nonresectable or if the disease presents with metastases. The function of the serine/threonine kinase Mirk was investigated in this cancer. Mirk has both growth arrest and survival functions in terminally differentiating skeletal myoblasts. Maintenance of Mirk growth arrest properties would cause down-regulation of Mirk in transformed myoblasts. Alternatively, Mirk expression would be retained if rhabdomyosarcoma cells used Mirk survival capability. Mirk expression was significant in 12 of 16 clinical cases of rhabdomyosarcoma. Mirk was detected in each rhabdomyosarcoma cell line examined. Mirk was a functional kinase in each of three rhabdomyosarcoma cell lines, where it proved to be more active than in C2C12 skeletal myoblasts. Mirk mediated survival of the majority of clonogenic rhabdomyosarcoma cells. Knockdown of Mirk by RNA interference reduced the fraction of RD and of Rh30 rhabdomyosarcoma cells capable of colony formation 3- to 4-fold in multiple experiments. Depletion of Mirk induced cell death by apoptosis, as shown by increased numbers of terminal deoxynucleotidyl transferase-mediated nick-end labeling-positive cells and by increased binding of Annexin V. Mirk is a stress-activated kinase that mediates expression of contractile proteins in differentiating myoblasts, but Mirk is not essential for muscle formation in the embryo. It is likely that Mirk also facilitates survival of satellite cell-derived rhabdomyoblasts in regenerating skeletal muscle and aids their differentiation. This survival function is maintained in rhabdomyosarcoma, where Mirk may be a novel therapeutic target.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/fisiologia , Rabdomiossarcoma Alveolar/enzimologia , Rabdomiossarcoma Embrionário/enzimologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Criança , Pré-Escolar , Citoplasma/enzimologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mioblastos/citologia , Mioblastos/patologia , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Rabdomiossarcoma Alveolar/patologia , Rabdomiossarcoma Embrionário/patologia , Fatores de Transcrição , Quinases DyrkRESUMO
Ductal adenocarcinoma of the pancreas is almost uniformly lethal as this cancer is invariably detected at an advanced stage and is resistant to treatment. The serine/threonine kinase Mirk/Dyrk1B has been shown to be antiapoptotic in rhabdomyosarcomas. We have now investigated whether Mirk might mediate survival in another cancer in which Mirk is widely expressed, pancreatic ductal adenocarcinoma. Mirk was an active kinase in each pancreatic cancer cell line where it was detected. Mirk knockdown by RNA interference (RNAi) reduced the clonogenicity of Panc1 pancreatic cancer cells 4-fold and decreased tumor cell number, showing that Mirk mediates survival in these cells. Mirk knockdown by synthetic duplex RNAis in Panc1, AsPc1, and SU86.86 pancreatic cancer cells induced apoptosis and enhanced the apoptosis induced by gemcitibine. Mirk knockdown did not increase the abundance or activation of Akt. However, four of five pancreatic carcinoma cell lines exhibited either elevated Mirk activity or elevated Akt activity, suggesting that pancreatic cancer cells primarily rely on Mirk or Akt for survival signaling. Mirk protein was detected by immunohistochemistry in 25 of 28 cases (89%) of pancreatic ductal adenocarcinoma, with elevated expression in 11 cases (39%). Increased expression of Mirk was seen in pancreatic carcinomas compared with primary cultures of normal ductal epithelium by serial analysis of gene expression and by immunohistochemistry. Thus, Mirk is a survival factor for pancreatic ductal adenocarcinoma. Because knockout of Mirk does not cause embryonic lethality, Mirk is not essential for normal cell growth and may represent a novel therapeutic target.
Assuntos
Carcinoma Ductal Pancreático/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias Pancreáticas/enzimologia , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Sobrevivência Celular/fisiologia , Citoplasma/enzimologia , Ativação Enzimática , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Proteínas Quinases Ativadas por Mitógeno/deficiência , Proteínas Quinases Ativadas por Mitógeno/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Fatores de Transcrição , Quinases DyrkRESUMO
The kinase Mirk/dyrk1B is essential for the differentiation of C2C12 myoblasts. Mirk reinforces the G0/G1 arrest state in which differentiation occurs by directly phosphorylating and stabilizing p27(Kip1) and destabilizing cyclin D1. We now demonstrate that Mirk is anti-apoptotic in myoblasts. Knockdown of endogenous Mirk by RNA interference activated caspase 3 and decreased myoblast survival by 75%, whereas transient overexpression of Mirk increased cell survival. Mirk exerts its anti-apoptotic effects during muscle differentiation at least in part through effects on the cell cycle inhibitor and pro-survival molecule p21(Cip1). Overexpression and RNA interference experiments demonstrated that Mirk phosphorylates p21 within its nuclear localization domain at Ser-153 causing a portion of the typically nuclear p21 to localize in the cytoplasm. Phosphomimetic GFP-p21-S153D was pancellular in both cycling C2C12 myoblasts and NIH3T3 cells. Endogenous Mirk in myotubes and overexpressed Mirk in NIH3T3 cells were able to cause the pancellular localization of wild-type GFP-p21 but not the nonphosphorylatable mutant GFP-p21-S153A. Translocation to the cytoplasm enables p21 to block apoptosis through inhibitory interaction with pro-apoptotic molecules. Phosphomimetic p21-S153D was more effective than wild-type p21 in blocking the activation of caspase 3. Transient expression of p21-S153D also increased myoblast viability in colony forming assays, whereas the p21-S153A mutant had no effect. This Mirk-dependent change in p21 intracellular localization is a natural part of myoblast differentiation. Endogenous p21 localized exclusively to the nuclei of proliferating myoblasts but was also found in the cytoplasm of post-mitotic multinucleated myotubes and adult human skeletal myofibers.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/fisiologia , Animais , Apoptose/fisiologia , Caspase 3 , Caspases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular , Sobrevivência Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p21 , Citoplasma/metabolismo , Humanos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/genética , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Fosforilação , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Regeneração/fisiologia , Serina/metabolismo , Fatores de Transcrição , Transfecção , Quinases DyrkRESUMO
Mirk/dyrk1B is a member of the dyrk/minibrain family of serine/threonine kinases that mediate the transition from growth to differentiation in lower eukaryotes and mammals. Depletion of endogenous Mirk from C2C12 myoblasts by RNA interference blocks skeletal muscle differentiation (Deng, X., Ewton, D., Pawlikowski, B., Maimone, M., and Friedman, E. (2003) J. Biol. Chem. 278, 41347-41354). We now demonstrate that knockdown of Mirk blocks transcription of the muscle regulatory factor myogenin. Co-expression of Mirk with MEF2C, but not MyoD or Myf5, enhanced activation of the myogenin promoter in a Mirk kinase-dependent manner. Mirk activated MEF2 not through direct phosphorylation of MEF2 but by phosphorylation of its inhibitors, the class II histone deacetylases (HDACs). MEF2 is sequestered by class II HDACs such as HDAC5 and MEF2-interacting transcriptional repressor (MITR). Mirk antagonized the inhibition of MEF2C by MITR, whereas kinase-inactive Mirk was ineffective. Mirk phosphorylates class II HDACs at a conserved site within the nuclear localization region, reducing their nuclear accumulation in a dose-dependent and kinase-dependent manner. Moreover, less mutant MITR phosphomimetic at the Mirk phosphorylation site localized in the nucleus than wild-type MITR. Regulation of class II HDACs occurs by multiple mechanisms. Others have shown that calcium signaling leads to phosphorylation of HDACs at 14-3-3-binding sites, blocking their association with MEF2 within the nucleus. Mirk provides another level of regulation. Mirk is induced within the initial 24 h of myogenic differentiation and enables MEF2 to transcribe the myogenin gene by decreasing the nuclear accumulation of class II HDACs.
Assuntos
Núcleo Celular/metabolismo , Regulação Enzimológica da Expressão Gênica , Histona Desacetilases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Músculo Esquelético/citologia , Miogenina/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Tirosina Quinases/biossíntese , Sequência de Aminoácidos , Animais , Sítios de Ligação , Northern Blotting , Butiratos/farmacologia , Diferenciação Celular , Linhagem Celular , DNA/química , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase/metabolismo , Luciferases/metabolismo , Fatores de Transcrição MEF2 , Camundongos , Dados de Sequência Molecular , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteína MyoD/metabolismo , Fator Regulador Miogênico 5 , Fatores de Regulação Miogênica/metabolismo , Miogenina/genética , Células NIH 3T3 , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Frações Subcelulares , Transativadores/metabolismo , Fatores de Transcrição , Transcrição Gênica , Transfecção , Regulação para Cima , Quinases DyrkRESUMO
The phosphorylation of cyclin D1 at threonine 286 by glycogen synthase kinase 3beta (GSK3beta) has been shown to be required for the ubiquitination and nuclear export of cyclin D1 and its subsequent degradation in the proteasome. The mutation of the nearby residue, threonine 288, to nonphosphorylatable alanine has also been shown to reduce the ubiquitination of cyclin D1, suggesting that phosphorylation at threonine 288 may also lead to degradation of cyclin D1. We now demonstrate that the G(0)/G(1)-active arginine-directed protein kinase Mirk/dyrk1B binds to cyclin D1 and phosphorylates cyclin D1 at threonine 288 in vivo and that the cyclin D1-T288A construct is more stable than wild-type cyclin D1. Transient overexpression of Mirk in nontransformed Mv1Lu lung epithelial cells blocked cells in G(0)/G(1). Depletion of endogenous Mirk by RNA interference increased cyclin D1 protein levels but not mRNA levels, indicating that Mirk destabilizes cyclin D1 protein. Destabilization was confirmed by induction of a stable Mirk transfectant of Mv1Lu cells, which blocked cell migration (Zou, Y., Lim, S., Lee, K., Deng, X., and Friedman, E. (2003) J. Biol. Chem. 278, 49573-49581), and caused a decrease in the half-life of endogenous cyclin D1, concomitant with an increase in Mirk expression. In vitro cyclin D1 was phosphorylated in an additive fashion by Mirk and GSK3beta. Mirk-phosphorylated cyclin D1 mutated at the GSK3beta phosphorylation site and was capable of phosphorylating cyclin D1 in the presence of the GSK3beta inhibitor LiCl. Mirk may function together with GSK3beta to assist cell arrest in G(0)/G(1) by destabilizing cyclin D1.
Assuntos
Ciclina D1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Treonina/metabolismo , Animais , Divisão Celular/fisiologia , Linhagem Celular , Ciclina D1/genética , Cicloeximida/farmacologia , Indução Enzimática/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Isopropiltiogalactosídeo/farmacologia , Pulmão/citologia , Camundongos , Vison , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Proteínas Quinases Ativadas por Mitógeno/genética , Mioblastos/citologia , Mioblastos/enzimologia , Fosforilação , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fase S/fisiologia , Fatores de Transcrição , Quinases DyrkRESUMO
Elevated levels of the cyclin-dependent kinase (CDK) inhibitor p27 block the cell in G(0)/G(1) until mitogenic signals activate G(1) cyclins and initiate proliferation. Post-translational regulation of p27 by different phosphorylation events is critical in allowing cells to proceed through the cell cycle. We now demonstrate that the arginine-directed kinase, Mirk/dyrk1B, is maximally active in G(0) in NIH3T3 cells, when it stabilizes p27 by phosphorylating it at Ser-10. The phospho-mimetic mutant p27-S10D was more stable, and the non-phosphorylatable mutant p27-S10A was less stable than wild-type when expressed in G(0)-arrested cells. Following phosphorylation by Mirk, p27 remains a functional CDK inhibitor, capable of binding to CDK2. Mirk did not induce the translocation of p27 from the nucleus in G(0), but instead co-localized with nuclear p27. Depletion of Mirk by RNA interference decreased the phosphorylation of p27 at Ser-10 and the stability of endogenous p27. RNA(i) to Mirk increased cell entry from G(0) into G(1) as shown by increased expression of proliferating cell nuclear antigen and decreased expression of p27. These data suggest a model in which Mirk increases the amount of nuclear p27 by stabilizing it during G(0) when Mirk is most abundant. Mitogen stimulation then causes cells to enter G(1), reduces Mirk levels (Deng, X., Ewton, D., Pawlikowski, B., Maimone, M., and Friedman, E. (2003) J. Biol. Chem. 278, 41347-41354), and initiates the translocation of p27 to the cytoplasm. In addition, depletion of Mirk by RNA(i) in postmitotic C2C12 myoblasts decreased protein but not mRNA levels of p27, suggesting that stabilization of p27 by Mirk also occurs during differentiation.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Animais , Northern Blotting , Ciclo Celular , Diferenciação Celular , Divisão Celular , Linhagem Celular , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p27 , Cicloeximida/farmacologia , Inibidores Enzimáticos/farmacologia , Glutationa Transferase/metabolismo , Camundongos , Microscopia de Fluorescência , Mitose , Mutação , Células NIH 3T3 , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Antígeno Nuclear de Célula em Proliferação/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico , Interferência de RNA , Proteínas Recombinantes/química , Fase de Repouso do Ciclo Celular , Fatores de Tempo , Fatores de Transcrição , Transfecção , Quinases DyrkRESUMO
The Rho family of small GTPases regulates numerous signaling pathways that control the organization of the cytoskeleton, transcription factor activity, and many aspects of the differentiation of skeletal myoblasts. We now demonstrate that the kinase Mirk (minibrain-related kinase)/dyrk1B is induced by members of the Rho-family in myoblasts and that Mirk is active in skeletal muscle differentiation. Mirk is an arginine-directed serine/threonine kinase which is expressed at elevated levels in skeletal muscle compared with other normal tissues. A Mirk promoter construct was activated when C2C12 myoblasts were switched from growth to differentiation medium and was also activated by the Rho family members RhoA, Cdc42, and to a lesser degree Rac1, but not by MyoD or Myf5. Mirk protein levels increased following transient expression of constitutively active Cdc42-QL, RhoA-QL, or Rac1-QL in C2C12 cells. High concentrations of serum mitogens down-regulated Mirk through activation of the Ras-MEK-Erk pathway. As a result, Mirk transcription was induced by the MEK1 inhibitor PD98059 and by the switch from growth to differentiation medium. Mirk was induced with similar kinetics to another Rho-induced differentiation gene, myogenin. Mirk protein levels increased 10-fold within 24-48 h after primary cultured muscle cells; C2C12 mouse myoblasts or L6 rat myoblasts were induced to differentiate. Thus Mirk was induced following the commitment stage of myogenesis. Stable overexpression of Mirk enabled myoblasts to fuse more rapidly when placed in differentiation medium. The function of Mirk in muscle differentiation was established by depletion of endogenous Mirk by small interfering RNA, which prevented myoblast fusion into myotubes and inhibited induction of markers of differentiation, including myogenin, fast twitch troponin T, and muscle myosin heavy chain. Other members of the dyrk/minibrain/HIPK family of kinases in lower organisms have been shown to regulate the transition from growth to differentiation, and Mirk is now shown to participate in skeletal muscle development.
Assuntos
Músculo Esquelético/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Deleção de Genes , Cinética , Camundongos , Músculo Esquelético/metabolismo , Músculos/metabolismo , Miogenina/metabolismo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Interferência de RNA , RNA Mensageiro/metabolismo , Ratos , Fatores de Tempo , Distribuição Tecidual , Transfecção , Quinases DyrkRESUMO
Mirk/dyrk1B is an arginine-directed protein kinase, which functions as a transcriptional activator and mediates serum-free growth of colon carcinoma cells by an unknown mechanism. We now report that turnover of the cdk inhibitor p27(kip1) and the G(1)-phase cyclin cyclin D1 is enhanced in each of 4 Mirk stable transfectants compared to vector control transfectants and Mirk kinase-inactive mutant transfectants. This enhanced turnover is proteasome-dependent and leads to lower protein levels of both p27(kip1) and cyclin D1. Lower protein levels of the cdk inhibitor p21(cip1) were also observed in the 4 Mirk stable transfectants. Mirk did not alter the activity of a p27(kip1) promoter construct or p27(kip1) mRNA levels by stable expression, indicating that the decrease in p27(kip1) protein levels was due to a posttranscriptional mechanism. These data are consistent with mirk enhancing the expression of some component common to the proteolysis of both p27(kip1) and cyclin D1.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclina D1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Transfecção , Células Tumorais Cultivadas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Células 3T3 , Animais , Northern Blotting , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Ciclina D1/genética , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/genética , Ciclinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Meia-Vida , Humanos , Immunoblotting , Luciferases/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição , Proteínas Supressoras de Tumor/genética , Quinases DyrkRESUMO
Stress signals activate the SAPK/JNK and p38 MAPK classes of protein kinases, which mediate cellular responses, including steps in apoptosis and the maturation of some cell types. We now show that stress signals initiated by transforming growth factor-beta 1 (TGF-beta 1) induce G(1) arrest through protein stabilization of the CDK inhibitor p21(Cip1). TGF-beta 1 was previously shown to increase p21 protein levels, which in turn mediated G(1) arrest through inactivation of the CDK2-cyclin E complex in HD3 cells (Yan, Z., Kim, G.-Y., Deng, X., and Friedman, E. (2002) J. Biol. Chem. 277, 9870-9879). We now demonstrate that the increase in p21 abundance is caused by a post-transcriptional, SMAD-independent mechanism. TGF-beta1 activated p38 alpha and JNK1, which initiated the phosphorylation of p21. TGF-beta1 treatment increased the half-life of p21 by 3-4-fold. The increase in p21 stability was detected following activation of p38 alpha and JNK1, and treatment of cells with the p38 inhibitor SB203580 prevented this increase in p21 stability. p38 alpha and JNK1 phosphorylated p21 in vivo, and both p38 alpha and JNK1 phosphorylated p21 at Ser(130) in vitro. Peptide mapping demonstrated that both TGF-beta 1 and p38 alpha induced phosphorylation of p21 at Ser(130) in vivo, and mutation of Ser(130) to alanine rendered p21 less stable than wild-type p21. TGF-beta 1 increased the stability of wild-type p21, but not the p21-S130A mutant. These findings demonstrate that SAPKs can mediate cell cycle arrest through post-translational modification of p21.
Assuntos
Ciclinas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Imidazóis/farmacologia , Proteína Quinase 14 Ativada por Mitógeno , Proteína Quinase 8 Ativada por Mitógeno , Fosforilação , Piridinas/farmacologia , Serina/metabolismo , Transdução de Sinais , Treonina/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Células Tumorais Cultivadas , Regulação para CimaRESUMO
IGF-I stimulates intestinal cell differentiation after initiating a short proliferative burst, similar to its effect on muscle cell differentiation. Levels of IGF-I attainable in serum (10-20 ng/ml) induced transient growth stimulation of colon carcinoma cells, then growth arrest. When IGF-I functioned as a mitogen, it blocked differentiation. Intestinal cell differentiation occurred once cells had undergone the IGF-I-initiated growth arrest and IGF-I and butyrate acted synergistically to induce maturation markers. IGF-I induces NIH-3T3 cell proliferation and survival by activating the kinase akt, which in turn inhibits various apoptotic mediators and the forkhead family of transcription factors, which mediate expression of p27(kip1). Promoter reporter assays demonstrated that forkhead1 mediates transcription of p27(kip1) in colon carcinoma cells. The mitogenic effects of IGF-I on 4 colon carcinoma cell lines were transient because the inactivating phosphorylation of forkhead1 by akt was short-lived. This allowed transcriptional upregulation of the cdk inhibitor p27(kip1), with a resulting growth arrest. In contrast, in NIH-3T3 cells treated in parallel with identical IGF-I levels, forkhead phosphorylation levels were sustained; thus, no increase in p27(kip1) levels was seen and cells continued to proliferate. Intestinal epithelial cells in vivo undergo a limited number of divisions, then growth arrest and completion of their maturation. IGFs found in intestinal tissue may control the timing of this process. In addition, colon cancers may have developed strategies to overcome IGF-I-mediated growth arrest. Earlier (Kansra et al., Int J Cancer 2000;87:373-8), we found that levels of IGFBP-3 were elevated at least 2-fold in 70% of resected colon cancers compared with adjacent normal tissue. In the current study, growth inhibition by IGF-I and IGF-II was blocked by concurrent addition of IGFBP-3, implying that colon cancers with elevated IGFBP-3 levels would be selected for in vivo because they could bind and inactivate high serum IGF-I levels and continue to proliferate.