RESUMO
The Akt-Rheb-mTORC1 pathway plays a crucial role in regulating cell growth, but the mechanisms underlying the activation of Rheb-mTORC1 by Akt remain unclear. In our previous study, we found that CBAP was highly expressed in human T-ALL cells and primary tumors, and its deficiency led to reduced phosphorylation of TSC2/S6K1 signaling proteins as well as impaired cell proliferation and leukemogenicity. We also demonstrated that CBAP was required for Akt-mediated TSC2 phosphorylation in vitro. In response to insulin, CBAP was also necessary for the phosphorylation of TSC2/S6K1 and the dissociation of TSC2 from the lysosomal membrane. Here we report that CBAP interacts with AKT and TSC2, and knockout of CBAP or serum starvation leads to an increase in TSC1 in the Akt/TSC2 immunoprecipitation complexes. Lysosomal-anchored CBAP was found to override serum starvation and promote S6K1 and 4EBP1 phosphorylation and c-Myc expression in a TSC2-dependent manner. Additionally, recombinant CBAP inhibited the GAP activity of TSC2 complexes in vitro, leading to increased Rheb-GTP loading, likely due to the competition between TSC1 and CBAP for binding to the HBD domain of TSC2. Overexpression of the N26 region of CBAP, which is crucial for binding to TSC2, resulted in a decrease in mTORC1 signaling and an increase in TSC1 association with the TSC2/AKT complex, ultimately leading to increased GAP activity toward Rheb and impaired cell proliferation. Thus, we propose that CBAP can modulate the stability of TSC1-TSC2 as well as promote the translocation of TSC1/TSC2 complexes away from lysosomes to regulate Rheb-mTORC1 signaling.
Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas de Membrana , Proteínas Proto-Oncogênicas c-akt , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Humanos , Proliferação de Células , Guanosina Trifosfato/metabolismo , Imunoprecipitação , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismoRESUMO
Microsomal triglyceride transfer protein (MTTP) is an endoplasmic reticulum resident protein that is essential for the assembly and secretion of triglyceride (TG)-rich, apoB-containing lipoproteins. Although the function and structure of mammalian MTTP have been extensively studied, how exactly MTTP transfers lipids to lipid acceptors and whether there are other biomolecules involved in MTTP-mediated lipid transport remain elusive. Here we identify a role in this process for the poorly characterized protein PRAP1. We report that PRAP1 and MTTP are partially colocalized in the endoplasmic reticulum. We observe that PRAP1 directly binds to TG and facilitates MTTP-mediated lipid transfer. A single amino acid mutation at position 85 (E85V) impairs PRAP1's ability to form a ternary complex with TG and MTTP, as well as impairs its ability to facilitate MTTP-mediated apoB-containing lipoprotein assembly and secretion, suggesting that the ternary complex formation is required for PRAP1 to facilitate MTTP-mediated lipid transport. PRAP1 is detectable in chylomicron/VLDL-rich plasma fractions, suggesting that MTTP recognizes PRAP1-bound TG as a cargo and transfers TG along with PRAP1 to lipid acceptors. Both PRAP1-deficient and E85V knock-in mutant mice fed a chow diet manifested an increase in the length of their small intestines, likely to compensate for challenges in absorbing lipid. Interestingly, both genetically modified mice gained significantly less body weight and fat mass when on high-fat diets compared with littermate controls and were prevented from hepatosteatosis. Together, this study provides evidence that PRAP1 plays an important role in MTTP-mediated lipid transport and lipid absorption.
Assuntos
Proteínas de Transporte/metabolismo , Metabolismo dos Lipídeos , Proteínas da Gravidez/metabolismo , Animais , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismo , Transporte Biológico , Dieta Hiperlipídica , Fígado Gorduroso/genética , Lipoproteínas/metabolismo , Camundongos , Camundongos Knockout , Proteínas da Gravidez/genética , Ligação Proteica , Triglicerídeos/metabolismoRESUMO
Replication of sufficient mitochondrial DNA (mtDNA) is essential for maintaining mitochondrial functions in mammalian cells. During mtDNA replication, RNA primers must be removed before the nascent circular DNA strands rejoin. This process involves mitochondrial RNase H1, which removes most of the RNA primers but leaves two ribonucleotides attached to the 5' end of nascent DNA. A subsequent 5'-exonuclease is required to remove the residual ribonucleotides, however, it remains unknown if any mitochondrial 5'-exonuclease could remove two RNA nucleotides from a hybrid duplex DNA. Here, we report that human mitochondrial Exonuclease G (ExoG) may participate in this particular process by efficiently cleaving at RNA-DNA junctions to remove the 5'-end RNA dinucleotide in an RNA/DNA hybrid duplex. Crystal structures of human ExoG bound respectively with DNA, RNA/DNA hybrid and RNA-DNA chimeric duplexes uncover the underlying structural mechanism of how ExoG specifically recognizes and cleaves at RNA-DNA junctions of a hybrid duplex with an A-form conformation. This study hence establishes the molecular basis of ExoG functioning as a unique 5'-exonuclease to mediate the flap-independent RNA primer removal process during mtDNA replication to maintain mitochondrial genome integrity.
Assuntos
Replicação do DNA , DNA Mitocondrial/genética , Endonucleases/metabolismo , Exonucleases/metabolismo , Ribonuclease H/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Endonucleases/genética , Exonucleases/genética , Humanos , Mitocôndrias/genética , Nucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , RNA/genética , Ribonucleotídeos/metabolismoRESUMO
IL-15 is an essential survival factor for CD8αα(+) intestinal intraepithelial lymphocytes (iIELs) in vitro and in vivo. However, the IL-15-induced survival signals in primary CD8αα(+) iIELs remains elusive. Although Bcl-2 level in CD8αα(+) iIELs positively correlates with IL-15Rα expression in the intestinal epithelial cells, overexpression of Bcl-2 only moderately restores CD8αα(+) γδ iIELs in Il15(-/-) mice. Here, we found that IL-15 promptly activated a Jak3-Jak1-PI3K-Akt pathway that led to the upregulation of Bcl-2 and Mcl-1. This pathway also induced a delayed but sustained ERK1/2 activation, which not only was necessary for the maintenance of Bcl-2 but also resulted in the phosphorylation of extra-long Bim at Ser(65) . The latter event facilitated the dissociation of Bim from Bcl-2 without affecting Bim abundance in IL-15-treated CD8αα(+) iIELs. Using an adoptive cell transfer approach, we found that either overexpression of Bcl-2 or removal of Bim from CD8αα(+) iIELs promoted their survival in Il15ra(-/-) mice. Taken together, IL-15 promotes CD8αα(+) iIEL survival by both increasing Bcl-2 levels and dissociating Bim from Bcl-2 through activation of a Jak3-Jak1-PI3K-Akt-ERK1/2 pathway, which differs from a previously reported IL-15-induced survival signal.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Interleucina-15/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Subpopulações de Linfócitos T/imunologia , Transferência Adotiva , Animais , Proteína 11 Semelhante a Bcl-2 , Antígenos CD8/metabolismo , Sobrevivência Celular , Células Epiteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Intestinos/citologia , Intestinos/imunologia , Janus Quinase 3/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Interleucina-15/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Translationally controlled tumor protein (TCTP), a repressor for Na,K-ATPase has been implicated in the development of systemic hypertension, as proved by TCTP-over-expressing transgenic (TCTP-TG) mice. Aorta of TCTP-TG exhibited hypercontractile response compared to that of non-transgenic mice (NTG) suggesting dys-regulation of signaling pathways involved in the vascular contractility by TCTP. Because dys-regulation of RhoA/Rho kinase pathway is implicated in increased vascular contractility, we examined whether TCTP induces alterations in RhoA pathway in vascular smooth muscle cells (VSMCs). We found that TCTP over-expression by adenovirus infection up-regulated RhoA pathway including the expression of RhoA, and its downstream signalings, phosphorylation of myosin phosphatase target protein (MYPT-1), and myosin light chain (MLC). Conversely, lentiviral silencing of TCTP reduced the RhoA expression and Rho kinase signalings. Using immunohistochemical and Western blotting studies on aortas from TCTP-TG confirmed the elevated expression of RhoA and increase in p-MLC (phosphorylated MLC). In contrast, down-regulation of RhoA and p-MLC were found in aortas from heterozygous mice with deleted allele of TCTP (TCTP+/-). We conclude that up-regulation of TCTP induces RhoA-mediated pathway, and that TCTP-induced RhoA plays a role in the regulation in vasculature. Modulation of TCTP may offer a therapeutic target for hypertension and in vascular contractility dysfunction.
Assuntos
Biomarcadores Tumorais/metabolismo , Miócitos de Músculo Liso/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Células Cultivadas , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve , Fosforilação , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteína Tumoral 1 Controlada por Tradução , Regulação para CimaRESUMO
The role of ß-catenin in thymocyte development has been extensively studied, however, the function of ß-catenin in thymic epithelial cells (TECs) remains largely unclear. Here, we demonstrate a requirement for ß-catenin in keratin 5 (K5)-expressing TECs, which comprise the majority of medullary TECs (mTECs) and a progenitor subset for cortical TECs (cTECs) in the young adult thymus. We found that conditionally ablated ß-catenin in K5(+)-TECs and their progeny cells resulted in thymic atrophy. The composition of TECs was also aberrantly affected. Percentages of K5(hi)K8(+)-TECs, K5(+)K8(-)-TECs and UEA1(+)-mTECs were significantly decreased and the percentage of K5(lo)K8(+)-TECs and Ly51(+)-cTECs were increased in ß-catenin-deficient thymi compared with that in the control thymi. We also observed that ß-catenin-deficient TEC lineage could give rise to K8(+)-cTECs more efficiently than wild-type TECs using lineage-tracing approach. Importantly, the expression levels of several transcription factors (p63, FoxN1 and Aire), which are essential for TEC differentiation, were altered in ß-catenin-deficient thymi. Under the aberrant differentiation of TECs, development of all thymocytes in ß-catenin-deficient thymi was impaired. Interleukin-7 (IL-7) and chemokines (Ccl19, Ccl25 and Cxcl12) levels were also downregulated in the thymic stromal cells in the mutants. Finally, introducing a BCL2 transgene in lymphoid lineages, which has been shown to rescue IL-7-deficient thymopoiesis, partially rescued the thymic atrophy and thymocyte development defects caused by induced ablation of ß-catenin in K5(+)-TECs. Collectively, these findings suggest that ß-catenin is required for the differentiation of TECs, thereby contributing to thymocyte development in the postnatal thymus.
Assuntos
Epitélio/metabolismo , Células Precursoras de Linfócitos T/imunologia , Linfócitos T/imunologia , Timócitos/imunologia , Timo/patologia , beta Catenina/metabolismo , Animais , Atrofia/genética , Células Cultivadas , Citocinas/metabolismo , Epitélio/imunologia , Genes bcl-2/genética , Queratina-5/genética , Queratina-5/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Timo/crescimento & desenvolvimento , Transcrição Gênica/genética , beta Catenina/genéticaRESUMO
BACKGROUND & AIMS: Mcl-1-deficient hepatocytes are prone to undergo apoptosis. The tumor suppressor protein p53 plays an important role in apoptosis control as well as other cellular responses. This study was initially aimed to examine whether p53 was involved in Mcl-1 deficiency-induced apoptosis of hepatocytes. METHODS: Hepatocyte-specific Mcl-1 knockout (Alb-Mcl-1(-/-)) mice and Alb-Mcl-1(-/-) mice in wild-type or p53-deficient background were generated and characterized. RESULTS: Alb-Mcl-1(-/-) mice were viable, but their liver cells were prone to undergo apoptosis and manifested a slightly elevated level of p53. To examine the role of p53 in Alb-Mcl-1(-/-) livers, Alb-Mcl-1(-/-) mice without p53 (DKO mice) were characterized. Unexpectedly, although p53-deficient mice appeared to be developmentally normal, DKO mice were highly susceptible to neonatal death (â¼60%). Further analysis revealed that such an early lethality was likely due to hepatic failure caused by a marked reduction of fully-differentiated hepatocytes at the perinatal/neonatal stage. Moreover, those DKO mice that did survive to adulthood manifested more severe liver damage than Alb-Mcl-1(-/-) mice, suggesting that p53 was activated in Alb-Mcl-1(-/-) livers to promote cell survival. Microarray followed by quantitative PCR analysis suggested that p21(Waf1/Cip1), one p53 target gene with apoptosis-inhibitory function, is likely involved in the protective role of p53 in Alb-Mcl-1(-/-) livers. Moreover, we demonstrated that loss of p53 promoted liver fibrosis and tumor development in Alb-Mcl-1(-/-) mice. CONCLUSIONS: This study revealed an unexpected synergism between Mcl-1 and p53 in protecting from hepatic injury, fibrosis, and cancer.
Assuntos
Cirrose Hepática Experimental/prevenção & controle , Neoplasias Hepáticas Experimentais/prevenção & controle , Fígado/lesões , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Apoptose/genética , Apoptose/fisiologia , Sequência de Bases , Proliferação de Células , Primers do DNA/genética , Feminino , Genes p53 , Hepatócitos/patologia , Hepatócitos/fisiologia , Fígado/patologia , Fígado/fisiopatologia , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/patologia , Cirrose Hepática Experimental/fisiopatologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Hepáticas Experimentais/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Gravidez , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
We previously demonstrated that IL-3 stimulates transcription of the antiapoptotic gene mcl-1 via two promoter elements designated as the SIE and CRE-2 sites. To further study the functional role of these two DNA elements, mutant mice with targeted mutations of both SIE and CRE-2 sites (SC mutants) were generated. Homozygous SC mutants manifested a markedly reduced level of Mcl-1 in thymus but not in other major organs such as spleen, liver, lung, or heart. Reduced expression of Mcl-1 in SC mutant thymus resulted in attenuated positive selection of double-positive thymocytes into both CD4 and CD8 lineages, a result likely due to reduced survival of SC mutant double-positive thymocytes that were supposed to be positively selected. In contrast, in the peripheral lymphoid organs, only CD8(+) but not CD4(+) T cells were significantly reduced in homozygous SC mutant mice, a result consistent with a more dramatic decrease both of Mcl-1 expression and cell viability in mutant CD8(+) compared with mutant CD4(+) T cells. Impaired T cell development and peripheral CD8(+) lymphopenia in homozygous SC mutant mice were both cell autonomous and could be rescued by enforced expression of human Mcl-1. Together, the promoter-knock-in mouse model generated in this study not only revealed a role of Mcl-1 in thymocyte-positive selection, but also uncovered that Mcl-1 expression is regulated in a tissue or cell lineage-specific manner.
Assuntos
Linhagem da Célula/genética , Regulação da Expressão Gênica/imunologia , Técnicas de Introdução de Genes , Mutação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Subpopulações de Linfócitos T/imunologia , Timo/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Feminino , Técnicas de Introdução de Genes/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Timo/citologia , Timo/metabolismoRESUMO
Translationally controlled tumor protein (TCTP) is expressed throughout T cell development and prominently induced following T cell activation. However, its function(s) during these processes is unclear. Here, we demonstrated that conditional deletion of TCTP before the beta selection checkpoint resulted into a partial block of thymocyte development at the double-negative (DN) 3 stage. Deletion of TCTP in the double-positive (DP) stage did not cause any significant phenotype in the thymus except a slight increase of mature CD8 single-positive (SP) thymocytes. In contrast to the very modest phenotype observed in the thymus, a significant reduction of mature T cells was observed in the peripheral lymphoid organs of these two conditional null TCTP mutant mice. Detailed analysis revealed that the latter phenotype (peripheral T cell lymphopenia) was largely due to a decreased viability of mature TCTP-deficient (TCTP(-/-)) T cells. Transgenic expression of the anti-apoptotic protein Bcl-2 rescued the partial block of early thymocyte development, but not peripheral T cell lymphopenia of T-lineage-specific TCTP(-/-) mice, suggesting that the signaling networks of TCTP in these two processes are not identical. Last, we demonstrated that TCTP(-/-) T cells manifested a significant defect in T cell Ag receptor (TCR)-mediated cell proliferation. Further analysis revealed that such defect was due to a marked delay in the initial cell-cycle entry of TCTP(-/-) T cells following TCR stimulation. Together, these results indicate that TCTP plays a very modest role in thymocyte development, but is critical for peripheral T cell maintenance and TCR-mediated cell proliferation.
Assuntos
Biomarcadores Tumorais/fisiologia , Proliferação de Células , Homeostase/imunologia , Receptores de Antígenos de Linfócitos T/fisiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Biomarcadores Tumorais/deficiência , Biomarcadores Tumorais/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Homeostase/genética , Imunofenotipagem , Ligantes , Linfopenia/genética , Linfopenia/imunologia , Linfopenia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/citologia , Timo/citologia , Timo/imunologia , Timo/metabolismo , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Translationally controlled Tumor Protein (TCTP) is an evolutionally highly conserved protein which has been implicated in many cellular functions that are related to cell growth, death, and even the allergic response of the host. To address the physiological roles of TCTP, we generated TCTP knockout mice by targeted gene disruption. Heterozygous mutants appeared to be developmentally normal. However, homozygous mutants (TCTP(-/-)) were embryonic lethal. TCTP(-/-) embryos were smaller in size than the control littermates at all postimplantation stages examined. Although TCTP is widely expressed in both extraembryonic and embryonic tissues, the most prominent defect of the TCTP(-/-) embryo at embryonic stage day 5.5 (E5.5) was in its epiblast, which had a reduced number of cells compared with wild-type controls. The knockout embryos also suffered a higher incidence of apoptosis in epiblast starting about E6.5 and subsequently died around E9.5-10.5 with a severely disorganized structure. Last, we demonstrated that TCTP(-/-) and control mouse embryonic fibroblasts manifested similar proliferation activities and apoptotic sensitivities to various death stimuli. Taken together, our results suggest that despite that TCTP is widely expressed in many tissues or cell types, it appears to regulate cell proliferation and survival in a tissue- or cell type-specific manner.
Assuntos
Biomarcadores Tumorais/deficiência , Biomarcadores Tumorais/metabolismo , Animais , Apoptose , Proliferação de Células , Sobrevivência Celular , Ciclinas/metabolismo , Embrião de Mamíferos/anormalidades , Feminino , Morte Fetal , Fibroblastos/citologia , Fase G1 , Fase G2 , Gástrula/metabolismo , Marcação de Genes , Masculino , Camundongos , Camundongos Knockout , Mitose , Mutação/genética , Especificidade de Órgãos , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Deficiency of the palmitoyl-acyl transferase ZDHHC13 compromises skin barrier permeability and renders mice susceptible to environmental bacterial infection and inflammatory dermatitis. It had been unclear how the lack of ZDHHC13 proteins resulted in cutaneous abnormalities. In this study, we first demonstrate that enzymatic palmitoylation activity, rather than protein scaffolding, by ZDHHC13 is essential for skin barrier integrity, showing that knock-in mice bearing an enzymatically dead DQ-to-AA ZDHHC13 mutation lost their hair after weaning cyclically, recapitulating knockout phenotypes of skin inflammation and dermatitis. To establish the ZDHHC13 substrates responsible for skin barrier development, we employed quantitative proteomic approaches to identify protein molecules whose palmitoylation is tightly controlled by ZDHHC13. We identified over 300 candidate proteins that could be classified into four biological categories: immunological disease, skin development and function, dermatological disease, and lipid metabolism. Palmitoylation of three of these candidates-loricrin, peptidyl arginine deiminase type III, and keratin fiber crosslinker transglutaminase 1-by ZDHHC13 was confirmed by biochemical assay. Palmitoylation was critical for in vivo protein stability of the latter two candidates. Our findings reveal the importance of protein palmitoylation in skin barrier development, partly by promoting envelope protein crosslinking and the filaggrin processing pathway.
Assuntos
Aciltransferases/metabolismo , Dermatite/metabolismo , Pele/metabolismo , Aciltransferases/genética , Animais , Dermatite/genética , Proteínas Filagrinas , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Queratinas/metabolismo , Lipoilação/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Mutação/genética , Estabilidade Proteica , Proteína-Arginina Desiminase do Tipo 3/metabolismo , Proteômica , Transdução de Sinais , Pele/patologia , Transglutaminases/metabolismoRESUMO
Mcl-1 functions at an apical step in many regulatory programs that control cell death. Although the mitochondrion is one major subcellular organelle where Mcl-1 functions, the molecular mechanism by which Mcl-1 is targeted to mitochondria remains unclear. Here, we demonstrate that Mcl-1 is loosely associated with the outer membrane of mitochondria. Furthermore, we demonstrate that Mcl-1 interacts with the mitochondrial import receptor Tom70, and such interaction requires an internal domain of Mcl-1 that contains an EELD motif. A Tom70 antibody that blocks Mcl-1-Tom70 interaction blocks mitochondrial import of Mcl-1 in vitro. Furthermore, Mcl-1 is significantly less targeted to mitochondria in Tom70 knockdown than in the control cells. Similar targeting preference is also observed for the DM mutant of Mcl-1 whose mutation at the EELD motif markedly attenuates its Tom70 binding activity. Together, our results indicate that the internal EELD domain facilitates mitochondrial targeting of Mcl-1 via a Tom70-dependent pathway.
Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Apoptose , Células Cultivadas , Humanos , Células K562 , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Transporte da Membrana Mitocondrial , Membranas Mitocondriais/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mutantes/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico , TermodinâmicaRESUMO
High-frequency relapse remains a clinical hurdle for complete remission of T-cell acute lymphoblastic leukemia (T-ALL) patients, with heterogeneous dysregulated signaling profiles-including of Raf-MEK-ERK and Akt-mTORC1-S6K signaling pathways-recently being implicated in disease outcomes. Here we report that GM-CSF/IL-3/IL-5 receptor common ß-chain-associated protein (CBAP) is highly expressed in human T-ALL cell lines and many primary tumor tissues and is required to bolster leukemia cell proliferation in tissue culture and for in vivo leukemogenesis in a xenograft mouse model. Downregulation of CBAP markedly restrains expansion of leukemia cells and alleviates disease aggravation of leukemic mice. Transcriptomic profiling and molecular biological analyses suggest that CBAP acts upstream of Ras and Rac1, and functions as a modulator of both Raf-MEK-ERK and Akt-mTORC1 signaling pathways to control leukemia cell growth. Specifically, CBAP facilitated Akt-dependent TSC2 phosphorylation in cell-based assays and in vitro analysis, decreased lysosomal localization of TSC2, and elevated Rheb-GTP loading and subsequent activation of mTORC1 signaling. Taken together, our findings reveal a novel oncogenic contribution of CBAP in T-ALL leukemic cells, in addition to its original pro-apoptotic function in cytokine-dependent cell lines and primary hematopoietic cells, by demonstrating its functional role in the regulation of Akt-TSC2-mTORC1 signaling for leukemia cell proliferation. Thus, CBAP represents a novel therapeutic target for many types of cancers and metabolic diseases linked to PI3K-Akt-mTORC1 signaling.
Assuntos
Proteínas de Membrana/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo/genética , Proteína 2 do Complexo Esclerose Tuberosa/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Fosforilação , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Linfócitos T/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aged hepatocyte-specific-Mcl-1 knockout (MKO-hep) mice are prone to develop liver tumors mimicking human hepatocellular carcinoma (HCC). Here we reported that a protein named UDP-N-acetylglucosamine pyrophosphorylase-1-like-1 (Uap1l1) is upregulated in the liver of young MKO-hep mice without any macroscopically detectable tumor nodules and is prominently expressed in the hepatic tumors developed in the aged MKO-hep mice. Intriguingly, human UAP1L1 is also significantly upregulated in a distinct subset of HCC tissues and patients with upregulated expression of UAP1L1 appeared to have poor prognosis. Overexpression of UAP1L1 significantly promoted, whereas UAP1L1 knockdown markedly reduced the proliferation of human hepatoma cells both in vitro and in vivo. UAP1L1 shows ~59% sequence identity to UDP-N-acetylglucosamine pyrophosphorylase-1 (UAP1), which is directly involved in the synthesis of the sugar donor (UDP-GlcNac) for N-acetylglucosamine modification (O-GlcNAcylation) of proteins. However, unlike UAP1, UAP1L1 harbors very limited UDP-GlcNAc synthesis activity. Moreover, although both UAP1 and UAP1L1 are required for O-GlcNAc transferase (OGT)-mediated protein O-GlcNAcylation, they appear to function distinctly from each other. UAP1L1 directly interacts with OGT, but does not seem to be an OGT substrate. In addition, UAP1L1 alone is not sufficient to activate OGT activity in vitro, suggesting that UAP1L1 may function together with other proteins to modulate OGT activity in vivo. Lastly, UAP1L1 knockdown attenuated c-MYC O-GlcNAcylation and protein stability, and overexpression of c-MYC significantly rescued the proliferation defect of UAP1L1 knockdown HepG2 cells, suggesting that c-MYC is one downstream target of UAP1L1 that contributes to UAP1L1-mediated cell proliferation, at least in HepG2 cells.
Assuntos
Carcinoma Hepatocelular/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/enzimologia , Proteínas de Neoplasias/fisiologia , Nucleotidiltransferases/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Carcinoma Hepatocelular/patologia , Divisão Celular , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glicosilação , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Hepáticas Experimentais/patologia , Camundongos , N-Acetilglucosaminiltransferases/metabolismo , Nucleotidiltransferases/genética , Prognóstico , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transcriptoma , Regulação para CimaRESUMO
Mcl-1 is one Bcl-2 family member that plays a pivotal role in animal development. The extremely labile nature of the Mcl-1 protein itself and the fact that the Mcl-1 level is a critical determinant in various cell survival pathways suggest that cellular processes that regulate Mcl-1 stability are as important as those that regulate Mcl-1 synthesis. Although transcriptional stimulation of Mcl-1 synthesis in response to various stimuli has been well documented, regulation of Mcl-1 stability has been hardly explored. In this study, we identified that the translationally controlled tumor protein (TCTP) was one cellular factor that interacted with Mcl-1 and modulated Mcl-1 stability. While overexpression of TCTP augmented the protein stability of Mcl-1, knockdown expression of TCTP by RNA interference destabilized Mcl-1. Furthermore, TCTP stabilized Mcl-1 through interfering with Mcl-1's degradation by the ubiquitin-dependent proteasome degradation pathway, and the TCTP binding-defective mutant of Mcl-1 (K257V) was much more susceptible to degradation and manifested a compromised antiapoptotic activity. Taken together, these results suggest that TCTP modulates Mcl-1's antiapoptotic activity by modulating its protein stability. The possible mechanism(s) involved in TCTP's modulation process is discussed.
Assuntos
Apoptose/fisiologia , Biomarcadores Tumorais/fisiologia , Proteínas de Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular , Regulação para Baixo , Lisina/genética , Lisina/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides , Complexo de Endopeptidases do Proteassoma/metabolismo , Mapeamento de Interação de Proteínas , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Ativação Transcricional , Proteína Tumoral 1 Controlada por Tradução , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismoRESUMO
We have previously demonstrated that the antiapoptotic gene mcl-1 is activated by interleukin-3 (IL-3) in Ba/F3 pro-B cells through two promoter elements designated the CRE-2 and SIE motifs. While the CRE-2-binding complex contains the CREB protein and is activated by IL-3 through the phosphatidylinositol 3-kinase/Akt-dependent pathway, the identity and cytokine activation pathway of the SIE-binding complex remains unclear. In this report, we demonstrated that PU.1 is one component of the SIE-binding complex. A chromatin immunoprecipitation assay further confirmed that PU.1 binds to the mcl-1 promoter region containing the SIE motif in vivo. While IL-3 stimulation does not significantly alter the SIE-binding activity of PU.1, it markedly increases PU.1's transactivation activity. The latter effect coincides with the increased phosphorylation of PU.1 following IL-3 activation of a p38 mitogen-activated protein kinase (p38(MAPK))-dependent pathway. A serine-to-alanine substitution at position 142 significantly weakens PU.1's ability to be phosphorylated by the p38(MAPK) immunocomplex. Furthermore, this S142A mutant is impaired in the ability to be further stimulated by IL-3 to transactivate the mcl-1 reporter through the SIE motif. Taken together, our results demonstrate that IL-3 stimulation of mcl-1 gene transcription through the SIE motif involves phosphorylation of PU.1 at serine 142 by a p38(MAPK)-dependent pathway.
Assuntos
Interleucina-3/farmacologia , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/fisiologia , Transativadores/fisiologia , Transcrição Gênica/efeitos dos fármacos , Androstadienos/farmacologia , Animais , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Inibidores Enzimáticos/farmacologia , Genes Precoces/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imidazóis/farmacologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Mutagênese Sítio-Dirigida , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/biossíntese , Fosfatidilinositol 3-Quinases/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Piridinas/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Sequências Reguladoras de Ácido Nucleico , Transativadores/química , Transativadores/genética , Wortmanina , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
JAK2 activation is crucial for cytokine receptor signal transduction and leukemogenesis. However, the underlying processes that lead to full activation of JAK2 are unclear. Here, we report a positive role for ubiquitination of JAK2 during GM-CSF-induced activation. Upon GM-CSF stimulation, JAK2 ubiquitination is significantly enhanced through K63-linked poly-ubiquitination. Studies employing both knockout and overexpression of Cbl, an E3 ubiquitin ligase, led to the conclusion that Cbl specifically promotes JAK2 ubiquitination, and this was further confirmed in vitro using a Cbl ubiquitination assay. Moreover, following GM-CSF stimulation, the levels of phospho-JAK2 and -STAT5 and a STAT5 luciferase reporter assay were all reduced in Cbl knockout cells and this effect could be rescued by Cbl expression. Mechanistically, Cbl can interact with, and ubiquitinate JAK2 FERM and kinase domains via the Cbl TKB domain. Using lysine-to-arginine site-directed mutagenesis, K970 in the kinase domain of JAK2 was identified as the ubiquitination site important for promoting full JAK2 activation by Cbl via K63-conjugated poly-ubiquitination. Our study suggests that GM-CSF-induced JAK2 activation is enhanced by Cbl-mediated ubiquitination of JAK2. Targeting ubiquitination of JAK2 might offer a novel therapeutic strategy against JAK2-mediated disorders.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Janus Quinase 2/metabolismo , Lisina/metabolismo , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Janus Quinase 2/química , Janus Quinase 2/genética , Mutagênese Sítio-Dirigida , Fosforilação/efeitos dos fármacos , Ligação Proteica , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacosRESUMO
BACKGROUND: The integrity of the gut barrier in patients with inflammatory bowel disease is known to be impaired but the exact mechanisms remain mostly unknown. SHANK3 mutations are associated with autism, and patients with autism are known to have higher proportions of inflammatory bowel disease. Here, we explore the role of SHANK3 in inflammatory bowel disease, both in vivo and in vitro. METHODS: Dextran sulfate sodium colitis was induced in SHANK3 knockout mice. Transepithelial electrical resistance, paracellular permeability, and Salmonella invasion assays were used to evaluate epithelial barrier function, in vitro and in vivo. Expression of tight junction proteins, protein kinases, and MAP kinase phosphorylation changes were analyzed by immunoblotting after overexpression or knockdown of SHANK3 expression. SHANK3 expression in intestinal tissue from patients with Crohn's disease was analyzed by quantitative polymerase chain reaction and immunohistochemistry. RESULTS: SHANK3 knockout mice were more susceptible to dextran sulfate sodium. SHANK3 knockout resulted in a leaky epithelial barrier phenotype, as demonstrated by decreased transepithelial electrical resistance, increased paracellular permeability, and increased Salmonella invasion. Overexpression of SHANK3 enhanced ZO-1 expression, and knockdown of SHANK3 resulted in decreased expression of ZO-1. Regulation of ZO-1 expression by SHANK3 seems to be mediated through a PKCε-dependent pathway. SHANK3 expression correlated with ZO-1 and PKCε in colonic tissue of patients with Crohn's disease. CONCLUSIONS: The expression level of SHANK3 affects ZO-1 expression and the barrier function in intestinal epithelial cells. This may provide novel insights in Crohn's disease pathogenesis and treatment.