ABSTRACT
CKIα ablation induces p53 activation, and CKIα degradation underlies the therapeutic effect of lenalidomide in a pre-leukemia syndrome. Here we describe the development of CKIα inhibitors, which co-target the transcriptional kinases CDK7 and CDK9, thereby augmenting CKIα-induced p53 activation and its anti-leukemic activity. Oncogene-driving super-enhancers (SEs) are highly sensitive to CDK7/9 inhibition. We identified multiple newly gained SEs in primary mouse acute myeloid leukemia (AML) cells and demonstrate that the inhibitors abolish many SEs and preferentially suppress the transcription elongation of SE-driven oncogenes. We show that blocking CKIα together with CDK7 and/or CDK9 synergistically stabilize p53, deprive leukemia cells of survival and proliferation-maintaining SE-driven oncogenes, and induce apoptosis. Leukemia progenitors are selectively eliminated by the inhibitors, explaining their therapeutic efficacy with preserved hematopoiesis and leukemia cure potential; they eradicate leukemia in MLL-AF9 and Tet2-/-;Flt3ITD AML mouse models and in several patient-derived AML xenograft models, supporting their potential efficacy in curing human leukemia.
Subject(s)
Casein Kinase Ialpha/antagonists & inhibitors , Leukemia, Myeloid, Acute/drug therapy , Animals , Apoptosis/drug effects , Casein Kinase Ialpha/physiology , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Cyclin-Dependent Kinase 9/physiology , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/physiology , DNA-Binding Proteins , Disease Models, Animal , Enhancer Elements, Genetic/genetics , Hematopoiesis , Humans , Mice , Mice, Inbred C57BL , Oncogene Proteins, Fusion/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins , Tumor Suppressor Protein p53/physiology , Xenograft Model Antitumor AssaysABSTRACT
In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF(Skp2) substrate p27(Kip1). CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.
Subject(s)
Amino Acids/pharmacology , Biphenyl Compounds/pharmacology , Ubiquitin-Protein Ligase Complexes/antagonists & inhibitors , Allosteric Site , Amino Acid Sequence , Anaphase-Promoting Complex-Cyclosome , DNA Mutational Analysis , Humans , Models, Molecular , Molecular Sequence Data , Sequence Alignment , Ubiquitin-Conjugating Enzymes , Ubiquitin-Protein Ligase Complexes/chemistry , Ubiquitin-Protein Ligase Complexes/geneticsABSTRACT
The nuclear factor-κB (NF-κB) transcription factor family has been considered the central mediator of the inflammatory process and a key participant in innate and adaptive immune responses. Coincident with the molecular cloning of NF-κB/RelA and identification of its kinship to the v-Rel oncogene, it was anticipated that NF-κB itself would be involved in cancer development. Oncogenic activating mutations in NF-κB genes are rare and have been identified only in some lymphoid malignancies, while most NF-κB activating mutations in lymphoid malignancies occur in upstream signaling components that feed into NF-κB. NF-κB activation is also prevalent in carcinomas, in which NF-κB activation is mainly driven by inflammatory cytokines within the tumor microenvironment. Importantly, however, in all malignancies, NF-κB acts in a cell type-specific manner: activating survival genes within cancer cells and inflammation-promoting genes in components of the tumor microenvironment. Yet, the complex biological functions of NF-κB have made its therapeutic targeting a challenge.
Subject(s)
Inflammation/metabolism , NF-kappa B/metabolism , Neoplasms/metabolism , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Humans , Inflammation/genetics , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , Neoplasms/drug therapy , Neoplasms/genetics , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Signal Transduction/drug effects , Tumor Microenvironment , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
IMiDs immunomodulatory drugs, including lenalidomide and pomalidomide represent a novel class of small molecule anticancer and anti-inflammatory drugs with broad biologic activities. However, the molecular mechanism through which these drugs exert their effects is largely undefined. Using pomalidomide and primary human monocytes, we report that pomalidomide rapidly and selectively activated RhoA and Rac1, but not Cdc42 or Ras, in the absence of any costimulation. Consistent with the activation of Rho GTPases, we found that pomalidomide enhanced F-actin formation, stabilized microtubules, and increased cell migration, all of which were blocked by selective inhibitors of ROCK1 and Rac1. Further, we showed that in Swiss 3T3 cells, pomalidomide only activated RhoA, not Rac1 or Cdc42, and potently induced stress fiber formation. The pomalidomide effect on actin cytoskeleton was blocked by the ROCK1 inhibitor, but not Rac1 inhibitor. Finally, we demonstrated that pomalidomide was able to regulate the activity of Rho GTPases and the formation of F-actin in primary human T cells as it did in monocytes and showed that the activation of RhoA was essential for pomalidomide-induced interleukin-2 expression in T cells. These novel activities provide what we believe a critical mechanism by which IMiDs drugs function as therapeutic immunomodulatory agents.
Subject(s)
Cytoskeleton/drug effects , Cytoskeleton/enzymology , Immunosuppressive Agents/pharmacology , Thalidomide/analogs & derivatives , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/metabolism , Actins/metabolism , Animals , Cell Movement/drug effects , Cells, Cultured , Cytoskeleton/immunology , Enzyme Activation/drug effects , Gene Expression Regulation/drug effects , Humans , Interleukin-2/genetics , Interleukin-2/immunology , Interleukin-2/metabolism , Mice , Monocytes/cytology , Monocytes/drug effects , Monocytes/enzymology , T-Lymphocytes/drug effects , T-Lymphocytes/enzymology , T-Lymphocytes/immunology , Thalidomide/pharmacologyABSTRACT
Decreased p27(Kip1) levels are a poor prognostic factor in many malignancies, and can occur through up-regulation of SCF(Skp2) E3 ligase function, resulting in enhanced p27 ubiquitination and proteasome-mediated degradation. While proteasome inhibitors stabilize p27(Kip1), agents inhibiting SCF(Skp2) may represent more directly targeted drugs with the promise of enhanced efficacy and reduced toxicity. Using high-throughput screening, we identified Compound A (CpdA), which interfered with SCF(Skp2) ligase function in vitro, and induced specific accumulation of p21 and other SCF(Skp2) substrates in cells without activating a heat-shock protein response. CpdA prevented incorporation of Skp2 into the SCF(Skp2) ligase, and induced G(1)/S cell-cycle arrest as well as SCF(Skp2)- and p27-dependent cell killing. This programmed cell death was caspase-independent, and instead occurred through activation of autophagy. In models of multiple myeloma, CpdA overcame resistance to dexamethasone, doxorubicin, and melphalan, as well as to bortezomib, and also acted synergistically with this proteasome inhibitor. Importantly, CpdA was active against patient-derived plasma cells and both myeloid and lymphoblastoid leukemia blasts, and showed preferential activity against neoplastic cells while relatively sparing other marrow components. These findings provide a rational framework for further development of SCF(Skp2) inhibitors as a novel class of antitumor agents.
Subject(s)
Antineoplastic Agents/pharmacology , Autophagy , Cell Cycle , Cyclin-Dependent Kinase Inhibitor p27/physiology , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , Ubiquitin-Protein Ligases/antagonists & inhibitors , Cell Line, Tumor , Drug Delivery Systems/methods , Drug Resistance, Neoplasm , Humans , Multiple Myeloma/drug therapyABSTRACT
Small molecules targeting the cereblon-containing E3 ubiquitin ligase including thalidomide, lenalidomide, and pomalidomide modulate turnover of downstream client proteins and demonstrate pre-clinical and clinical anti-myeloma activity. Different drugs that engage with cereblon hold the potential of unique phenotypic effects, and we therefore studied the novel protein homeostatic modulator (PHM™) BTX306 with a unique thiophene-fused scaffold bearing a substituted phenylurea and glutarimide. This agent much more potently reduced human-derived myeloma cell line viability, with median inhibitory concentrations in the single nanomolar range versus micromolar values for lenalidomide or pomalidomide, and more potently activated caspases 3/8/9. While lenalidomide and pomalidomide induced greater degradation of Ikaros and Aiolos in myeloma cells, BTX306 more potently reduced levels of GSPT1, eRF1, CK1α, MCL-1, and c-MYC. Suppression of cereblon or overexpression of Aiolos or Ikaros induced relative resistance to BTX306, and this agent did not impact viability of murine hematopoietic cells in an in vivo model, demonstrating its specificity for human cereblon. Interestingly, BTX306 did show some reduced activity in lenalidomide-resistant cell line models but nonetheless retained its nanomolar potency in vitro, overcame bortezomib resistance, and was equipotent against otherwise isogenic cell line models with either wild-type or knockout TP53. Finally, BTX306 demonstrated strong activity against primary CD138-positive plasma cells, showed enhanced anti-proliferative activity in combination with bortezomib and dexamethasone, and was effective in an in vivo systemic model of multiple myeloma. Taken together, the data support further translational studies of BTX306 and its derivatives to the clinic for patients with relapsed and/or refractory myeloma. KEY MESSAGES: BTX306 has a unique thiophene-fused scaffold bearing phenylurea and glutarimide. BTX306 is more potent against myeloma cells than lenalidomide or pomalidomide. BTX306 overcomes myeloma cell resistance to lenalidomide or bortezomib in vitro. BTX306 is active against primary myeloma cells, and shows efficacy in vivo.
Subject(s)
Antineoplastic Agents/pharmacology , Bortezomib/pharmacology , Drug Resistance, Neoplasm/drug effects , Lenalidomide/pharmacology , Proteostasis/drug effects , Animals , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Biomarkers, Tumor , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Drug Resistance, Neoplasm/genetics , Humans , Mice , Multiple Myeloma , Ubiquitin-Protein Ligases/antagonists & inhibitorsABSTRACT
We have identified a novel pathway of ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) signaling that results in nuclear factor kappaB (NF-kappaB) activation and chemoresistance in response to DNA damage. We show that the anthracycline doxorubicin (DOX) and its congener N-benzyladriamycin (AD 288) selectively activate ATM and DNA-PK, respectively. Both ATM and DNA-PK promote sequential activation of the mitogen-activated protein kinase (MAPK)/p90(rsk) signaling cascade in a p53-independent fashion. In turn, p90(rsk) interacts with the IkappaB kinase 2 (IKK-2) catalytic subunit of IKK, thereby inducing NF-kappaB activity and cell survival. Collectively, our findings suggest that distinct members of the phosphatidylinositol kinase family activate a common prosurvival MAPK/IKK/NF-kappaB pathway that opposes the apoptotic response following DNA damage.
Subject(s)
DNA Damage , DNA-Binding Proteins , Mitogen-Activated Protein Kinase Kinases/metabolism , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Signal Transduction/physiology , Animals , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/metabolism , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins , Cell Survival , DNA-Activated Protein Kinase , Doxorubicin/analogs & derivatives , Doxorubicin/metabolism , Enzyme Activation , Enzyme Inhibitors/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , I-kappa B Kinase , Mice , NIH 3T3 Cells , Nuclear Proteins , Protein Subunits/metabolism , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor ProteinsABSTRACT
Nuclear factor-kappaB (NF-kappaB) plays a critical role during fetal liver development and hepatic oncogenesis. Here, we have assessed whether NF-kappaB activity is required for murine hepatocellular carcinoma cell survival. We show that adenoviral-mediated inhibition of inhibitor of NF-kappaB kinase-beta (IKK-2) activity in hepatocellular carcinomas derived from transforming growth factor (TGF)-alpha/c-myc bitransgenic mice leads to inhibition of NF-kappaB and promotes tumor necrosis factor (TNF)-alpha-mediated cell death of malignant hepatocytes but not the surrounding peritumorous tissue. Induction of apoptosis is accompanied by inhibition of Bcl-X(L) and XIAP, two pro-survival NF-kappaB target genes. In addition, we have identified the alpha-fetoprotein (AFP) as a novel downstream target of NF-kappaB. We show that repression of IKK-2 activity in hepatocellular carcinomas promotes down-regulation of AFP gene expression. Likewise, genetic disruption of the RelA subunit results in reduced AFP gene expression during embryonic liver development, at a time in which fetal hepatocytes are sensitized to TNF-alpha-mediated cell killing. In this regard, we show that AFP inhibits TNF-alpha-induced cell death of murine hepatocellular carcinomas through association with TNF-alpha and inhibition of TNFRI signaling. Thus, NF-kappaB-mediated regulation of AFP gene expression during liver tumor formation and embryonic development of the liver constitutes a potential novel mechanism used by malignant and fetal hepatocytes to evade immune surveillance.
Subject(s)
Hepatocytes/pathology , Liver Neoplasms, Experimental/pathology , Liver/embryology , NF-kappa B/physiology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , alpha-Fetoproteins/physiology , Adenoviridae/genetics , Animals , Antigens, CD/physiology , Apoptosis/physiology , Gene Expression Regulation, Developmental , Hepatocytes/metabolism , I-kappa B Kinase , Liver/enzymology , Liver/physiology , Liver Neoplasms, Experimental/enzymology , Male , Mice , Mice, Transgenic , NF-kappa B/genetics , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-myc , Receptors, Tumor Necrosis Factor/physiology , Receptors, Tumor Necrosis Factor, Type I , Transforming Growth Factor alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/physiology , X-Linked Inhibitor of Apoptosis Protein , alpha-Fetoproteins/genetics , alpha-Fetoproteins/metabolism , bcl-X ProteinABSTRACT
NF-kappaB has been implicated in the regulation of apoptosis, a key mechanism of normal and malignant growth control. Previously, we demonstrated that inhibition of NF-kappaB activity by TGF-beta1 leads directly to induction of apoptosis of murine B-cell lymphomas and hepatocytes. Thus, we were surprised to determine that NF-kappaB is transiently activated in response to TGF-beta1 treatment. Here we elucidate the mechanism of TGF-beta1-mediated regulation of NF-kappaB and induction of apoptosis in epithelial cells. We report that TGF-beta1 activates IKK kinase, which mediates IkappaB-alpha phosphorylation. In turn, the activation of IKK following TGF-beta1 treatment is mediated by the TAK1 kinase. As a result of NF-kappaB activation, IkappaB-alpha mRNA and protein levels are increased leading to postrepression of NF-kappaB and induction of cell death. Inhibition of NF-kappaB following TGF-beta1 treatment increased AP-1 complex transcriptional activity through sustained c-Jun phosphorylation, thereby potentiating AP-1/SMADs-mediated cell killing. Furthermore, TGF-beta1-mediated upregulation of Smad7 appeared independent of NF-kappaB. In hepatocellular carcinomas of TGF-beta1 or TGF-alpha/c-myc transgenic mice, we observed constitutive activation of NF-kappaB that led to inhibition of JNK signaling. Overall, our data illustrate an autocrine mechanism based on the ability of IKK/NF-kappaB/IkappaB-alpha signaling to negatively regulate NF-kappaB levels thereby permitting TGF-beta1-induced apoptosis through AP-1 activity.
Subject(s)
DNA-Binding Proteins/metabolism , MAP Kinase Kinase Kinases/metabolism , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/metabolism , Trans-Activators/metabolism , Transcription Factor AP-1/metabolism , Transforming Growth Factor beta/metabolism , Animals , Apoptosis/drug effects , Apoptosis/physiology , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cells, Cultured , DNA-Binding Proteins/genetics , Enzyme Activation , Hepatocytes/cytology , Hepatocytes/metabolism , I-kappa B Kinase , I-kappa B Proteins/metabolism , JNK Mitogen-Activated Protein Kinases , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , MAP Kinase Kinase Kinases/genetics , Mice , Mice, Transgenic , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/genetics , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Transport/drug effects , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Signal Transduction , Smad7 Protein , Trans-Activators/genetics , Transforming Growth Factor beta/pharmacology , Transforming Growth Factor beta1ABSTRACT
An increasing body of evidence indicates that constitutive activation of NF-kappaB contributes to tumorigenesis and inflammation. Ubiquitination and degradation of IkappaB plays an essential role in NF-kappaB activation. Here we describe an in vitro IkappaBalpha ubiquitination assay system in which purified E1, E2, SCF(beta-Trcp1) E3, IkappaBalpha, IKK2, and Ub were used to generate ubiquitinated IkappaBalpha. The ubiquitination of IkappaBalpha is strictly dependent on its phosphorylation by IKK2, as well as the presence of E1, E2, E3, and Ub. The assay was adapted into 384-well plate format in which an antibody against IkappaBalpha was used to capture IkappaBalpha, and the biotinylated ubiquitin attached to IkappaBalpha was detected with europium (Eu)-labeled streptavidin. This assay can be used to discover inhibitors of IkappaBalpha ubiquitination. Such inhibitors would block NF-kappaB activation by stabilizing IkappaB levels in cells and thus provide a new therapeutic approach to NF-kappaB-related human diseases.
Subject(s)
I-kappa B Proteins/metabolism , SKP Cullin F-Box Protein Ligases/metabolism , Ubiquitin/metabolism , Blotting, Western , Electrophoresis, Polyacrylamide Gel , In Vitro Techniques , NF-KappaB Inhibitor alpha , PhosphorylationABSTRACT
Thalidomide the first commercially available immune modulatory drug (IMiD), has activity in the treatment of Waldenstrom's macroglobulinemia (WM), as well as multiple myeloma, myelodysplastic syndrome, myelofibrosis with myeloid metaplasia, chronic lymphocytic leukemia (CLL), and B-cell lymphomas. Although its molecular mechanisms of action have not yet been elucidated, thalidomide and the IMiDs affect a variety of cytokines and inflammatory mediators including tumor necrosis factor-alpha (TNFalpha), interleukin (IL)-1beta, interferon gamma (IFNgamma), IL-6, IL-10, IL-12, and COX-2 and angiogenesis factors such as vascular endothelial growth factor (VEGF) and its receptor. The IMiDs also affect adhesion molecules such as ICAM-1, ICAM-2, and L-CAM, in addition to preferentially stimulating CD8 cells and expanding natural killer (NK) cell populations. Since most IMiDs share these properties, it would be expected that the second-generation IMiDs (REVIMID, ACTIMID) would have activity similar to thalidomide in WM with an improved safety profile. TNFalpha and angiogenesis most likely play a role in promoting the growth and development of WM. The selective cytokine inhibitory drugs (SelCIDs) are potent phosphodiesterase 4 (PDE-4) inhibitors that inhibit TNFalpha production and are highly antiangiogenic. In addition, inhibition of PDE-4 induces apoptosis in human CLL lymphocytes. It is therefore expected that the SelCIDs might have activity in Waldenstrom's tumors. Jun N-terminal kinase (JNK) is a component of signaling cascades that modulate apoptosis, the induction of an inflammatory response via the AP-1 pathway, and modulation of cellular proliferation. In a variety of tumors, including multiple myeloma, JNK is induced as part of a protective mechanism. It is hypothesized that inhibition of JNK activity might allow other chemotherapeutic agents to be more effective in a similar manner to corticosteroids. Work is in progress to evaluate this. Inhibitors of the E3 subunit of ubiquitin ligase may also selectively modulate the expression of receptors, growth factors, and transcription factors essential to the growth, survival, and spread of tumors. We hypothesize that the IMiDs, SelCIDs, JNK inhibitors, and ligase inhibitors will be the basis for a new nonchemotherapeutic approach to the treatment of WM and other related diseases.
Subject(s)
Adjuvants, Immunologic/therapeutic use , Cytokines/antagonists & inhibitors , Enzyme Inhibitors/therapeutic use , JNK Mitogen-Activated Protein Kinases , Ligases/antagonists & inhibitors , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Waldenstrom Macroglobulinemia/drug therapy , 3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , Clinical Trials as Topic , Cyclic Nucleotide Phosphodiesterases, Type 4 , Humans , Lenalidomide , MAP Kinase Kinase 4 , Thalidomide/analogs & derivatives , Thalidomide/therapeutic use , Ubiquitin-Protein LigasesABSTRACT
The human suppressor of morphogenesis in genitalia-1 (hSMG-1) protein kinase plays dual roles in mRNA surveillance and genotoxic stress response pathways in human cells. Here, we report that small interfering RNA-mediated depletion of hSMG-1, but not ATM, ATR, hUpf1, or hUpf2, in human U2OS osteosarcoma cells markedly increases the magnitude and accelerates the rate of apoptosis induced by tumor necrosis factor-alpha (TNFalpha) stimulation. The increase in TNFalpha-mediated cell killing observed in hSMG-1-depleted cells is not related to the suppression of nonsense-mediated mRNA decay or to the inhibition of TNFalpha-induced NF-kappaB activation. Rather, we observed that loss of hSMG-1 accelerates the degradation of the long form of the FLICE-inhibitory protein (FLIP(L)), an inhibitor of death-inducing signaling complex-mediated caspase-8 activation, in TNFalpha-treated cells. These results suggest that hSMG-1 plays an important role in cell survival during TNFalpha-induced stress.
Subject(s)
Apoptosis/drug effects , Cytoprotection/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Tumor Necrosis Factor-alpha/pharmacology , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Cell Line, Tumor , Death Domain Receptor Signaling Adaptor Proteins/metabolism , Humans , NF-kappa B/metabolism , Phosphatidylinositol 3-Kinases/genetics , Protein Biosynthesis/genetics , Protein Serine-Threonine Kinases , RNA, Messenger/genetics , RNA, Small Interfering/geneticsABSTRACT
SCF ubiquitin ligases recruit substrates for degradation via F box protein adaptor subunits. WD40 repeat F box proteins, such as Cdc4 and beta-TrCP, contain a conserved dimerization motif called the D domain. Here, we report that the D domain protomers of yeast Cdc4 and human beta-TrCP form a superhelical homotypic dimer. Disruption of the D domain compromises the activity of yeast SCF(Cdc4) toward the CDK inhibitor Sic1 and other substrates. SCF(Cdc4) dimerization has little effect on the affinity for Sic1 but markedly stimulates ubiquitin conjugation. A model of the dimeric holo-SCF(Cdc4) complex based on small-angle X-ray scatter measurements reveals a suprafacial configuration, in which substrate-binding sites and E2 catalytic sites lie in the same plane with a separation of 64 A within and 102 A between each SCF monomer. This spatial variability may accommodate diverse acceptor lysine geometries in both substrates and the elongating ubiquitin chain and thereby increase catalytic efficiency.
Subject(s)
Cell Cycle Proteins/metabolism , SKP Cullin F-Box Protein Ligases/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/chemistry , Amino Acid Sequence , Binding Sites , Catalytic Domain , Dimerization , F-Box Proteins , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Protein Binding , Protein Conformation , Protein Structure, Tertiary , SKP Cullin F-Box Protein Ligases/metabolism , Sequence Homology, Amino AcidABSTRACT
p27, an important cell cycle regulator, blocks the G(1)/S transition in cells by binding and inhibiting Cdk2/cyclin A and Cdk2/cyclin E complexes (Cdk2/E). Ubiquitination and subsequent degradation play a critical role in regulating the levels of p27 during cell cycle progression. Here we provide evidence suggesting that both Cdk2/E and phosphorylation of Thr(187) on p27 are essential for the recognition of p27 by the SCF(Skp2/Cks1) complex, the ubiquitin-protein isopeptide ligase (E3). Cdk2/E provides a high affinity binding site, whereas the phosphorylated Thr(187) provides a low affinity binding site for the Skp2/Cks1 complex. Furthermore, binding of phosphorylated p27/Cdk2/E to the E3 complex showed positive cooperativity. Consistently, p27 is also ubiquitinated in a similarly cooperative manner. In the absence of p27, Cdk2/E and Cks1 increase Skp2 phosphorylation. This phosphorylation enhances Skp2 auto-ubiquitination, whereas p27 inhibits both phosphorylation and auto-ubiquitination of Skp2.
Subject(s)
Carrier Proteins/chemistry , Cyclin-Dependent Kinases/chemistry , Multiprotein Complexes/chemistry , Protein Processing, Post-Translational , S-Phase Kinase-Associated Proteins/chemistry , Ubiquitin-Protein Ligases/chemistry , Animals , CDC2-CDC28 Kinases , Carrier Proteins/metabolism , Cell-Free System/chemistry , Cell-Free System/metabolism , Cyclin A/chemistry , Cyclin A/metabolism , Cyclin E/chemistry , Cyclin E/metabolism , Cyclin-Dependent Kinase 2/chemistry , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase Inhibitor p27/chemistry , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Cyclin-Dependent Kinases/metabolism , G1 Phase/physiology , Humans , Multiprotein Complexes/metabolism , Phosphorylation , Protein Processing, Post-Translational/physiology , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , S Phase/physiology , S-Phase Kinase-Associated Proteins/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
IkappaB kinase (IKK) complex plays a key regulatory role in macrophages for NF-kappaB activation during both innate and adaptive immune responses. Because IKK1-/- mice died at birth, we differentiated functional macrophages from embryonic day 15.5 IKK1 mutant embryonic liver. The embryonic liver-derived macrophage (ELDM) showed enhanced phagocytotic clearance of bacteria, more efficient antigen-presenting capacity, elevated secretion of several key proinflammatory cytokines and chemokines, and known NFkappaB target genes. Increased NFkappaB activity in IKK1 mutant ELDM was the result of prolonged degradation of IkappaBalpha in response to infectious pathogens. The delayed restoration of IkappaBalpha in pathogen-activated IKK1-/- ELDM was a direct consequence of uncontrolled IKK2 kinase activity. We hypothesize that IKK1 plays a checkpoint role in the proper control of IkappaBalpha kinase activity in innate and adaptive immunity.
Subject(s)
Macrophages/metabolism , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/deficiency , Animals , Antigen Presentation , Chemokines/biosynthesis , Cytokines/biosynthesis , Female , I-kappa B Kinase , I-kappa B Proteins/metabolism , In Vitro Techniques , Inflammation Mediators/metabolism , Liver/cytology , Liver/embryology , Liver/metabolism , Macrophages/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , NF-KappaB Inhibitor alpha , Phagocytosis , Phosphorylation , Pregnancy , Protein Serine-Threonine Kinases/geneticsABSTRACT
The Chk1 kinase is a major effector of S phase checkpoint signaling during the cellular response to genotoxic stress. Here, we report that replicative stress induces the polyubiquitination and degradation of Chk1 in human cells. This response is triggered by phosphorylation of Chk1 at Ser-345, a known target site for the upstream activating kinase ATR. The ubiquitination of Chk1 is mediated by E3 ligase complexes containing Cul1 or Cul4A. Treatment of cells with the anticancer agent camptothecin (CPT) triggers Chk1 destruction, which blocks recovery from drug-induced S phase arrest and leads to cell death. These findings indicate that ATR-dependent phosphorylation of Chk1 delivers a signal that both activates Chk1 and marks this protein for proteolytic degradation. Proteolysis of activated Chk1 may promote checkpoint termination under normal conditions, and may play an important role in the cytotoxic effects of CPT and related anticancer drugs.
Subject(s)
DNA Damage/physiology , Proteasome Endopeptidase Complex/physiology , Protein Kinases/metabolism , Ubiquitin/physiology , Bromodeoxyuridine , Camptothecin/toxicity , Cell Cycle Proteins/physiology , Cell Line , Cell Line, Tumor , Checkpoint Kinase 1 , Cullin Proteins/physiology , Down-Regulation/drug effects , Genes, Reporter , Humans , S Phase/drug effects , S Phase/physiologyABSTRACT
We report that the product of the inducible gene encoding the kinase known as IKKi/IKKepsilon (IKKi) is required for expression of a group of genes up-regulated by pro-inflammatory stimuli such as bacterial endotoxin (lipopolysaccharide (LPS)). Here, using murine embryonic fibroblasts obtained from mice bearing deletions in IKK2, p65, and IKKi genes, we provide evidence to support a link between signaling through the NF-kappaB and CCAAA/enhancer-binding protein (C/EBP) pathways. This link includes an NF-kappaB-dependent regulation of C/EBPbeta and C/EBPdelta gene transcription and IKKi-mediated activation of C/EBP. Disruption of the NF-kappaB pathway results in the blockade of the inducible up-regulation of C/EBPbeta, C/EBPdelta, and IKKi genes. Cells lacking IKKi are normal in activation of the canonical NF-kappaB pathway but fail to induce C/EBPdelta activity and transcription of C/EBP and C/EBP-NF-kappaB target genes in response to LPS. In addition we show that, in response to LPS or tumor necrosis factor alpha, both beta and delta subunits of C/EBP interact with IKKi promoter, suggesting a feedback mechanism in the regulation of IKKi-dependent cellular processes. These data are among the first to provide insights into the biological function of IKKi.
Subject(s)
Inflammation/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Base Sequence , CCAAT-Enhancer-Binding Proteins/metabolism , Cells, Cultured , Feedback , Humans , I-kappa B Kinase , Inflammation/genetics , Lipopolysaccharides/pharmacology , Mice , Mice, Knockout , NF-kappa B/metabolism , Promoter Regions, Genetic , Protein Serine-Threonine Kinases/genetics , RNA Processing, Post-Transcriptional , RNA, Small Interfering/genetics , Signal Transduction , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
The nuclear factor-kappa B (NF-kappaB) family of transcription factors plays a seminal role in inflammation, apoptosis, development, and cancer. Modulation of NF-kappaB-mediated gene expression in response to diverse signals is coordinated by the IkappaB kinase (IKK) complex. We identified ELKS, an essential regulatory subunit of the IKK complex. Silencing ELKS expression by RNA interference blocked induced expression of NF-kappaB target genes, including the NF-kappaB inhibitor IkappaBalpha and proinflammatory genes such as cyclo-oxygenase 2 and interleukin 8. These cells were also not protected from apoptosis in response to cytokines. ELKS likely functions by recruiting IkappaBalpha to the IKK complex and thus serves a regulatory function for IKK activation.
Subject(s)
Carrier Proteins/metabolism , NF-kappa B/metabolism , Nerve Tissue Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Adaptor Proteins, Signal Transducing , Animals , Apoptosis , Carrier Proteins/genetics , Cell Line , Cyclooxygenase 2 , Gene Expression , Genes, Reporter , HeLa Cells , Humans , I-kappa B Kinase , I-kappa B Proteins/genetics , I-kappa B Proteins/metabolism , Interleukin-1/pharmacology , Interleukin-8/genetics , Isoenzymes/genetics , Membrane Proteins , Mice , Mice, Knockout , Mitogen-Activated Protein Kinases/metabolism , Mutation , NF-KappaB Inhibitor alpha , Nerve Tissue Proteins/genetics , Phosphorylation , Precipitin Tests , Prostaglandin-Endoperoxide Synthases/genetics , RNA Interference , Tumor Necrosis Factor-alpha/pharmacology , rab GTP-Binding ProteinsABSTRACT
Rotavirus is the major etiologic agent of diarrhea in children and the most common cause of severe pediatric gastroenteritis. Rotavirus infection is limited to mature enterocytes that line the villi of the small intestine. Gut epithelial cells, upon infection and cytokine stimulation, are able to produce chemokines, a family of small chemotactic cytokines that regulate the migration and activation of leukocytes. We have previously shown that rotavirus infection of the intestinal epithelial cell line HT-29 induces increased expression of the CXC chemokine interleukin- (IL) 8. Mechanisms responsible for the transcriptional regulation of the IL-8 gene in intestinal epithelial cells during viral infections have not been fully elucidated. Therefore, the purpose of this study was to define the molecular mechanisms of IL-8 gene expression in HT-29 cells infected with rotavirus. Transient transfection analysis of 5' deletions and mutations of the IL-8 promoter driving expression of luciferase reporter gene indicates that the activating protein- (AP) 1 and nuclear factor- (NF) kappaB elements are necessary for IL-8 promoter activation during rotavirus infection. The importance of NF-kappaB activation for IL-8 gene expression was further demonstrated by the inhibition of rotavirus-induced IL-8 gene transcription and protein synthesis following blockade of degradation of the NF-kappaB cytoplasmic inhibitor IkappaB-alpha. Rotavirus infection of HT-29-induced IkappaB kinase (IKK) activation and overexpression of a dominant negative mutant of IKK-beta greatly reduced rotavirus-induced IL-8 promoter activation and NF-kappaB-driven transcription, indicating that IKK is involved in rotavirus-induced IL-8 gene expression and NF-kappaB activation.
Subject(s)
Epithelial Cells/virology , Interleukin-8/genetics , Intestines/virology , Protein Serine-Threonine Kinases/physiology , Rotavirus/immunology , Epithelial Cells/immunology , Gene Expression Regulation , Humans , I-kappa B Kinase , Interleukin-8/metabolism , Mutation , NF-kappa B/metabolism , Promoter Regions, Genetic , Protein Serine-Threonine Kinases/biosynthesis , Protein Serine-Threonine Kinases/genetics , Transcription Factor AP-1/metabolismABSTRACT
beta-TrCP/E3RS (E3RS) is the F-box protein that functions as the receptor subunit of the SCF(beta-TrCP) ubiquitin ligase (E3). Surprisingly, although its two recognized substrates, IkappaB(alpha) and beta-catenin, are present in the cytoplasm, we have found that E3RS is located predominantly in the nucleus. Here we report the isolation of the major E3RS-associated protein, hnRNP-U, an abundant nuclear phosphoprotein. This protein occupies E3RS in a specific and stoichiometric manner, stabilizes the E3 component, and is likely responsible for its nuclear localization. hnRNP-U binding was abolished by competition with a pIkappaB(alpha) peptide, or by a specific point mutation in the E3RS WD region, indicating an E3-substrate-type interaction. However, unlike pI(kappa)Balpha, which is targeted by SCF(beta-TrCP) for degradation, the E3-bound hnRNP-U is stable and is, therefore, a pseudosubstrate. Consequently, hnRNP-U engages a highly neddylated active SCF(beta-TrCP), which dissociates in the presence of a high-affinity substrate, resulting in ubiquitination of the latter. Our study points to a novel regulatory mechanism, which secures the localization, stability, substrate binding threshold, and efficacy of a specific protein-ubiquitin ligase.