ABSTRACT
The oligoadenylate synthetase (OAS)-RNase L system is an IFN-inducible antiviral pathway activated by viral infection. Viral double-stranded (ds) RNA activates OAS isoforms that synthesize the second messenger 2-5A, which binds and activates the pseudokinase-endoribonuclease RNase L. In cells, OAS activation is tamped down by ADAR1, an adenosine deaminase that destabilizes dsRNA. Mutation of ADAR1 is one cause of Aicardi-Goutières syndrome (AGS), an interferonopathy in children. ADAR1 deficiency in human cells can lead to RNase L activation and subsequent cell death. To evaluate RNase L as a possible therapeutic target for AGS, we sought to identify small-molecule inhibitors of RNase L. A 500-compound library of protein kinase inhibitors was screened for modulators of RNase L activity in vitro. We identified ellagic acid (EA) as a hit with 10-fold higher selectivity against RNase L compared with its nearest paralog, IRE1. SAR analysis identified valoneic acid dilactone (VAL) as a superior inhibitor of RNase L, with 100-fold selectivity over IRE1. Mechanism-of-action analysis indicated that EA and VAL do not bind to the pseudokinase domain of RNase L despite acting as ATP competitive inhibitors of the protein kinase CK2. VAL is nontoxic and functional in cells, although with a 1,000-fold decrease in potency, as measured by RNA cleavage activity in response to treatment with dsRNA activator or by rescue of cell lethality resulting from self dsRNA induced by ADAR1 deficiency. These studies lay the foundation for understanding novel modes of regulating RNase L function using small-molecule inhibitors and avenues of therapeutic potential.
Subject(s)
Adenosine Deaminase/deficiency , Autoimmune Diseases of the Nervous System/enzymology , Endoribonucleases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Nervous System Malformations/enzymology , Phenol/pharmacology , 2',5'-Oligoadenylate Synthetase/genetics , 2',5'-Oligoadenylate Synthetase/metabolism , Adenine Nucleotides/metabolism , Adenosine Deaminase/genetics , Autoimmune Diseases of the Nervous System/genetics , Autoimmune Diseases of the Nervous System/physiopathology , Cell Death/drug effects , Endoribonucleases/genetics , Endoribonucleases/metabolism , Enzyme Inhibitors/chemistry , Humans , Nervous System Malformations/genetics , Nervous System Malformations/physiopathology , Oligoribonucleotides/metabolism , Phenol/chemistry , RNA-Binding Proteins/geneticsABSTRACT
RNase L is an ankyrin repeat domain-containing dual endoribonuclease-pseudokinase that is activated by unusual 2,'5'-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferon-regulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here we present a functional characterization of 2.5 Å and 3.25 Å X-ray crystal and small-angle X-ray scattering structures of RNase L bound to a natural 2-5A activator with and without ADP or the nonhydrolysable ATP mimetic AMP-PNP. These studies reveal how recognition of 2-5A through interactions with the ankyrin repeat domain and the pseudokinase domain, together with nucleotide binding, imposes a rigid intertwined dimer configuration that is essential for RNase catalytic and antiviral functions. The involvement of the pseudokinase domain of RNase L in 2-5A sensing, nucleotide binding, dimerization, and ribonuclease functions highlights the evolutionary adaptability of the eukaryotic protein kinase fold.
Subject(s)
Adenine Nucleotides/chemistry , Endoribonucleases/chemistry , Oligoribonucleotides/chemistry , Adenosine Diphosphate/chemistry , Adenylyl Imidodiphosphate/chemistry , Animals , Ankyrin Repeat , Binding Sites , Crystallography, X-Ray , Dimerization , Encephalomyocarditis virus , Endoribonucleases/genetics , Endoribonucleases/physiology , HeLa Cells , Humans , Models, Molecular , Mutagenesis, Site-Directed , Picornaviridae , Protein Structure, Tertiary , Scattering, Radiation , Structure-Activity Relationship , Sus scrofaABSTRACT
Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.
Subject(s)
Cysteine Endopeptidases/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitinated Proteins/metabolism , Amino Acid Substitution , Cell Line , Crystallography, X-Ray , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/genetics , Deubiquitinating Enzymes , Humans , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Organisms, Genetically Modified , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Quaternary , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Ubiquitin/chemistry , Ubiquitin/metabolism , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitination , Yeasts/genetics , Yeasts/growth & developmentABSTRACT
MOB1 is a multifunctional protein best characterized for its integrative role in regulating Hippo and NDR pathway signaling in metazoans and the Mitotic Exit Network in yeast. Human MOB1 binds both the upstream kinases MST1 and MST2 and the downstream AGC group kinases LATS1, LATS2, NDR1, and NDR2. Binding of MOB1 to MST1 and MST2 is mediated by its phosphopeptide-binding infrastructure, the specificity of which matches the phosphorylation consensus of MST1 and MST2. On the other hand, binding of MOB1 to the LATS and NDR kinases is mediated by a distinct interaction surface on MOB1. By assembling both upstream and downstream kinases into a single complex, MOB1 facilitates the activation of the latter by the former through a trans-phosphorylation event. Binding of MOB1 to its upstream partners also renders MOB1 a substrate, which serves to differentially regulate its two protein interaction activities (at least in vitro). Our previous interaction proteomics analysis revealed that beyond associating with MST1 (and MST2), MOB1A and MOB1B can associate in a phosphorylation-dependent manner with at least two other signaling complexes, one containing the Rho guanine exchange factors (DOCK6-8) and the other containing the serine/threonine phosphatase PP6. Whether these complexes are recruited through the same mode of interaction as MST1 and MST2 remains unknown. Here, through a comprehensive set of biochemical, biophysical, mutational and structural studies, we quantitatively assess how phosphorylation of MOB1A regulates its interaction with both MST kinases and LATS/NDR family kinases in vitro Using interaction proteomics, we validate the significance of our in vitro studies and also discover that the phosphorylation-dependent recruitment of PP6 phosphatase and Rho guanine exchange factor protein complexes differ in key respects from that elucidated for MST1 and MST2. Together our studies confirm and extend previous work to delineate the intricate regulatory steps in key signaling pathways.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Protein Serine-Threonine Kinases/metabolism , Adaptor Proteins, Signal Transducing/genetics , Intracellular Signaling Peptides and Proteins , Phosphoprotein Phosphatases/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Proteomics , Rho Guanine Nucleotide Exchange Factors/metabolism , Serine-Threonine Kinase 3ABSTRACT
The Hippo tumor suppressor pathway regulates organ size and tissue homoeostasis in response to diverse signaling inputs. The core of the pathway consists of a short kinase cascade: MST1 and MST2 phosphorylate and activate LATS1 and LATS2, which in turn phosphorylate and inactivate key transcriptional coactivators, YAP1 and TAZ (gene WWTR1). The MOB1 adapter protein regulates both phosphorylation reactions firstly by concurrently binding to the upstream MST and downstream LATS kinases to enable the trans phosphorylation reaction, and secondly by allosterically activating the catalytic function of LATS1 and LATS2 to directly stimulate phosphorylation of YAP and TAZ. Studies of yeast Mob1 and human MOB1 revealed that the ability to recognize phosphopeptide sequences in their interactors, Nud1 and MST2 respectively, was critical to their roles in regulating the Mitotic Exit Network in yeast and the Hippo pathway in metazoans. However, the underlying rules of phosphopeptide recognition by human MOB1, the implications of binding specificity for Hippo pathway signaling, and the generality of phosphopeptide binding function to other human MOB family members remained elusive.Employing proteomics, peptide arrays and biochemical analyses, we systematically examine the phosphopeptide binding specificity of MOB1 and find it to be highly complementary to the substrate phosphorylation specificity of MST1 and MST2. We demonstrate that autophosphorylation of MST1 and MST2 on several threonine residues provides multiple MOB1 binding sites with varying binding affinities which in turn contribute to a redundancy of MST1-MOB1 protein interactions in cells. The crystal structures of MOB1A in complex with two favored phosphopeptide sites in MST1 allow for a full description of the MOB1A phosphopeptide-binding consensus. Lastly, we show that the phosphopeptide binding properties of MOB1A are conserved in all but one of the seven MOB family members in humans, thus providing a starting point for uncovering their elusive cellular functions.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Protein Serine-Threonine Kinases/metabolism , Adaptor Proteins, Signal Transducing/chemistry , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins , Phosphopeptides/metabolism , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Recombinant Proteins/metabolism , Serine-Threonine Kinase 3 , Signal TransductionABSTRACT
In this issue of Molecular Cell, Ngo et al. (2008) describe the crystal structure of the SRPK1 protein kinase in complex with its substrate, the spliceosome factor ASF/SF2, providing an unprecedented view of multiple targeting mechanisms in action on a single substrate.
Subject(s)
Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Animals , Humans , Mice , Nuclear Proteins/genetics , Phosphorylation , Protein Serine-Threonine Kinases/genetics , RNA-Binding Proteins , Serine-Arginine Splicing Factors , Substrate SpecificityABSTRACT
Kae1 is a universally conserved ATPase and part of the essential gene set in bacteria. In archaea and eukaryotes, Kae1 is embedded within the protein kinase-containing KEOPS complex. Mutation of KEOPS subunits in yeast leads to striking telomere and transcription defects, but the exact biochemical function of KEOPS is not known. As a first step to elucidating its function, we solved the atomic structure of archaea-derived KEOPS complexes involving Kae1, Bud32, Pcc1, and Cgi121 subunits. Our studies suggest that Kae1 is regulated at two levels by the primordial protein kinase Bud32, which is itself regulated by Cgi121. Moreover, Pcc1 appears to function as a dimerization module, perhaps suggesting that KEOPS may be a processive molecular machine. Lastly, as Bud32 lacks the conventional substrate-recognition infrastructure of eukaryotic protein kinases including an activation segment, Bud32 may provide a glimpse of the evolutionary history of the protein kinase family.
Subject(s)
Archaeal Proteins/chemistry , Multiprotein Complexes/chemistry , Protein Kinases/chemistry , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Carrier Proteins/chemistry , Crystallography, X-Ray , Escherichia coli/genetics , Humans , Intracellular Signaling Peptides and Proteins , Methanococcus/genetics , Methanococcus/metabolism , Models, Molecular , Multiprotein Complexes/physiology , Nuclear Magnetic Resonance, Biomolecular , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Structure, Tertiary , Protein Subunits/chemistry , Sequence Homology, Amino Acid , Telomere/metabolism , Thermoplasma/genetics , Thermoplasma/metabolism , Transcription, GeneticABSTRACT
RAF kinases have a prominent role in cancer. Their mode of activation is complex but critically requires dimerization of their kinase domains. Unexpectedly, several ATP-competitive RAF inhibitors were recently found to promote dimerization and transactivation of RAF kinases in a RAS-dependent manner and, as a result, undesirably stimulate RAS/ERK pathway-mediated cell growth. The mechanism by which these inhibitors induce RAF kinase domain dimerization remains unclear. Here we describe bioluminescence resonance energy transfer-based biosensors for the extended RAF family that enable the detection of RAF dimerization in living cells. Notably, we demonstrate the utility of these tools for profiling kinase inhibitors that selectively modulate RAF dimerization and for probing structural determinants of RAF dimerization in vivo. Our findings, which seem generalizable to other kinase families allosterically regulated by kinase domain dimerization, suggest a model whereby ATP-competitive inhibitors mediate RAF dimerization by stabilizing a rigid closed conformation of the kinase domain.
Subject(s)
Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-raf/chemistry , Biosensing Techniques , Crystallization , DNA, Complementary/metabolism , Dimerization , Energy Transfer , HEK293 Cells , Humans , Luminescence , Mutation , Neoplasms/metabolism , Protein Structure, Tertiary , Proto-Oncogene Proteins c-raf/metabolism , Time Factors , UltracentrifugationABSTRACT
The universally conserved Kae1/Qri7/YgjD and Sua5/YrdC protein families have been implicated in growth, telomere homeostasis, transcription and the N6-threonylcarbamoylation (t(6)A) of tRNA, an essential modification required for translational fidelity by the ribosome. In bacteria, YgjD orthologues operate in concert with the bacterial-specific proteins YeaZ and YjeE, whereas in archaeal and eukaryotic systems, Kae1 operates as part of a larger macromolecular assembly called KEOPS with Bud32, Cgi121, Gon7 and Pcc1 subunits. Qri7 orthologues function in the mitochondria and may represent the most primitive member of the Kae1/Qri7/YgjD protein family. In accordance with previous findings, we confirm that Qri7 complements Kae1 function and uncover that Qri7 complements the function of all KEOPS subunits in growth, t(6)A biosynthesis and, to a partial degree, telomere maintenance. These observations suggest that Kae1 provides a core essential function that other subunits within KEOPS have evolved to support. Consistent with this inference, Qri7 alone is sufficient for t(6)A biosynthesis with Sua5 in vitro. In addition, the 2.9 Å crystal structure of Qri7 reveals a simple homodimer arrangement that is supplanted by the heterodimerization of YgjD with YeaZ in bacteria and heterodimerization of Kae1 with Pcc1 in KEOPS. The partial complementation of telomere maintenance by Qri7 hints that KEOPS has evolved novel functions in higher organisms.
Subject(s)
Adenosine/analogs & derivatives , DNA-Binding Proteins/metabolism , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , RNA, Transfer/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Adenosine/biosynthesis , Adenosine/metabolism , Dimerization , Metalloendopeptidases/physiology , Mitochondrial Proteins/physiology , Models, Molecular , Protein Subunits/physiology , RNA, Transfer/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/physiology , Telomere HomeostasisABSTRACT
Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) helps control various endolysosome functions including organelle morphology, membrane recycling, and ion transport. Further highlighting its importance, PtdIns(3,5)P2 misregulation leads to the development of neurodegenerative diseases like Charcot-Marie-Tooth disease. The Fab1/PIKfyve lipid kinase phosphorylates PtdIns(3)P into PtdIns(3,5)P2 whereas the Fig4/Sac3 lipid phosphatase antagonizes this reaction. Interestingly, Fab1 and Fig4 form a common protein complex that coordinates synthesis and degradation of PtdIns(3,5)P2 by a poorly understood process. Assembly of the Fab1 complex requires Vac14/ArPIKfyve, a multimeric scaffolding adaptor protein that coordinates synthesis and turnover of PtdIns(3,5)P2. However, the properties and function of Vac14 multimerization remain mostly uncharacterized. Here we identify several conserved C-terminal motifs on Vac14 required for self-interaction and provide evidence that Vac14 likely forms a dimer. We also show that monomeric Vac14 mutants do not support interaction with Fab1 or Fig4, suggesting that Vac14 multimerization is likely the first molecular event in the assembly of the Fab1 complex. Finally, we show that cells expressing monomeric Vac14 mutants have enlarged vacuoles that do not fragment after hyperosmotic shock, which indicates that PtdIns(3,5)P2 levels are greatly abated. Therefore, our observations support an essential role for the Vac14 homocomplex in controlling PtdIns(3,5)P2 levels.
Subject(s)
Gene Expression Regulation, Fungal , Membrane Proteins/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Motifs , Chromatography, Liquid/methods , Dimerization , Mutation , Neurodegenerative Diseases/metabolism , Phosphatidylinositol Phosphates/chemistry , Plasmids/metabolism , Protein Binding , Protein Multimerization , Protein Structure, Tertiary , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Vacuoles/metabolismABSTRACT
Alzheimer's disease (AD) is a neurodegenerative disease with a projected significant increase in incidence. Therefore, this study analyzed single-nucleus AD data to provide a theoretical basis for the clinical development and treatment of AD. We downloaded AD-related monocyte data from the Gene Expression Omnibus database, annotated cells, compared cell abundance between groups, and investigated glial and neuronal cell biological processes and pathways through functional enrichment analysis. Furthermore, we constructed a global regulatory network for AD based on cell communication and ecological analyses. Our findings revealed increased abundance of Capping Protein Regulator And Myosin 1 linker 1 (CARMIL1)+ astrocytes (AST), Immunoglobulin Superfamily Member 21 (IGSF21)+ microglia (MIC), SRY-Box Transcription Factor 6 (SOX6)+ inhibitory neurons (InNeu), and laminin alpha-2 chain (LAMA2)+ oligodendrocytes (OLI) cell subgroups in tissues of patients with AD, while prostaglandin D2 synthase (PTGDS)+ AST, Src Family Tyrosine Kinase (FYN)+ MIC, and Proteolipid Protein 1 (PLP1)+ InNeu subgroups specifically decreased. We found that the cell phenotype of patients with AD shifted from a simpler to a more complex state compared to the control group. Cell communication analysis revealed strong communication between MIC and NEU. Furthermore, AST, MIC, NEU, and OLI were involved in oxidative stress- and inflammation-related pathways, potentially contributing to disease development. This study provides a theoretical basis for further exploring the specific mechanisms underlying AD.
ABSTRACT
The atomic-resolution structural information that X-ray crystallography can provide on the binding interface between a Fab and its cognate antigen is highly valuable for understanding the mechanism of interaction. However, many Fab:antigen complexes are recalcitrant to crystallization, making the endeavor a considerable effort with no guarantee of success. Consequently, there have been significant steps taken to increase the likelihood of Fab:antigen complex crystallization by altering the Fab framework. In this investigation, we applied the surface entropy reduction strategy coupled with phage-display technology to identify a set of surface substitutions that improve the propensity of a human Fab framework to crystallize. In addition, we showed that combining these surface substitutions with previously reported Crystal Kappa and elbow substitutions results in an extraordinary improvement in Fab and Fab:antigen complex crystallizability, revealing a strong synergistic relationship between these sets of substitutions. Through comprehensive Fab and Fab:antigen complex crystallization screenings followed by structure determination and analysis, we defined the roles that each of these substitutions play in facilitating crystallization and how they complement each other in the process.
Subject(s)
Antigen-Antibody Complex , Immunoglobulin Fab Fragments , Humans , Crystallization/methods , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/chemistry , Antigen-Antibody Complex/chemistry , Antigens/chemistry , Crystallography, X-Ray , Protein ConformationABSTRACT
PKMYT1 is a regulator of CDK1 phosphorylation and is a compelling therapeutic target for the treatment of certain types of DNA damage response cancers due to its established synthetic lethal relationship with CCNE1 amplification. To date, no selective inhibitors have been reported for this kinase that would allow for investigation of the pharmacological role of PKMYT1. To address this need compound 1 was identified as a weak PKMYT1 inhibitor. Introduction of a dimethylphenol increased potency on PKMYT1. These dimethylphenol analogs were found to exist as atropisomers that could be separated and profiled as single enantiomers. Structure-based drug design enabled optimization of cell-based potency. Parallel optimization of ADME properties led to the identification of potent and selective inhibitors of PKMYT1. RP-6306 inhibits CCNE1-amplified tumor cell growth in several preclinical xenograft models. The first-in-class clinical candidate RP-6306 is currently being evaluated in Phase 1 clinical trials for treatment of various solid tumors.
Subject(s)
Neoplasms , Protein-Tyrosine Kinases , Cell Line, Tumor , Cell Proliferation , Humans , Membrane Proteins , Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein Serine-Threonine KinasesABSTRACT
The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway-Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3' CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential.
Subject(s)
Archaeal Proteins/chemistry , Methanocaldococcus/metabolism , Multiprotein Complexes/chemistry , RNA, Transfer/chemistry , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Crystallography, X-Ray , Methanocaldococcus/genetics , Models, Molecular , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Nucleic Acid Conformation , Protein Binding , Protein Domains , RNA, Transfer/genetics , RNA, Transfer/metabolism , RNA, Transfer, Lys/chemistry , RNA, Transfer, Lys/genetics , RNA, Transfer, Lys/metabolismABSTRACT
Using an expression cloning approach, we identify CUL7, a member of the cullin family, as a functional inhibitor of Myc-induced apoptosis. Deregulated expression of the Myc oncogene drives cellular proliferation yet also sensitizes cells to undergo p53-dependent and p53-independent apoptosis. Here, we report that CUL7 exerts its antiapoptotic function through p53. CUL7 binds directly to p53, and small interfering RNA-mediated knockdown of CUL7 results in the elevation of p53 protein levels. This antiapoptotic role of CUL7 enables this novel oncogene to cooperate with Myc to drive transformation. Deregulated ectopic expression of c-Myc and CUL7 promotes Rat1a cell growth in soft agar, and knockdown of CUL7 significantly blocks human neuroblastoma SHEP cell growth in an anchorage-independent manner. Furthermore, using public microarray data sets, we show that CUL7 mRNA is significantly overexpressed in non-small cell lung carcinoma and is associated with poor patient prognosis. We provide experimental evidence to show CUL7 is a new oncogene that cooperates with Myc in transformation by blocking Myc-induced apoptosis in a p53-dependent manner.
Subject(s)
Apoptosis/genetics , Cullin Proteins/genetics , Oncogenes , Animals , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Cell Growth Processes/genetics , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Cullin Proteins/metabolism , Humans , Osteosarcoma/genetics , Osteosarcoma/metabolism , Osteosarcoma/pathology , Proto-Oncogene Proteins c-myc/genetics , RNA, Small Interfering/genetics , Rats , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
The ability of p53 to promote apoptosis and cell cycle arrest is believed to be important for its tumor suppression function. Besides activating the expression of cell cycle arrest and proapoptotic genes, p53 also represses a number of genes. Previous studies have shown an association between p53 activation and down-regulation of c-myc expression. However, the mechanism and physiological significance of p53-mediated c-myc repression remain unclear. Here, we show that c-myc is repressed in a p53-dependent manner in various mouse and human cell lines and mouse tissues. Furthermore, c-myc repression is not dependent on the expression of p21(WAF1). Abrogating the repression of c-myc by ectopic c-myc expression interferes with the ability of p53 to induce G(1) cell cycle arrest and differentiation but enhances the ability of p53 to promote apoptosis. We propose that p53-dependent cell cycle arrest is dependent not only on the transactivation of cell cycle arrest genes but also on the transrepression of c-myc. Chromatin immunoprecipitation assays indicate that p53 is bound to the c-myc promoter in vivo. We report that trichostatin A, an inhibitor of histone deacetylases, abrogates the ability of p53 to repress c-myc transcription. We also show that p53-mediated transcriptional repression of c-myc is accompanied by a decrease in the level of acetylated histone H4 at the c-myc promoter and by recruitment of the corepressor mSin3a. These data suggest that p53 represses c-myc transcription through a mechanism that involves histone deacetylation.
Subject(s)
G1 Phase , Proto-Oncogene Proteins c-myc/genetics , Transcription, Genetic/genetics , Tumor Suppressor Protein p53/metabolism , Acetylation , Animals , Base Sequence , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Differentiation , Cell Line , Cyclin-Dependent Kinase Inhibitor p21 , Gene Expression Regulation , Histones/metabolism , Humans , Mice , Molecular Sequence Data , Promoter Regions, Genetic/genetics , Protein Binding , Proto-Oncogene Proteins c-myc/metabolismABSTRACT
The human NDR family kinases control diverse aspects of cell growth, and are regulated through phosphorylation and association with scaffolds such as MOB1. Here, we report the crystal structure of the human NDR1 kinase domain in its non-phosphorylated state, revealing a fully resolved atypically long activation segment that blocks substrate binding and stabilizes a non-productive position of helix αC. Consistent with an auto-inhibitory function, mutations within the activation segment of NDR1 dramatically enhance in vitro kinase activity. Interestingly, NDR1 catalytic activity is further potentiated by MOB1 binding, suggesting that regulation through modulation of the activation segment and by MOB1 binding are mechanistically distinct. Lastly, deleting the auto-inhibitory activation segment of NDR1 causes a marked increase in the association with upstream Hippo pathway components and the Furry scaffold. These findings provide a point of departure for future efforts to explore the cellular functions and the mechanism of NDR1.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Epithelial Cells/enzymology , Hepatocyte Growth Factor/chemistry , Microtubule-Associated Proteins/chemistry , Protein Serine-Threonine Kinases/chemistry , Proto-Oncogene Proteins/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Binding Sites , Cell Cycle Proteins , Cell Line, Tumor , Cloning, Molecular , Crystallography, X-Ray , Epithelial Cells/cytology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Gene Expression Regulation , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HEK293 Cells , Hepatocyte Growth Factor/genetics , Hepatocyte Growth Factor/metabolism , Humans , Kinetics , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Models, Molecular , Mutation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Serine-Threonine Kinase 3 , Signal Transduction , Substrate SpecificityABSTRACT
The c-myc proto-oncogene encodes a transcription factor, c-Myc, which is deregulated and/or overexpressed in many human cancers. Despite c-Myc's importance, the identity of Myc-regulated genes and the mechanism by which Myc regulates these genes remain unclear. By combining chromatin immunoprecipitation with CpG island arrays, we identified 177 human genomic loci that are bound by Myc in vivo. Analyzing a cohort of known and novel Myc target genes showed that Myc-associated protein X, Max, also bound to these regulatory regions. Indeed, Max is bound to these loci in the presence or absence of Myc. The Myc:Max interaction is essential for Myc-dependent transcriptional activation; however, we show that Max bound targets also include Myc-repressed genes. Moreover, we show that the interaction between Myc and Max is essential for gene repression to occur. Taken together, the identification and analysis of Myc bound target genes supports a model whereby Max plays an essential and universal role in the mechanism of Myc-dependent transcriptional regulation.
Subject(s)
CpG Islands/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Genes, myc/genetics , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Genes, Regulator/genetics , HL-60 Cells , Humans , Oligonucleotide Array Sequence Analysis/methods , Proto-Oncogene Mas , Repressor Proteins/genetics , Repressor Proteins/metabolismABSTRACT
The N terminus of the c-Myc oncoprotein interacts with Bin1, a ubiquitously expressed nucleocytoplasmic protein with features of a tumor suppressor. The c-Myc/Bin1 interaction is dependent on the highly conserved Myc Box 1 (MB1) sequence of c-Myc. The c-Myc/Bin1 interaction has potential regulatory significance as c-Myc-mediated transformation and apoptosis can be modulated by the expression of Bin1. Multiple splicing of the Bin1 transcript results in ubiquitous, tissue-specific and tumor-specific populations of Bin1 proteins in vivo. We report on the structural features of the interaction between c-Myc and Bin1, and describe two mechanisms by which the binding of different Bin1 isoforms to c-Myc may be regulated in cells. Our findings identify a consensus class II SH3-binding motif in c-Myc and the C-terminal SH3 domain of Bin1 as the primary structure determinants of their interaction. We present biochemical and structural evidence that tumor-specific isoforms of Bin1 are precluded from interaction with c-Myc through an intramolecular polyproline-SH3 domain interaction that inhibits the Bin1 SH3 domain from binding to c-Myc. Furthermore, c-Myc/Bin1 interaction can be inhibited by phosphorylation of c-Myc at Ser62, a functionally important residue found within the c-Myc SH3-binding motif. Our data provide a structure-based model of the c-Myc/Bin1 interaction and suggest a mode of regulation that may be important for c-Myc function as a regulator of gene transcription.
Subject(s)
Alternative Splicing , Carrier Proteins/metabolism , Models, Molecular , Nuclear Proteins/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Tumor Suppressor Proteins/metabolism , Adaptor Proteins, Signal Transducing , Binding Sites , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cell Line , Humans , Neoplasm Proteins/chemistry , Neoplasm Proteins/metabolism , Nuclear Magnetic Resonance, Biomolecular , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Phosphorylation , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Proto-Oncogene Proteins c-myc/chemistry , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/genetics , src Homology DomainsABSTRACT
The c-Myc transcription factor represses the mRNA expression of the platelet-derived growth factor receptor beta gene (PDGFRB). Using chromatin immunoprecipitation, we show that c-Myc binds to the proximal promoter of the PDGFRB gene in proliferating rat fibroblasts. Interestingly, mutant c-Myc proteins that are unable to repress PDGFRB gene expression, c-Myc(dBR) and c-Myc(d106-143), are still able to bind to the promoter in vivo. Hence, promoter-binding and repression of PDGFRB by c-Myc are separable activities. We also show that Myc repression of PDGFRB is not dependent on previously described or known transactivator-binding regions, suggesting Myc may be recruited to the promoter by multiple or yet unidentified transcription factors. In the presence of intact promoter-binding by Myc, trichostatin A (TSA) can block Myc repression of PDGFRB in vivo, again demonstrating that promoter-binding and repression are separable. Taken together, we hypothesize that Myc repression of PDGFRB expression occurs by a multi-step mechanism in which repression is initiated after Myc is recruited to the promoter.