RESUMEN
Mature differentiated macrophages can self-maintain by local proliferation in tissues and can be extensively expanded in culture under specific conditions, but the mechanisms of this phenomenon remain only partially defined. Here, we show that SIRT1, an evolutionary conserved regulator of life span, positively affects macrophage self-renewal ability in vitro and in vivo Overexpression of SIRT1 during bone marrow-derived macrophage differentiation increased their proliferative capacity. Conversely, decrease of SIRT1 expression by shRNA inactivation, CRISPR/Cas9 mediated deletion and pharmacological inhibition restricted macrophage self-renewal in culture. Furthermore, pharmacological SIRT1 inhibition in vivo reduced steady state and cytokine-induced proliferation of alveolar and peritoneal macrophages. Mechanistically, SIRT1 inhibition negatively regulated G1/S transition, cell cycle progression and a network of self-renewal genes. This included inhibition of E2F1 and Myc and concomitant activation of FoxO1, SIRT1 targets mediating cell cycle progression and stress response, respectively. Our findings indicate that SIRT1 is a key regulator of macrophage self-renewal that integrates cell cycle and longevity pathways. This suggests that macrophage self-renewal might be a relevant parameter of ageing.
Asunto(s)
Proliferación Celular , Autorrenovación de las Células , Macrófagos/fisiología , Sirtuina 1/metabolismo , Animales , Ciclo Celular , Expresión Génica , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Ratones , Sirtuina 1/genéticaRESUMEN
The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) regulates cell cycle arrest and terminal differentiation of neutrophils and adipocytes. Mutations in the basic leucine zipper domain (bZip) of C/EBPα are associated with acute myeloid leukemia. A widely used murine transforming C/EBPα basic region mutant (BRM2) entails two bZip point mutations (I294A/R297A). BRM2 has been discordantly described as defective for DNA binding or defective for interaction with E2F. We have separated the two BRM2 mutations to shed light on the intertwined reciprocity between C/EBPα-E2F-DNA interactions. Both, C/EBPα I294A and R297A retain transactivation capacity and interaction with E2F-DP. The C/EBPα R297A mutation destabilized DNA binding, whereas the C/EBPα I294A mutation enhanced binding to DNA. The C/EBPα R297A mutant, like BRM2, displayed enhanced interaction with E2F-DP but failed to repress E2F-dependent transactivation although both mutants were readily suppressed by E2F1 for transcription through C/EBP cis-regulatory sites. In contrast, the DNA binding enhanced C/EBPα I294A mutant displayed increased repression of E2F-DP mediated transactivation and resisted E2F-DP mediated repression. Thus, the efficient repression of E2F dependent S-phase genes and the activation of differentiation genes reside in the balanced DNA binding capacity of C/EBPα.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , ADN/metabolismo , Factores de Transcripción E2F/metabolismo , Dominios y Motivos de Interacción de Proteínas , Secuencia de Aminoácidos , Animales , Proteínas Potenciadoras de Unión a CCAAT/química , Proteínas Potenciadoras de Unión a CCAAT/genética , Células Cultivadas , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica/fisiología , Dominios y Motivos de Interacción de Proteínas/genética , Estructura Secundaria de Proteína/genética , Homología de Secuencia de AminoácidoRESUMEN
Cellular signalling cascades regulate the activity of transcription factors that convert extracellular information into gene regulation. C/EBPbeta is a ras/MAPkinase signal-sensitive transcription factor that regulates genes involved in metabolism, proliferation, differentiation, immunity, senescence, and tumourigenesis. The protein arginine methyltransferase 4 PRMT4/CARM1 interacts with C/EBPbeta and dimethylates a conserved arginine residue (R3) in the C/EBPbeta N-terminal transactivation domain, as identified by mass spectrometry of cell-derived C/EBPbeta. Phosphorylation of the C/EBPbeta regulatory domain by ras/MAPkinase signalling abrogates the interaction between C/EBPbeta and PRMT4/CARM1. Differential proteomic screening, protein interaction studies, and mutational analysis revealed that methylation of R3 constraines interaction with SWI/SNF and Mediator complexes. Mutation of the R3 methylation site alters endogenous myeloid gene expression and adipogenic differentiation. Thus, phosphorylation of the transcription factor C/EBPbeta couples ras signalling to arginine methylation and regulates the interaction of C/EBPbeta with epigenetic gene regulatory protein complexes during cell differentiation.
Asunto(s)
Arginina/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteínas Cromosómicas no Histona/genética , Metilación , Ratones , Datos de Secuencia Molecular , Fosforilación , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Factores de Transcripción/genéticaRESUMEN
CCAAT/enhancer-binding protein beta (C/EBPß) induces primary v-Abl immortalized mouse B cells to transdifferentiate (BT, B cell transdifferentiation) into granulocyte-macrophage progenitor-like cells (GMPBTs). GMPBTs maintain cytokine-independent self-renewal, lineage choice, and multilineage differentiation. Single-cell transcriptomics demonstrated that GMPBTs comprise a continuum of myelomonopoietic differentiation states that seamlessly fit into state-to-fate maps of normal granulocyte-macrophage progenitors (GMPs). Inactivating v-Abl kinase revealed the dependence on activated CSF2-JAK2-STAT5 signaling. Deleting IRF8 diminished monopoiesis and enhanced granulopoiesis while removing C/EBPß-abrogated self-renewal and granulopoiesis but permitted macrophage differentiation. The GMPBT culture system is easily scalable to explore the basics of GMP biology and lineage commitment and largely reduces ethically and legislatively debatable, labor-intensive, and costly animal experiments.
Asunto(s)
Granulocitos , Monocitos , Ratones , Animales , Granulocitos/metabolismo , Transdiferenciación Celular , Hematopoyesis , Diferenciación Celular , BiologíaRESUMEN
Here, we describe how the speed of C/EBPα-induced B cell to macrophage transdifferentiation (BMT) can be regulated, using both mouse and human models. The identification of a mutant of C/EBPα (C/EBPαR35A) that greatly accelerates BMT helped to illuminate the mechanism. Thus, incoming C/EBPα binds to PU.1, an obligate partner expressed in B cells, leading to the release of PU.1 from B cell enhancers, chromatin closing and silencing of the B cell program. Released PU.1 redistributes to macrophage enhancers newly occupied by C/EBPα, causing chromatin opening and activation of macrophage genes. All these steps are accelerated by C/EBPαR35A, initiated by its increased affinity for PU.1. Wild-type C/EBPα is methylated by Carm1 at arginine 35 and the enzyme's perturbations modulate BMT velocity as predicted from the observations with the mutant. Increasing the proportion of unmethylated C/EBPα in granulocyte/macrophage progenitors by inhibiting Carm1 biases the cell's differentiation toward macrophages, suggesting that cell fate decision velocity and lineage directionality are closely linked processes.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT , Transdiferenciación Celular , Transactivadores , Animales , Humanos , Ratones , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Diferenciación Celular/genética , Cromatina , Metilación , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Chromosomal rearrangements of the mixed-lineage leukemia gene MLL1 are the hallmark of infant acute leukemia. The granulocyte-macrophage progenitor state forms the epigenetic basis for myelomonocytic leukemia stemness and transformation by MLL-type oncoproteins. Previously, it was shown that the establishment of murine myelomonocytic MLL-ENL transformation, but not its maintenance, depends on the transcription factor C/EBPα, suggesting an epigenetic hit-and-run mechanism of MLL-driven oncogenesis. Here, we demonstrate that compound deletion of Cebpa/Cebpb almost entirely abrogated the growth and survival of MLL-ENL-transformed cells. Rare, slow-growing, and apoptosis-prone MLL-ENL-transformed escapees were recovered from compound Cebpa/Cebpb deletions. The escapees were uniformly characterized by high expression of the resident Cebpe gene, suggesting inferior functional compensation of C/EBPα/C/EBPß deficiency by C/EBPε. Complementation was augmented by ectopic C/EBPß expression and downstream activation of IGF1 that enhanced growth. Cebpe gene inactivation was accomplished only in the presence of complementing C/EBPß, but not in its absence, confirming the Cebpe dependency of the Cebpa/Cebpb double knockouts. Our data show that MLL-transformed myeloid cells are dependent on C/EBPs during the initiation and maintenance of transformation.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/deficiencia , Proteína beta Potenciadora de Unión a CCAAT/deficiencia , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Transformación Celular Neoplásica/genética , Células Precursoras de Granulocitos/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Animales , Apoptosis/genética , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/genética , Proliferación Celular/genética , Supervivencia Celular/genética , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Ratones , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Transducción de Señal/genética , TransfecciónRESUMEN
C/EBPα represents a paradigm intrinsically disordered transcription factor containing short linear motifs and post-translational modifications (PTM). Unraveling C/EBPα protein interaction networks is a prerequisite for understanding the multi-modal functions of C/EBPα in hematopoiesis and leukemia. Here, we combined arrayed peptide matrix screening (PRISMA) with BioID to generate an in vivo validated and isoform specific interaction map of C/EBPα. The myeloid C/EBPα interactome comprises promiscuous and PTM-regulated interactions with protein machineries involved in gene expression, epigenetics, genome organization, DNA replication, RNA processing, and nuclear transport. C/EBPα interaction hotspots coincide with homologous conserved regions of the C/EBP family that also score as molecular recognition features. PTMs alter the interaction spectrum of C/EBP-motifs to configure a multi-valent transcription factor hub that interacts with multiple co-regulatory components, including BAF/SWI-SNF or Mediator complexes. Combining PRISMA and BioID is a powerful strategy to systematically explore the PTM-regulated interactomes of intrinsically disordered transcription factors.
RESUMEN
CCAAT enhancer-binding protein beta (C/EBPß) is a pioneer transcription factor that specifies cell differentiation. C/EBPß is intrinsically unstructured, a molecular feature common to many proteins involved in signal processing and epigenetics. The structure of C/EBPß differs depending on alternative translation initiation and multiple post-translational modifications (PTM). Mutation of distinct PTM sites in C/EBPß alters protein interactions and cell differentiation, suggesting that a C/EBPß PTM indexing code determines epigenetic outcomes. Herein, we systematically explored the interactome of C/EBPß using an array technique based on spot-synthesized C/EBPß-derived linear tiling peptides with and without PTM, combined with mass spectrometric proteomic analysis of protein interactions. We identified interaction footprints of â¼1,300 proteins in nuclear extracts, many with chromatin modifying, chromatin remodeling, and RNA processing functions. The results suggest that C/EBPß acts as a multi-tasking molecular switchboard, integrating signal-dependent modifications and structural plasticity to orchestrate interactions with numerous protein complexes directing cell fate and function.
RESUMEN
The lymphoid-myeloid transdifferentiation potentials of members of the C/EBP family (C/EBPα, ß, δ, and ε) were compared in v-Abl-immortalized primary B cells. Conversion of B cells to macrophages was readily induced by the ectopic expression of any C/EBP, and enhanced by endogenous C/EBPα and ß activation. High transgene expression of C/EBPß or C/EBPε, but not of C/EBPα or C/EBPδ, also induced the formation of granulocytes. Granulocytes and macrophages emerged in a mutually exclusive manner. C/EBPß-expressing B cells produced granulocyte-macrophage progenitor (GMP)-like progenitors when subjected to selective pressure to eliminate lymphoid cells. The GMP-like progenitors remained self-renewing and cytokine-independent, and continuously produced macrophages and granulocytes. In addition to their suitability to study myelomonocytic lineage bifurcation, lineage-switched GMP-like progenitors could reflect the features of the lympho-myeloid lineage switch observed in leukemic progression.
Asunto(s)
Linfocitos B/citología , Linfocitos B/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Transdiferenciación Celular/genética , Células Progenitoras de Granulocitos y Macrófagos/citología , Células Progenitoras de Granulocitos y Macrófagos/metabolismo , Animales , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Diferenciación Celular , Linaje de la Célula/genética , Proliferación Celular , Dosificación de Gen , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Familia de Multigenes , FenotipoRESUMEN
The transcription factor C/EBPß controls differentiation, proliferation, and functionality of many cell types, including innate immune cells. A detailed molecular understanding of how C/EBPß directs alternative cell fates remains largely elusive. A multitude of signal-dependent post-translational modifications (PTMs) differentially affect the protean C/EBPß functions. In this study we apply an assay that converts primary mouse B lymphoid progenitors into myeloid cells in order to answer the question how C/EBPß regulates (trans-) differentiation and determines myeloid cell fate. We found that structural alterations and various C/EBPß PTMs determine the outcome of trans-differentiation of lymphoid into myeloid cells, including different types of monocytes/macrophages, dendritic cells, and granulocytes. The ability of C/EBPß to recruit chromatin remodeling complexes is required for the granulocytic trans-differentiation outcome. These novel findings reveal that PTMs and structural plasticity of C/EBPß are adaptable modular properties that integrate and rewire epigenetic functions to direct differentiation to diverse innate immune system cells, which are crucial for the organism survival.
Asunto(s)
Linfocitos B/fisiología , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Transdiferenciación Celular , Células Mieloides/fisiología , Procesamiento Proteico-Postraduccional , Sustitución de Aminoácidos , Animales , Proteína beta Potenciadora de Unión a CCAAT/química , Proteína beta Potenciadora de Unión a CCAAT/genética , Células Cultivadas , Inmunidad Innata , Ratones Endogámicos C57BL , Ratones Noqueados , Estructura Terciaria de Proteína , Activación Transcripcional , TranscriptomaRESUMEN
Post-translational modifications (PTMs) of transcription factors alter interactions with co-regulators and epigenetic modifiers. For example, members of the C/EBP transcription factor family are extensively methylated on arginine and lysine residues in short, conserved, modular domains, implying modification-dependent cofactor docking. Here we describe array peptide screening (APS), a systematic and differential approach to detect PTM-dependent interactions in the human proteome using chemically synthesized, biotinylated peptides coupled to fluorophore-labeled streptavidin. Peptides with and without a modified residue are applied in parallel to bacterial expression libraries in an arrayed format. Interactions are detected and quantified by laser scanning to reveal proteins that differentially bind to nonmodified or modified peptides. We have previously used this method to investigate the effect of arginine methylation of C/EBPß peptides. The method enables determination of PTM-dependent transcription factor interactions, quantification of relative binding affinities and rapid protein classification, all independently of the transactivation potential of peptides or cellular abundance of interactors. The protocol provides a cost-effective alternative to mass spectrometric approaches and takes 3-4 d to complete.
Asunto(s)
Unión Proteica/fisiología , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Factores de Transcripción/metabolismo , Arginina/metabolismo , Humanos , Metilación , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/química , Péptidos/metabolismo , Proteínas/metabolismo , Proteoma/análisis , Proteoma/metabolismo , Factores de Transcripción/genéticaRESUMEN
C/EBPs are implied in an amazing number of cellular functions: C/EBPs regulate tissue and cell type specific gene expression, proliferation, and differentiation control. C/EBPs assist in energy metabolism, female reproduction, innate immunity, inflammation, senescence, and the development of neoplasms. How can C/EBPs fulfill so many functions? Here we discuss that C/EBPs are extensively modified by methylation of arginine and lysine side chains and that regulated methylation profoundly affects the activity of C/EBPs.
Asunto(s)
Arginina/metabolismo , Proteína alfa Potenciadora de Unión a CCAAT/fisiología , Proteína beta Potenciadora de Unión a CCAAT/fisiología , Fenómenos Fisiológicos Celulares/fisiología , Lisina/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/genética , Sitios de Unión/genética , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Fenómenos Fisiológicos Celulares/genética , Femenino , Humanos , Lisina/genética , Metilación , Datos de Secuencia Molecular , Mutación , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Homología de Secuencia de AminoácidoRESUMEN
The functional capacity of the transcriptional regulatory CCAAT/enhancer-binding protein-beta (C/EBPbeta) is governed by protein interactions and post-translational protein modifications. In a proteome-wide interaction screen, the histone-lysine N-methyltransferase, H3 lysine 9-specific 3 (G9a), was found to directly interact with the C/EBPbeta transactivation domain (TAD). Binding between G9a and C/EBPbeta was confirmed by glutathione S-transferase pulldown and co-immunoprecipitation. Metabolic labeling showed that C/EBPbeta is post-translationally modified by methylation in vivo. A conserved lysine residue in the C/EBPbeta TAD served as a substrate for G9a-mediated methylation. G9a, but not a methyltransferase-defective G9a mutant, abrogated the transactivation potential of wild type C/EBPbeta. A C/EBPbeta TAD mutant that contained a lysine-to-alanine exchange was resistant to G9a-mediated inhibition. Moreover, the same mutation conferred super-activation of a chromatin-embedded, endogenous C/EBPbeta target gene. Our data identify C/EBPbeta as a direct substrate of G9a-mediated post-translational modification that alters the functional properties of C/EBPbeta during gene regulation.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Proteoma/metabolismo , Activación Transcripcional/fisiología , Proteína beta Potenciadora de Unión a CCAAT/genética , Células HeLa , Antígenos de Histocompatibilidad/genética , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Mutación , Estructura Terciaria de Proteína/fisiología , Proteoma/genéticaRESUMEN
The c-Myb transcription factor coordinates proliferation and differentiation of hematopoietic precursor cells. Myb has three consecutive N-terminal SANT-type repeat domains (R1, R2, R3), two of which (R2, R3) form the DNA-binding domain (DBD). Three amino acid substitutions in R2 alter the way Myb regulates genes and determine the leukemogenicity of the retrovirally transduced v-Myb oncogene. The molecular mechanism of how these mutations unleash the leukemogenic potential of Myb is unknown. Here we demonstrate that the c-Myb-DBD binds to the N-terminal histone tails of H3 and H3.3. C-Myb binding facilitates histone tail acetylation, which is mandatory during activation of prevalent differentiation genes in conjunction with CCAAT enhancer-binding proteins (C/EBP). Leukemogenic mutations in v-Myb eliminate the interaction with H3 and acetylation of H3 tails and abolish activation of endogenous differentiation genes. In primary v-myb-transformed myeloblasts, pharmacologic enhancement of H3 acetylation restored activation of differentiation genes and induced cell differentiation. Our data link a novel chromatin function of c-Myb with lineage-specific expression of differentiation genes and relate the loss of this function with the leukemic conversion of Myb.
Asunto(s)
Sustitución de Aminoácidos/genética , Cromatina/metabolismo , Histonas/metabolismo , Proteínas Oncogénicas v-myb/metabolismo , Mutación Puntual/genética , Proteínas Proto-Oncogénicas c-myb/metabolismo , Acetilación , Animales , Secuencia de Bases , Factor de Unión a CCAAT/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Pollos , Cromatina/genética , Regulación de la Expresión Génica/genética , Regulación Leucémica de la Expresión Génica/genética , Células Precursoras de Granulocitos/metabolismo , Humanos , Leucemia/genética , Leucemia/metabolismo , Datos de Secuencia Molecular , Proteínas Oncogénicas v-myb/genética , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myb/genética , Retroviridae , Transducción Genética/métodosRESUMEN
C/EBPbeta is an intrinsically repressed transcription factor that regulates genes involved in differentiation, proliferation, tumorigenesis, and apoptosis. C/EBPbeta acts as a repressor that is turned into an activator by the Ras oncoprotein through phosphorylation of a MAPK site. C/EBPbeta activation is accompanied by a conformational change. Active and repressive C/EBPbeta interacts with multisubunit Mediator complexes through the CRSP130/Sur2 subunit. The CRSP130/Sur2 subunit is common to two distinct types of Mediator complexes, characterized by CRSP70 and CDK8 proteins as transcriptionally active and inactive Mediator, respectively. Knockdown of CRSP130/Sur2 prevents Mediator binding and transactivation through C/EBPbeta. Oncogenic Ras signaling or activating mutations in C/EBPbeta selects the transcriptionally active Mediator complex that also associates with RNA polymerase II. These results show that a Ras-induced structural alteration of C/EBPbeta determines differential gene activation through selective interaction with distinct Mediator complexes.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteínas ras/fisiología , Apoptosis , Cromatina/metabolismo , Quinasa 8 Dependiente de Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Activación Enzimática , Genes Reporteros , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Sistema de Señalización de MAP Quinasas , Modelos Genéticos , Mutación , Pruebas de Precipitina , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , ARN/metabolismo , ARN Polimerasa II/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Transcripción Genética , Activación TranscripcionalRESUMEN
The viral Myb (v-Myb) oncoprotein of the avian myeloblastosis virus (AMV) is an activated form of the cellular transcription factor c-Myb causing acute monoblastic leukemia in chicken. Oncogenic v-Myb alterations include N- and C-terminal deletions as well as point mutations. Whereas truncations in Myb cause loss of various protein modifications, none of the point mutations in v-Myb has been directly linked to protein modifications. Here we show that the DNA-binding domain of c-Myb can be phosphorylated on serine 116 by the catalytic subunit of protein kinase A. Phosphorylation of Ser(116) differentially destabilizes a subtype of c-Myb-DNA complexes. The V117D mutation of the AMV v-Myb oncoprotein abolishes phosphorylation of the adjacent Ser(116) residue. Modification of Ser(116) was also detected in live cells in c-Myb, but not in AMV v-Myb. Phosphorylation-mimicking mutants of c-Myb failed to activate the resident mim-1 gene. Our data imply that protein kinase A or a kinase with similar specificity negatively regulates c-Myb function, including collaboration with C/EBP, and that the leukemogenic AMV v-Myb version evades inactivation by a point mutation that abolishes a phosphoacceptor consensus site. This suggests a novel link between Myb, a signal transduction pathway, cooperativity with C/EBP, and a point mutation in the myb oncogene.