ABSTRACT
PARP is a multifunctional protein that can affect genome stability, transcription control, telomere length and cell death. Recently we have reported that PARP binds to and enhances B-MYB transactivating potential. B-MYB is a potentially oncogenic transcription factor involved in mammalian cell proliferation, survival and differentiation. B-MYB gene expression is growth regulated and B-MYB protein is phosphorylated during S phase by cyclin A or E/cdk2 kinase, resulting in augmented transactivating potential. Here we show that PARP induces phosphorylation of B-MYB protein at cdk2 phosphorylation sites, since a B-MYB protein with mutated cdk2 phosphorylation sites is refractory to PARP-induced phosphorylation and co-activation in mammalian cells. We propose that PARP functions as a B-MYB co-factor by promoting cyclin/cdk2-dependent B-MYB phosphorylation. These results highlight a novel role for PARP as a factor that integrates cyclin-dependent kinases signaling with gene transcription.
Subject(s)
CDC2-CDC28 Kinases , Cell Cycle Proteins , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Poly(ADP-ribose) Polymerases/physiology , Protein Serine-Threonine Kinases/metabolism , Trans-Activators/chemistry , Trans-Activators/metabolism , Cell Line , Cells, Cultured , Cyclin A/metabolism , Cyclin-Dependent Kinase 2 , DNA-Binding Proteins/genetics , Genes, Reporter , Humans , Mutation , Phosphorylation , Poly(ADP-ribose) Polymerases/genetics , Protein Structure, Tertiary , Trans-Activators/genetics , Transcriptional Activation , Transfection , Tumor Cells, CulturedABSTRACT
B-MYB is an ubiquitous protein required for mammalian cell growth. In this report we show that B-MYB transactivates its own promoter through a 120 bp segment proximal to the transcription start site. The B-MYB-responsive element does not contain myb-binding sites and gel-shift analysis shows that SP1, but not B-MYB, protein contained in SAOS2 cell extracts binds to the 120 bp B-myb promoter fragment. B-MYB-dependent transactivation is cooperatively increased in the presence of SP1, but not SP3 overexpression. When the SP1 elements of the B-myb promoter are transferred in front of a heterologous promoter, an increased response to B-MYB results. In contrast, c-MYB, the prototype member of the Myb family, is not able to activate the luciferase construct containing the SP1 elements. With the use of an SP1-GAL4 fusion protein, we have determined that the cooperative activation occurs through the domain A of SP1. These observations suggest that B-MYB functions as a coactivator of SP1, and that diverse combinations of myb and SP1 sites may dictate the responsiveness of myb-target genes to the various members of the myb family.
Subject(s)
Cell Cycle Proteins , DNA-Binding Proteins/genetics , Promoter Regions, Genetic , Saccharomyces cerevisiae Proteins , Sp1 Transcription Factor/metabolism , Trans-Activators/genetics , Transcriptional Activation , Adenovirus E1A Proteins/metabolism , Binding Sites , Cell Line , DNA-Binding Proteins/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Herpes Simplex Virus Protein Vmw65/metabolism , Humans , NF-kappa B/metabolism , Oligonucleotides/pharmacology , Protein Binding , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-myb , Recombinant Fusion Proteins , Sp1 Transcription Factor/genetics , Sp3 Transcription Factor , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Transcriptional Activation/drug effects , TransfectionABSTRACT
B-MYB is implicated in cell growth control, differentiation, and cancer and belongs to the MYB family of nuclear transcription factors. Evidence exists that cellular proteins bind directly to B-MYB, and it has been hypothesized that B-MYB transcriptional activity may be modulated by specific cofactors. In an attempt to isolate proteins that interact with the B-MYB DNA-binding domain, a modular domain that has the potential to mediate protein-protein interaction, we performed pull-down experiments with a glutathione S-transferase-B-MYB protein and mammalian protein extracts. We isolated a 110-kDa protein associated endogenously with B-MYB in the nuclei of HL60 cells. Microsequence analysis and immunoprecipitation experiments determined that the bound protein was poly(ADP-ribose) polymerase (PARP). Transient transfection assays showed that PARP enhanced B-MYB transactivation and that PARP enzymatic activity is not required for B-MYB-dependent transactivation. These results suggest that PARP, as a transcriptional cofactor of a potentially oncogenic protein, may play a role in growth control and cancer.