Abstract
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.
Highlights
B-MYB is a nuclear transcription factor belonging to the MYB family, which is expressed ubiquitously and is involved in cell growth control, differentiation, and cancer [1, 2]
Protein lysates were incubated with glutathione S-transferase protein (GST)-B-MYB fusion protein, and cellular proteins binding to GST-B-MYB were analyzed by SDS-PAGE and autoradiography
In this report we show that poly(ADP-ribose) polymerase (PARP) binds to the B-MYB-DNA-binding domain and enhances its transactivating activity
Summary
B-MYB is a nuclear transcription factor belonging to the MYB family, which is expressed ubiquitously and is involved in cell growth control, differentiation, and cancer [1, 2]. The leading hypothesis describes B-MYB as a constitutively repressed molecule that requires post-translational modifications to disclose its activity In this regard, several groups [9, 10] have shown that phosphorylation of the B-MYB protein induced by the CDK2/cyclin A kinase results in activation of the B-MYB transactivating function. This suggests that, in addition to relieving intramolecular repression, phosphorylation may enhance B-MYB cross-talk with putative co-activators or may inhibit binding of co-repressors This hypothesis is corroborated by evidence suggesting that the B-MYB transactivating function depends on the cellular context and that it correlates with the binding of cellular proteins to specific BMYB domains [8]. As a basis of the present study, we hypothesized that proteins binding to the B-MYB DNA-binding domain may function as regulatory molecules, contributing to B-MYB transcriptional activity. Overexpression of PARP and B-MYB results in synergistic activation of a MYB-responsive promoter, suggesting that PARP is a B-MYB-specific co-activator
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