The oncogenic transcription factor B-Myb is an essential regulator of late cell cycle genes whose activation by phosphorylation is still poorly understood. We describe a stepwise phosphorylation mechanism of B-Myb, which involves sequential phosphorylations mediated by cyclin-dependent kinase (Cdk) and Polo-like kinase 1 (Plk1) and Pin1-facilitated peptidyl-prolyl cis/trans isomerization. Our data suggest a model in which initial Cdk-dependent phosphorylation of B-Myb enables subsequent Pin1 binding and Pin1-induced conformational changes of B-Myb. This, in turn, initiates further phosphorylation of Cdk-phosphosites, enabling Plk1 docking and subsequent Plk1-mediated phosphorylation of B-Myb to finally allow B-Myb to stimulate transcription of late cell cycle genes. Our observations reveal novel mechanistic hierarchies of B-Myb phosphorylation and activation and uncover regulatory principles that might also apply to other Myb family members. Strikingly, overexpression of B-Myb and of factors mediating its activation strongly correlates with adverse prognoses for tumor patients, emphasizing B-Myb's role in tumorigenesis.

B-MYB, a highly conserved member of the MYB transcription factor family, is expressed ubiquitously in proliferating cells and plays key roles in important cell cycle-related processes, such as control of G2/Mphase transcription, cytokinesis, G1/S-phase progression and DNA-damage reponse. Deregulation of B-MYB function is characteristic of several types of tumor cells, underlining its oncogenic potential. To gain a better understanding of the functions of B-MYB we have employed affinity purification coupled to mass spectrometry to discover novel B-MYB interacting proteins. Here we have identified the zinc-finger proteins ZMYM2 and ZMYM4 as novel B-MYB binding proteins. ZMYM4 is a poorly studied protein whose initial characterization reported here shows that it is highly SUMOylated and that its interaction with B-MYB is stimulated upon induction of DNA damage. Unlike knockdown of B-MYB, which causes G2/M arrest and defective cytokinesis in HEK293 cells, knockdown of ZMYM2 or ZMYM4 have no obvious effects on the cell cycle of these cells. By contrast, knockdown of ZMYM2 strongly impaired the G1/S-phase progression of HepG2 cells, suggesting that ZMYM2, like B-MYB, is required for entry into S-phase in these cells. Overall, our work identifies two novel B-MYB binding partners with possible functions in the DNA-damage response and the G1/S-transition. The highly conserved MYB proto-oncogene family member B-MYB is ubiquitously expressed in proliferating cells where it acts as an essential cell cycle-regulated transcription factor 1,2. Mammalian B-MYB, like its Drosophila melanogaster homolog, are important interaction partners of the MYB-MuvB/DREAM complex, an evolutionarily conserved multiprotein complex that controls the transcription of genes that are relevant for mitosis 3,4. In resting cells, the MuvB core complex, consisting of Lin-9, Lin-37, Lin-54, Lin-52 and RBBP4, associates with E2F4 and either p130 or p107 to form the DREAM complex, which acts as a repressor of E2F target genes. In S-phase, the MuvB core complex dissociates from E2F4/p130/p107 and recruits B-MYB to form the MYB-MuvB complex, which is then targeted to the promoters of genes required for the G2/M transition and mitosis 5-11. B-MYB activity itself is highly regulated during the cell cycle by transcriptional and post-transcriptional mechanisms 12-17. Notably, a stepwise phosphorylation mechanism of B-MYB has been described, which involves sequential phosphorylations mediated by cyclin-dependent kinase (Cdk) and Polo-like kinase 1 (Plk1) and, together with Pin1-facilitated peptidyl-prolyl cis/trans isomerization, triggers conformational changes of B-MYB to finally allow it to stimulate transcription of its target genes 18. In addition to its role as a cell cycle regulated transcription factor B-MYB has also "non-transcriptional" functions in proliferating cells. During mitosis, B-MYB interacts with the MYB-Clafi complex and thereby participates in the formation of the mitotic spindle 19. B-MYB also stimulates G1/S transition in a manner that is ...

Interactions of long non-coding RNAs (lncRNA) with proteins play important roles in the regulation of many cellular processes. PANDAR (Promotor of CDKN1A Antisense DNA damage Activated RNA) is a lncRNA that is transcribed in a p53-dependent manner from the CDKN1A promoter and is involved in the regulation of proliferation and senescence. Overexpression of PANDAR has been observed in several tumor species and correlated with a poor prognosis for patient survival rate. Depending on the cellular state, PANDAR is known to interact with proteins such as the nuclear transcription factor Y subunit A (NF-YA) and the scaffold attachment factor A (SAF-A). However, a comprehensive analysis of the PANDAR interactome was missing so far. Therefore, we applied peptide nucleic acid (PNA)-based pull-downs combined with quantitative mass spectrometry to identify new protein binding partners. We confirmed potential candidates like U2AF65 and PTBP1, known to be involved in RNA processing. Furthermore, we observed that overexpression of PANDAR leads to a reduced level of the short pro-apoptotic BCL-X splice variant (BCL-XS) which is regulated by PTBP1. Simultaneous overexpression of PTBP1 was able to rescue this effect. Overall, our data suggest a role for PANDAR in the regulation of splicing events via its interaction partner PTBP1.