ABSTRACT
The transcription factor B-MYB is an important regulator of cell cycle-related processes that is activated by step-wise phosphorylation of multiple sites by cyclin-dependent kinases (CDKs) and conformational changes induced by the peptidylprolyl cis/trans isomerase Pin1. Here, we show that a conserved amino acid sequence around Ser-577 in the C-terminal part of B-MYB is able to interact with the B-MYB DNA-binding domain. Phosphorylation of Ser-577 disrupts this interaction and is regulated by the interplay of CDKs and the phosphatase CDC14B. Deletion of sequences surrounding Ser-577 hyperactivates the transactivation potential of B-MYB, decreases its proteolytic stability, and causes cell cycle defects. Overall, we show for the first time that B-MYB can undergo an intramolecular interaction that is controlled by the phosphorylation state of Ser-577.
Subject(s)
Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Phosphoserine/metabolism , Serine/metabolism , Trans-Activators/chemistry , Trans-Activators/metabolism , Amino Acid Sequence , Animals , Cell Cycle/genetics , Cell Cycle Proteins/genetics , Cyclin-Dependent Kinases/metabolism , DNA/genetics , DNA/metabolism , Dual-Specificity Phosphatases/metabolism , HEK293 Cells , Humans , Phosphorylation , Protein Binding/genetics , Protein Domains/genetics , Proteolysis , Serine/genetics , Trans-Activators/genetics , Transcriptional Activation/geneticsABSTRACT
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.
Subject(s)
Cell Cycle Proteins/genetics , NIMA-Interacting Peptidylprolyl Isomerase/genetics , Neoplasms/genetics , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/genetics , Trans-Activators/genetics , Carcinogenesis/drug effects , Cell Cycle/genetics , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/chemistry , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/genetics , Gene Expression Regulation, Neoplastic/drug effects , HeLa Cells , Hep G2 Cells , Humans , Mitosis/drug effects , NIMA-Interacting Peptidylprolyl Isomerase/antagonists & inhibitors , NIMA-Interacting Peptidylprolyl Isomerase/chemistry , Neoplasms/drug therapy , Neoplasms/pathology , Nocodazole/pharmacology , Peptidylprolyl Isomerase/genetics , Phosphorylation/drug effects , Protein Conformation/drug effects , Protein Multimerization/drug effects , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/chemistry , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/chemistry , Roscovitine/pharmacology , Thymidine/pharmacology , Trans-Activators/chemistry , Transcription, Genetic/drug effects , Polo-Like Kinase 1ABSTRACT
B-Myb, a highly conserved member of the Myb transcription factor family, is expressed ubiquitously in proliferating cells and controls the cell cycle dependent transcription of G2/M-phase genes. Deregulation of B-Myb has been implicated in oncogenesis and loss of genomic stability. We have identified B-Myb as a novel interaction partner of the Mre11-Rad50-Nbs1 (MRN) complex, a key player in the repair of DNA double strand breaks. We show that B-Myb directly interacts with the Nbs1 subunit of the MRN complex and is recruited transiently to DNA-damage sites. In response to DNA-damage B-Myb is phosphorylated by protein kinase GSK3ß and released from the MRN complex. A B-Myb mutant that cannot be phosphorylated by GSK3ß disturbs the regulation of pro-mitotic B-Myb target genes and leads to inappropriate mitotic entry in response to DNA-damage. Overall, our work suggests a novel function of B-Myb in the cellular DNA-damage signalling.
Subject(s)
Cell Cycle Proteins/metabolism , DNA Damage , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/metabolism , MRE11 Homologue Protein/metabolism , Nuclear Proteins/metabolism , Signal Transduction , Trans-Activators/metabolism , Acid Anhydride Hydrolases , Amino Acid Sequence , Ataxia Telangiectasia Mutated Proteins/metabolism , Binding Sites , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Line , DNA Breaks, Double-Stranded , DNA Repair , DNA Repair Enzymes/chemistry , DNA-Binding Proteins/chemistry , Gene Expression Regulation , Glycogen Synthase Kinase 3 beta/metabolism , Humans , MRE11 Homologue Protein/chemistry , Mitosis/genetics , Models, Biological , Multiprotein Complexes/metabolism , Mutation , Nuclear Proteins/chemistry , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Trans-Activators/chemistry , Trans-Activators/geneticsABSTRACT
The tumor suppressor protein programmed cell death 4 (Pdcd4) is a highly conserved RNA-binding protein that inhibits the translation of specific mRNAs. Here, we have identified the homeobox-interacting protein kinase-2 (Hipk2) mRNA as a novel translational target of Pdcd4. Unlike most other protein kinases Hipk2 is constitutively active after being synthesized by the ribosome and its expression and activity are thought to be mainly controlled by modulation of the half-life of the kinase. Our work provides the first evidence that Hipk2 expression is also controlled on the level of translation. We show that Hipk2 stimulates the translation of its own mRNA and that Pdcd4 suppresses the translation of Hipk2 mRNA by interfering with this auto-regulatory feedback mechanism. We also show that the translation of the related kinase Hipk1 is controlled by a similar feedback loop and that Hipk2 also stimulates the translation of Hipk1 mRNA. Taken together, our work describes a novel mechanism of translational suppression by Pdcd4 and shows for the first time that Hipk2 controls its own synthesis by an auto-regulatory feedback mechanism. Furthermore, the effect of Hipk2 on the translation of Hipk1 RNA suggests that Hipk2 and Pdcd4 can act in similar manner to control the translation of other mRNAs.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Carrier Proteins/genetics , Protein Biosynthesis , Protein Serine-Threonine Kinases/genetics , RNA-Binding Proteins/metabolism , Animals , Apoptosis Regulatory Proteins/chemistry , Carrier Proteins/metabolism , Cell Line , Chickens , Eukaryotic Initiation Factor-4A/metabolism , Gene Expression Regulation , Humans , Models, Biological , Mutant Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Quail , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/chemistry , Structure-Activity RelationshipABSTRACT
B-Myb is a highly conserved member of the Myb transcription factor family, which plays an essential role in cell cycle progression by regulating the transcription of genes at the G 2/M-phase boundary. The role of B-Myb in other parts of the cell cycle is less well-understood. By employing siRNA-mediated silencing of B-Myb expression, we found that B-Myb is required for efficient entry into S-phase. Surprisingly, a B-Myb mutant that lacks sequence-specific DNA-binding activity and is unable to activate transcription of B-Myb target genes is able to rescue the S-phase defect observed after B-Myb knockdown. Moreover, we have identified polymerase delta-interacting protein 1 (Pdip1), a BTB domain protein known to bind to the DNA replication and repair factor PCNA as a novel B-Myb interaction partner. We have shown that Pdip1 is able to interact with B-Myb and PCNA simultaneously. In addition, we found that a fraction of endogenous B-Myb can be co-precipitated via PCNA, suggesting that B-Myb might be involved in processes related to DNA replication or repair. Taken together, our work suggests a novel role for B-Myb in S-phase that appears to be independent of its sequence-specific DNA-binding activity and its ability to stimulate the expression of bona fide B-Myb target genes.
