Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover.
Notch signaling releases the Notch receptor intracellular domain (ICD), which complexes with CBF1 and Mastermind (MAM) to activate responsive genes. We previously reported that MAM interacts with CBP/p300 and promotes hyperphosphorylation and degradation of the Notch ICD in vivo. Here we show that CycC:CDK8 and CycT1:CDK9/P-TEFb are recruited with Notch and associated coactivators (MAM, SKIP) to the HES1 promoter in signaling cells. MAM interacts directly with CDK8 and can cause it to localize to subnuclear foci. Purified recombinant CycC:CDK8 phosphorylates the Notch ICD within the TAD and PEST domains, and expression of CycC:CDK8 strongly enhances Notch ICD hyperphosphorylation and PEST-dependent degradation by the Fbw7/Sel10 ubiquitin ligase in vivo. Point mutations affecting conserved Ser residues within the ICD PEST motif prevent hyperphosphorylation by CycC:CDK8 and stabilize the ICD in vivo. These findings suggest a role for MAM and CycC:CDK8 in the turnover of the Notch enhancer complex at target genes.
Fryer
Christy J
CJ
Regulatory Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA.
White
J Brandon
JB
Jones
Katherine A
KA
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism; D018920:Coculture Techniques; D056850:Cyclin-Dependent Kinase 8; D042863:Cyclin-Dependent Kinase 9 / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D029721:Drosophila Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D017930:Genes, Reporter; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D007739:L Cells; D008156:Luciferases / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D012516:Osteosarcoma / Q000473:pathology; D010766:Phosphorylation; D017354:Point Mutation; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D051880:Receptors, Notch; D015398:Signal Transduction; D015534:Trans-Activators; D000072056:Transcription Factor HES-1; D014157:Transcription Factors; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051792:Basic Helix-Loop-Helix Proteins; D002843:Chromatin; D056850:Cyclin-Dependent Kinase 8; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D008156:Luciferases; D008565:Membrane Proteins; D009687:Nuclear Proteins; D051880:Receptors, Notch; D015534:Trans-Activators; D000072056:Transcription Factor HES-1; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D044767:Ubiquitin-Protein Ligases; C430872:MAML1 protein, human; C466999:SPHKAP protein, human; C078493:HES1 protein, human; C095686:CDK8 protein, human; C467969:CDK9 protein, human; C498308:Cdk8 protein, Drosophila