title: Hierarchical phosphorylation within the ankyrin repeat domain defines a phosphoregulatory loop that regulates Notch transcriptional activity.
abstract: The Notch signal transduction pathway mediates important cellular functions through direct cell-to-cell contact. Deregulation of Notch activity can lead to an altered cell proliferation and has been linked to many human cancers. Casein kinase 2 (CK2), a ubiquitous kinase, regulates several cellular processes by phosphorylating proteins involved in signal transduction, gene expression, and protein synthesis. In this report we identify Notch(ICD) as a novel target of phosphorylation by CK2. Using mapping and mutational studies, we identified serine 1901, located in the ankyrin domain of Notch, as the target amino acid. Interestingly, phosphorylation of serine 1901 by CK2 appears to generate a second phosphorylation site at threonine 1898. Furthermore, threonine 1898 phosphorylation only occurs when Notch forms a complex with Mastermind and CSL. Phosphorylation of both threonine 1898 and serine 1901 resulted in decreased binding of the Notch-Mastermind-CSL ternary complex to DNA and consequently lower transcriptional activity. These data indicate that the phosphorylation of serine 1901 and threonine 1898 negatively regulates Notch function by dissociating the complex from DNA. This study identifies a new component involved in regulation of Notch(ICD) transcriptional activity, reinforcing the notion that a precise and tight regulation is required for this essential signaling pathway.
authors.affiliation: Molecular Oncology Program, Division of Surgical Oncology, Dewitt Daughtry Family of Surgery and Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami Florida 33136.
authors.forename: Prathibha
authors.initials: P
authors.lastname: Ranganathan
authors.affiliation: Molecular Oncology Program, Division of Surgical Oncology, Dewitt Daughtry Family of Surgery and Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami Florida 33136.
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Vasquez-Del Carpio
authors.affiliation: Molecular Oncology Program, Division of Surgical Oncology, Dewitt Daughtry Family of Surgery and Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami Florida 33136.
authors.forename: Fred M
authors.initials: FM
authors.lastname: Kaplan
authors.affiliation: Molecular Oncology Program, Division of Surgical Oncology, Dewitt Daughtry Family of Surgery and Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami Florida 33136.
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.affiliation: Molecular Oncology Program, Division of Surgical Oncology, Dewitt Daughtry Family of Surgery and Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami Florida 33136.
authors.forename: Ashu
authors.initials: A
authors.lastname: Gupta
authors.affiliation: Molecular Oncology Program, Division of Surgical Oncology, Dewitt Daughtry Family of Surgery and Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami Florida 33136.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: VanWye
authors.affiliation: Molecular Oncology Program, Division of Surgical Oncology, Dewitt Daughtry Family of Surgery and Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami Florida 33136. Electronic address: TCapobianco@med.miami.edu.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Capobianco
mesh_terms: D017089:Ankyrin Repeat / Q000502:physiology; D047390:Casein Kinase II / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods; D010766:Phosphorylation / Q000502:physiology; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*
chemical_list: D004268:DNA-Binding Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; C430872:MAML1 protein, human; D046912:Multiprotein Complexes; C497570:RBPJ protein, human; D051880:Receptors, Notch; D014157:Transcription Factors; D047390:Casein Kinase II