title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1.
abstract: BACKGROUND
  We have previously identified kinase suppressor of ras-1 (KSR1) as a potential regulatory gene in breast cancer. KSR1, originally described as a novel protein kinase, has a role in activation of mitogen-activated protein kinases. Emerging evidence has shown that KSR1 may have dual functions as an active kinase as well as a scaffold facilitating multiprotein complex assembly. Although efforts have been made to study the role of KSR1 in certain tumour types, its involvement in breast cancer remains unknown.
  
  
  METHODS
  A quantitative mass spectrometry analysis using stable isotope labelling of amino acids in cell culture (SILAC) was implemented to identify KSR1-regulated phosphoproteins in breast cancer. In vitro luciferase assays, co-immunoprecipitation as well as western blotting experiments were performed to further study the function of KSR1 in breast cancer.
  
  
  RESULTS
  Of significance, proteomic analysis reveals that KSR1 overexpression decreases deleted in breast cancer-1 (DBC1) phosphorylation. Furthermore, we show that KSR1 decreases the transcriptional activity of p53 by reducing the phosphorylation of DBC1, which leads to a reduced interaction of DBC1 with sirtuin-1 (SIRT1); this in turn enables SIRT1 to deacetylate p53.
  
  
  CONCLUSIONS
  Our findings integrate KSR1 into a network involving DBC1 and SIRT1, which results in the regulation of p53 acetylation and its transcriptional activity.
authors.affiliation: Division of Cancer, Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 ONN, UK.
authors.forename: H
authors.initials: H
authors.lastname: Zhang
authors.forename: Y
authors.initials: Y
authors.lastname: Xu
authors.forename: A
authors.initials: A
authors.lastname: Filipovic
authors.forename: L C
authors.initials: LC
authors.lastname: Lit
authors.forename: C-Y
authors.initials: CY
authors.lastname: Koo
authors.forename: J
authors.initials: J
authors.lastname: Stebbing
authors.forename: G
authors.initials: G
authors.lastname: Giamas
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods; D015536:Down-Regulation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D045325:HCT116 Cells; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D010750:Phosphoproteins / Q000032:analysis; D011494:Protein Kinases / Q000502:physiology*; D040901:Proteomics / Q000379:methods*; D056564:Sirtuin 1 / Q000378:metabolism; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000596:Amino Acids; C527450:CCAR2 protein, human; D010750:Phosphoproteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D011494:Protein Kinases; C097199:KSR-1 protein kinase; C447939:SIRT1 protein, human; D056564:Sirtuin 1