title: Dissection of TBK1 signaling via phosphoproteomics in lung cancer cells.
abstract: TANK-binding kinase 1 (TBK1) has emerged as a novel therapeutic target for unspecified subset of lung cancers. TBK1 reportedly mediates prosurvival signaling by activating NF-κB and AKT. However, we observed that TBK1 knockdown also decreased viability of cells expressing constitutively active NF-κB and interferon regulatory factor 3. Basal phospho-AKT level was not reduced after TBK1 knockdown in TBK1-sensitive lung cancer cells, implicating that TBK1 mediates unknown survival mechanisms. To gain better insight into TBK1 survival signaling, we searched for altered phosphoproteins using mass spectrometry following RNAi-mediated TBK1 knockdown. In total, we identified 2,080 phosphoproteins (4,621 peptides), of which 385 proteins (477 peptides) were affected after TBK1 knockdown. A view of the altered network identified a central role of Polo-like kinase 1 (PLK1) and known PLK1 targets. We found that TBK1 directly phosphorylated PLK1 in vitro. TBK1 phosphorylation was induced at mitosis, and loss of TBK1 impaired mitotic phosphorylation of PLK1 in TBK1-sensitive lung cancer cells. Furthermore, lung cancer cell sensitivity to TBK1 was highly correlated with sensitivity to pharmacological PLK inhibition. We additionally found that TBK1 knockdown decreased metadherin phosphorylation at Ser-568. Metadherin was associated with poor outcome in lung cancer, and loss of metadherin caused growth inhibition and apoptosis in TBK1-sensitive lung cancer cells. These results collectively revealed TBK1 as a mitosis regulator through activation of PLK1 and also suggested metadherin as a putative TBK1 downstream effector involved in lung cancer cell survival.
authors.affiliation: Department ofThoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.
authors.forename: Jae-Young
authors.initials: JY
authors.lastname: Kim
authors.forename: Eric A
authors.initials: EA
authors.lastname: Welsh
authors.forename: Umut
authors.initials: U
authors.lastname: Oguz
authors.forename: Bin
authors.initials: B
authors.lastname: Fang
authors.forename: Yun
authors.initials: Y
authors.lastname: Bai
authors.forename: Fumi
authors.initials: F
authors.lastname: Kinose
authors.forename: Crystina
authors.initials: C
authors.lastname: Bronk
authors.forename: Lily L
authors.initials: LL
authors.lastname: Remsing Rix
authors.forename: Amer A
authors.initials: AA
authors.lastname: Beg
authors.forename: Uwe
authors.initials: U
authors.lastname: Rix
authors.forename: Steven A
authors.initials: SA
authors.lastname: Eschrich
authors.forename: John M
authors.initials: JM
authors.lastname: Koomen
authors.forename: Eric B
authors.initials: EB
authors.lastname: Haura
mesh_terms: D000595:Amino Acid Sequence; D011905:Genes, ras; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D040901:Proteomics; D015398:Signal Transduction
chemical_list: D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; C403191:TBK1 protein, human
keywords: astrocyte elevated gene-1 (AEG-1); non-canonical IκB kinase; stable isotope labeling by amino acids (SILAC)