title: Phosphorylation-Dependent Regulation of the DNA Damage Response of Adaptor Protein KIBRA in Cancer Cells.
abstract: Multifunctional adaptor proteins encompassing various protein-protein interaction domains play a central role in the DNA damage response pathway. In this report, we show that KIBRA is a physiologically interacting reversible substrate of ataxia telangiectasia mutated (ATM) kinase. We identified the site of phosphorylation in KIBRA as threonine 1006, which is embedded within the serine/threonine (S/T) Q consensus motif, by site-directed mutagenesis, and we further confirmed the same with a phospho-(S/T) Q motif-specific antibody. Results from DNA repair functional assays such as the γ-H2AX assay, pulsed-field gel electrophoresis (PFGE), Comet assay, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and clonogenic cell survival assay using stable overexpression clones of wild-type (wt.) KIBRA and active (T1006E) and inactive (T1006A) KIBRA phosphorylation mutants showed that T1006 phosphorylation on KIBRA is essential for optimal DNA double-strand break repair in cancer cells. Further, results from stable retroviral short hairpin RNA-mediated knockdown (KD) clones of KIBRA and KIBRA knockout (KO) model cells generated by a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that depleting KIBRA levels compromised the DNA repair functions in cancer cells upon inducing DNA damage. All these phenotypic events were reversed upon reconstitution of KIBRA into cells lacking KIBRA knock-in (KI) model cells. All these results point to the fact that phosphorylated KIBRA might be functioning as a scaffolding protein/adaptor protein facilitating the platform for further recruitment of other DNA damage response factors. In summary, these data demonstrate the imperative functional role of KIBRAper se(KIBRA phosphorylation at T1006 site as a molecular switch that regulates the DNA damage response, possibly via the nonhomologous end joining [NHEJ] pathway), suggesting that KIBRA could be a potential therapeutic target for modulating chemoresistance in cancer cells.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Madras (IITM), Chennai, India.
authors.forename: Jayadev
authors.initials: J
authors.lastname: Mavuluri
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Madras (IITM), Chennai, India.
authors.forename: Swarnalatha
authors.initials: S
authors.lastname: Beesetti
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Madras (IITM), Chennai, India Departments of Human Genetics, Sri Ramachandra University, Porur, Chennai, India.
authors.forename: Rohan
authors.initials: R
authors.lastname: Surabhi
authors.affiliation: Department of Nephrology, Hypertension and Rheumatology, University Hospital Muenster, Muenster, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Kremerskothen
authors.affiliation: Departments of Human Genetics, Sri Ramachandra University, Porur, Chennai, India ganeshv@sriramachandra.edu.in rayala@iitm.ac.in.
authors.forename: Ganesh
authors.initials: G
authors.lastname: Venkatraman
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Madras (IITM), Chennai, India ganeshv@sriramachandra.edu.in rayala@iitm.ac.in.
authors.forename: Suresh K
authors.initials: SK
authors.lastname: Rayala
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics / Q000378:metabolism; D001761:Bleomycin / Q000494:pharmacology; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage / Q000502:physiology; D004260:DNA Repair / Q000502:physiology*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D005260:Female; D055786:Gene Knockout Techniques; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; C513637:WWC1 protein, human; D001761:Bleomycin; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins