title: HIPK2-T566 autophosphorylation diversely contributes to UV- and doxorubicin-induced HIPK2 activation.
abstract: HIPK2 is a Y-regulated S/T kinase involved in various cellular processes, including cell-fate decision during development and DNA damage response. Cis-autophosphorylation in the activation-loop and trans-autophosphorylation at several S/T sites along the protein are required for HIPK2 activation, subcellular localization, and subsequent posttranslational modifications. The specific function of a few of these autophosphorylations has been recently clarified; however, most of the sites found phosphorylated by mass spectrometry in human and/or mouse HIPK2 are still uncharacterized. In the process of studying HIPK2 in human colorectal cancers, we identified a mutation (T566P) in a site we previously found autophosphorylated in mouse Hipk2. Biochemical and functional characterization of this site showed that compared to wild type (wt) HIPK2, HIPK2-T566P maintains nuclear-speckle localization and has only a mild reduction in kinase and growth arresting activities upon overexpression. Next, we assessed cell response following UV-irradiation or treatment with doxorubicin, two well-known HIPK2 activators, by evaluating cell number and viability, p53-Ser46 phosphorylation, p21 induction, and caspase cleavage. Interestingly, cells expressing HIPK2-T566P mutant did not respond to UV-irradiation, while behaved similarly to wt HIPK2 upon doxorubicin-treatment. Evaluation of HIPK2-T566 phosphorylation status by a T566-phospho-specific antibody showed constitutive phosphorylation in unstressed cells, which was maintained after doxorubicin-treatment but inhibited by UV-irradiation. Taken together, these data show that HIPK2-T566 phosphorylation contributes to UV-induced HIPK2 activity but it is dispensable for doxorubicin response.
authors.affiliation: Unit of Cellular Networks and Molecular Therapeutic Targets, Department of Research, Advanced Diagnostics, and Technological Innovation, Regina Elena National Cancer Institute - IRCCS, Rome, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Verdina
authors.affiliation: Unit of Cellular Networks and Molecular Therapeutic Targets, Department of Research, Advanced Diagnostics, and Technological Innovation, Regina Elena National Cancer Institute - IRCCS, Rome, Italy.
authors.forename: Giuliana
authors.initials: G
authors.lastname: Di Rocco
authors.affiliation: Unit of Cellular Networks and Molecular Therapeutic Targets, Department of Research, Advanced Diagnostics, and Technological Innovation, Regina Elena National Cancer Institute - IRCCS, Rome, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Virdia
authors.affiliation: Unit of Cellular Networks and Molecular Therapeutic Targets, Department of Research, Advanced Diagnostics, and Technological Innovation, Regina Elena National Cancer Institute - IRCCS, Rome, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Monteonofrio
authors.affiliation: Unit of Cellular Networks and Molecular Therapeutic Targets, Department of Research, Advanced Diagnostics, and Technological Innovation, Regina Elena National Cancer Institute - IRCCS, Rome, Italy.|Present address: Istituto di Biologia Cellulare e Neurobiologia, CNR, Monterotondo Scalo, Rome, Italy.
authors.forename: Veronica
authors.initials: V
authors.lastname: Gatti
authors.affiliation: Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Policicchio
authors.affiliation: Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bruselles
authors.affiliation: Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù - IRCCS, Rome, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Tartaglia
authors.affiliation: Unit of Cellular Networks and Molecular Therapeutic Targets, Department of Research, Advanced Diagnostics, and Technological Innovation, Regina Elena National Cancer Institute - IRCCS, Rome, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Soddu
mesh_terms: D000818:Animals; D001859:Bone Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D051379:Mice; D012516:Osteosarcoma / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014466:Ultraviolet Rays
chemical_list: D002352:Carrier Proteins; D004317:Doxorubicin; C115114:HIPK2 protein, human; C492003:Hipk2 protein, mouse; D017346:Protein Serine-Threonine Kinases
keywords: DNA-damage response; HIPK2; cancer stem cells; phosphorylation