HIPK2-T566 autophosphorylation diversely contributes to UV- and doxorubicin-induced HIPK2 activation

A. Verdina, G. Di Rocco, I. Virdia, L. Monteonofrio, V. Gatti, E. Policicchio, A. Bruselles, M. Tartaglia, S. Soddu

Research output: Contribution to journalArticlepeer-review


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.
Original languageEnglish
Pages (from-to)16744-16754
Number of pages11
Issue number10
Publication statusPublished - 2017


  • Cancer stem cells
  • DNA-damage response
  • HIPK2
  • Phosphorylation
  • caspase
  • doxorubicin
  • homeodomain interacting protein kinase 2
  • mutant protein
  • phosphotransferase
  • proline
  • protein p21
  • protein p53
  • serine
  • threonine
  • carrier protein
  • HIPK2 protein, human
  • Hipk2 protein, mouse
  • protein serine threonine kinase
  • antibody specificity
  • Article
  • autophosphorylation
  • cancer inhibition
  • cell count
  • cell nucleus
  • cell viability
  • cellular distribution
  • cellular stress response
  • colorectal cancer
  • controlled study
  • enzyme activation
  • enzyme active site
  • enzyme activity
  • enzyme analysis
  • enzyme localization
  • enzyme phosphorylation
  • human
  • human cell
  • mutation
  • protein cleavage
  • protein expression
  • radiation response
  • ultraviolet radiation
  • animal
  • Bone Neoplasms
  • drug effects
  • enzymology
  • genetic transfection
  • genetics
  • metabolism
  • mouse
  • osteosarcoma
  • phosphorylation
  • tumor cell line
  • Animals
  • Carrier Proteins
  • Cell Line, Tumor
  • Doxorubicin
  • Enzyme Activation
  • Humans
  • Mice
  • Osteosarcoma
  • Protein-Serine-Threonine Kinases
  • Transfection
  • Ultraviolet Rays


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