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

Alessandra Verdina, Giuliana Di Rocco, Ilaria Virdia, Laura Monteonofrio, Veronica Gatti, Eleonora Policicchio, Alessandro Bruselles, Marco Tartaglia, Silvia Soddu

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)16744-16754
Number of pages11
JournalOncotarget
Volume8
Issue number10
DOIs
Publication statusPublished - Jan 1 2017

Keywords

  • Cancer stem cells
  • DNA-damage response
  • HIPK2
  • Phosphorylation

ASJC Scopus subject areas

  • Oncology

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