HIPK2-T566 autophosphorylation diversely contributes to UV- and doxorubicin-induced HIPK2 activation
Metrics: PDF 694 views | HTML 1615 views | ?
Alessandra Verdina1,*, Giuliana Di Rocco1,*, Ilaria Virdia1, Laura Monteonofrio1, Veronica Gatti1,4, Eleonora Policicchio2, Alessandro Bruselles2, Marco Tartaglia3, Silvia Soddu1
1Unit of Cellular Networks and Molecular Therapeutic Targets, Department of Research, Advanced Diagnostics, and Technological Innovation, Regina Elena National Cancer Institute - IRCCS, Rome, Italy
2Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy
3Genetics and Rare Diseases Research Division, Ospedale Pediatrico Bambino Gesù – IRCCS, Rome, Italy
4Present address: Istituto di Biologia Cellulare e Neurobiologia, CNR, Monterotondo Scalo, Rome, Italy
*These authors have contributed equally to this work
Silvia Soddu, email: email@example.com
Keywords: HIPK2, phosphorylation, DNA-damage response, cancer stem cells
Received: October 04, 2016 Accepted: December 15, 2016 Published: January 02, 2017
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.
All site content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 License.