TY - JOUR
T1 - HIPK2-T566 autophosphorylation diversely contributes to UV- and doxorubicin-induced HIPK2 activation
AU - Verdina, Alessandra
AU - Di Rocco, Giuliana
AU - Virdia, Ilaria
AU - Monteonofrio, Laura
AU - Gatti, Veronica
AU - Policicchio, Eleonora
AU - Bruselles, Alessandro
AU - Tartaglia, Marco
AU - Soddu, Silvia
PY - 2017/1/1
Y1 - 2017/1/1
N2 - 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.
AB - 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.
KW - Cancer stem cells
KW - DNA-damage response
KW - HIPK2
KW - Phosphorylation
UR - http://www.scopus.com/inward/record.url?scp=85014703917&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85014703917&partnerID=8YFLogxK
U2 - 10.18632/oncotarget.14421
DO - 10.18632/oncotarget.14421
M3 - Article
C2 - 28060750
AN - SCOPUS:85014703917
VL - 8
SP - 16744
EP - 16754
JO - Oncotarget
JF - Oncotarget
SN - 1949-2553
IS - 10
ER -