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

TY - JOUR

T1 - HIPK2-T566 autophosphorylation diversely contributes to UV- and doxorubicin-induced HIPK2 activation

AU - Verdina, A.

AU - Di Rocco, G.

AU - Virdia, I.

AU - Monteonofrio, L.

AU - Gatti, V.

AU - Policicchio, E.

AU - Bruselles, A.

AU - Tartaglia, M.

AU - Soddu, S.

N1 - Cited By :1 Export Date: 6 April 2018 Correspondence Address: Soddu, S.; Unit of Cellular Networks and Molecular Therapeutic Targets, Department of Research, Advanced Diagnostics, and Technological Innovation, Regina Elena National Cancer Institute - IRCCSItaly; email: silvia.soddu@ifo.gov.it Chemicals/CAS: caspase, 186322-81-6; doxorubicin, 23214-92-8, 25316-40-9; phosphotransferase, 9031-09-8, 9031-44-1; proline, 147-85-3, 7005-20-1; protein p21, 85306-28-1; serine, 56-45-1, 6898-95-9; threonine, 36676-50-3, 72-19-5; carrier protein, 80700-39-6; protein serine threonine kinase; Carrier Proteins; Doxorubicin; HIPK2 protein, human; Hipk2 protein, mouse; Protein-Serine-Threonine Kinases Tradenames: adriamycin References: Calzado, M.A., De La Vega, L., Munoz, E., Schmitz, M.L., From top to bottom: the two faces of HIPK2 for regulation of the hypoxic response (2009) Cell Cycle, 8, pp. 1659-1664; D'Orazi, G., Rinaldo, C., Soddu, S., Updates on HIPK2: a resourceful oncosuppressor for clearing cancer (2012) J Exp Clin Cancer Res, 31, pp. 1-8; 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PY - 2017

Y1 - 2017

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

KW - caspase

KW - doxorubicin

KW - homeodomain interacting protein kinase 2

KW - mutant protein

KW - phosphotransferase

KW - proline

KW - protein p21

KW - protein p53

KW - serine

KW - threonine

KW - carrier protein

KW - HIPK2 protein, human

KW - Hipk2 protein, mouse

KW - protein serine threonine kinase

KW - antibody specificity

KW - Article

KW - autophosphorylation

KW - cancer inhibition

KW - cell count

KW - cell nucleus

KW - cell viability

KW - cellular distribution

KW - cellular stress response

KW - colorectal cancer

KW - controlled study

KW - enzyme activation

KW - enzyme active site

KW - enzyme activity

KW - enzyme analysis

KW - enzyme localization

KW - enzyme phosphorylation

KW - human

KW - human cell

KW - mutation

KW - protein cleavage

KW - protein expression

KW - radiation response

KW - ultraviolet radiation

KW - animal

KW - Bone Neoplasms

KW - drug effects

KW - enzymology

KW - genetic transfection

KW - genetics

KW - metabolism

KW - mouse

KW - osteosarcoma

KW - phosphorylation

KW - tumor cell line

KW - Animals

KW - Carrier Proteins

KW - Cell Line, Tumor

KW - Doxorubicin

KW - Enzyme Activation

KW - Humans

KW - Mice

KW - Osteosarcoma

KW - Protein-Serine-Threonine Kinases

KW - Transfection

KW - Ultraviolet Rays

M3 - Article

VL - 8

SP - 16744

EP - 16754

JO - Oncotarget

JF - Oncotarget

SN - 1949-2553

IS - 10

ER -