Homeodomain-interacting protein kinase 2 (HIPK2) is a Ser/Thr kinase controlling cell proliferation and survival, whose investigation has been hampered by the lack of specific inhibitors able to dissect its cellular functions. SB203580, a p38 MAP kinase inhibitor, has been used as a tool to inhibit HIPK2 in cells, but here we show that its efficacy as HIPK2 inhibitor is negligible (IC50>40 μM). In contrast by altering the scaffold of the promiscuous CK2 inhibitor TBI a new class of HIPK2 inhibitors has been generated. One of these, TBID, displays toward HIPK2 unprecedented efficacy (IC50 = 0.33 μM) and selectivity (Gini coefficient 0.592 out of a panel of 76 kinases). The two other members of the HIPK family, HIPK1 and HIPK3, are also inhibited by TBID albeit less efficiently than HIPK2. The mode of action of TBID is competitive with respect to ATP, consistent with modelling. We also provide evidence that TBID is cell permeable by showing that HIPK2 activity is reduced in cells treated with TBID, although with an IC50 two orders of magnitude higher (about 50 μM) than in vitro.
References
[1]
Sombroek D, Hofmann TG (2009) How cells switch HIPK2 on and off. Cell Death Differ 16: 187–194. doi: 10.1038/cdd.2008.154
[2]
Aikawa Y, Nguyen LA, Isono K, Takakura N, Tagata Y, et al. (2006) Roles of HIPK1 and HIPK2 in AML1- and p300-dependent transcription, hematopoiesis and blood vessel formation. EMBO J 25: 3955–3965. doi: 10.1038/sj.emboj.7601273
[3]
D'Orazi G, Cecchinelli B, Bruno T, Manni I, Higashimoto Y, et al. (2002) Homeodomain-interacting protein kinase-2 phosphorylates p53 at Ser 46 and mediates apoptosis. Nat Cell Biol 4: 11–19. doi: 10.1038/ncb714
[4]
Mayo LD, Seo YR, Jackson MW, Smith ML, Rivera Guzman J, et al. (2005) Phosphorylation of human p53 at serine 46 determines promoter selection and whether apoptosis is attenuated or amplified. J Biol Chem 280: 25953–25959. doi: 10.1074/jbc.m503026200
[5]
Zhang Q, Yoshimatsu Y, Hildebrand J, Frisch SM, Goodman RH (2003) Homeodomain interacting protein kinase 2 promotes apoptosis by downregulating the transcriptional corepressor CtBP. Cell 115: 177–186. doi: 10.1016/s0092-8674(03)00802-x
[6]
Hofmann TG, Stollberg N, Schmitz ML, Will H (2003) HIPK2 regulates transforming growth factor-beta-induced c-Jun NH(2)-terminal kinase activation and apoptosis in human hepatoma cells. Cancer Res 63: 8271–8277.
[7]
D'Orazi G, Rinaldo C, Soddu S (2012) Updates on HIPK2: a resourceful oncosuppressor for clearing cancer. J Exp Clin Cancer Res 31: 63. doi: 10.1186/1756-9966-31-63
[8]
Bain J, McLauchlan H, Elliott M, Cohen P (2003) The specificities of protein kinase inhibitors: an update. Biochem J 371: 199–204. doi: 10.1042/bj20021535
[9]
Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, et al. (2007) The selectivity of protein kinase inhibitors: a further update. Biochem J 408: 297–315. doi: 10.1042/bj20070797
[10]
Di Stefano V, Rinaldo C, Sacchi A, Soddu S, D'Orazi G (2004) Homeodomain-interacting protein kinase-2 activity and p53 phosphorylation are critical events for cisplatin-mediated apoptosis. Exp Cell Res 293: 311–320. doi: 10.1016/j.yexcr.2003.09.032
[11]
Roscic A, Moller A, Calzado MA, Renner F, Wimmer VC, et al. (2006) Phosphorylation-dependent control of Pc2 SUMO E3 ligase activity by its substrate protein HIPK2. Mol Cell 24: 77–89. doi: 10.1016/j.molcel.2006.08.004
[12]
Yamada D, Perez-Torrado R, Filion G, Caly M, Jammart B, et al. (2009) The human protein kinase HIPK2 phosphorylates and downregulates the methyl-binding transcription factor ZBTB4. Oncogene 28: 2535–2544. doi: 10.1038/onc.2009.109
[13]
Anastassiadis T, Deacon SW, Devarajan K, Ma H, Peterson JR (2011) Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol 29: 1039–1045. doi: 10.1038/nbt.2017
[14]
Meggio F, Pinna LA (2003) One-thousand-and-one substrates of protein kinase CK2? FASEB J 17: 349–368. doi: 10.1096/fj.02-0473rev
[15]
Salvi M, Sarno S, Cesaro L, Nakamura H, Pinna LA (2009) Extraordinary pleiotropy of protein kinase CK2 revealed by weblogo phosphoproteome analysis. Biochim Biophys Acta 1793: 847–859. doi: 10.1016/j.bbamcr.2009.01.013
[16]
Ahmed K, Issinger OG, Niefind K (2011) Protein kinase CK2: a catalyst for biology, medicine and structural biochemistry. Mol Cell Biochem 356: 1–3. doi: 10.1007/s11010-011-0940-0
[17]
Ruzzene M, Pinna LA (2010) Addiction to protein kinase CK2: a common denominator of diverse cancer cells? Biochim Biophys Acta 1804: 499–504. doi: 10.1016/j.bbapap.2009.07.018
[18]
Ruzzene M, Tosoni K, Zanin S, Cesaro L, Pinna LA (2011) Protein kinase CK2 accumulation in “oncophilic” cells: causes and effects. Mol Cell Biochem 356: 5–10. doi: 10.1007/s11010-011-0959-2
[19]
Pagano MA, Bain J, Kazimierczuk Z, Sarno S, Ruzzene M, et al. (2008) The selectivity of inhibitors of protein kinase CK2: an update. Biochem J 415: 353–365. doi: 10.1042/bj20080309
[20]
Sarno S, Papinutto E, Franchin C, Bain J, Elliott M, et al. (2011) ATP site-directed inhibitors of protein kinase CK2: an update. Curr Top Med Chem 11: 1340–1351. doi: 10.2174/156802611795589638
[21]
Egert-Schmidt AM, Dreher J, Dunkel U, Kohfeld S, Preu L, et al. (2010) Identification of 2-anilino-9-methoxy-5,7-dihydro-6H-pyrim？ido[5,4-d][1]benzazepin-6-onesas dual PLK1/VEGF-R2 kinase inhibitor chemotypes by structure-based lead generation. J Med Chem 53: 2433–2442. doi: 10.1021/jm901388c
[22]
Brandt W, Mologni L, Preu L, Lemcke T, Gambacorti-Passerini C, et al. (2010) Inhibitors of the RET tyrosine kinase based on a 2-(alkylsulfanyl)-4-(3-thienyl)nicotinon？itrilescaffold. Eur J Med Chem 45: 2919–2927. doi: 10.1016/j.ejmech.2010.03.017
[23]
Meggio F, Deana AD, Pinna LA (1981) Endogenous phosphate acceptor proteins for rat liver cytosolic casein kinases. J Biol Chem 256: 11958–11961.
[24]
Ferrari S, Marin O, Pagano MA, Meggio F, Hess D, et al. (2005) Aurora-A site specificity: a study with synthetic peptide substrates. Biochem J 390: 293–302. doi: 10.1042/bj20050343
[25]
Graczyk PP (2007) Gini coefficient: a new way to express selectivity of kinase inhibitors against a family of kinases. J Med Chem 50: 5773–5779. doi: 10.1021/jm070562u
[26]
Molecular Operating Environment (MOE 2009.10) CCG, Inc, 1255 University St., Suite 1600, Montreal, Quebec, Canada, H3B 3X3.
[27]
Cozza G, Gianoncelli A, Montopoli M, Caparrotta L, Venerando A, et al. (2008) Identification of novel protein kinase CK1 delta (CK1delta) inhibitors through structure-based virtual screening. Bioorg Med Chem Lett 18: 5672–5675. doi: 10.1016/j.bmcl.2008.08.072
[28]
Jones G, Willett P, Glen RC, Leach AR, Taylor R (1997) Development and validation of a genetic algorithm for flexible docking. J Mol Biol 267: 727–748. doi: 10.1006/jmbi.1996.0897
[29]
Di Maira G, Salvi M, Arrigoni G, Marin O, Sarno S, et al. (2005) Protein kinase CK2 phosphorylates and upregulates Akt/PKB. Cell Death Differ 12: 668–677. doi: 10.1038/sj.cdd.4401604
[30]
Ruzzene M, Penzo D, Pinna LA (2002) Protein kinase CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) induces apoptosis and caspase-dependent degradation of haematopoietic lineage cell-specific protein 1 (HS1) in Jurkat cells. Biochem J 364: 41–47.
[31]
Battistutta R, De Moliner E, Sarno S, Zanotti G, Pinna LA (2001) Structural features underlying selective inhibition of protein kinase CK2 by ATP site-directed tetrabromo-2-benzotriazole. Protein Sci 10: 2200–2206. doi: 10.1110/ps.19601
[32]
Battistutta R, Mazzorana M, Cendron L, Bortolato A, Sarno S, et al. (2007) The ATP-binding site of protein kinase CK2 holds a positive electrostatic area and conserved water molecules. Chembiochem 8: 1804–1809. doi: 10.1002/cbic.200700307
[33]
Pratt DS, Young CO (1918) Phthalic acid derivatives: constitutions and color, XIV. Some derivatives of tetrabromophthalimide. J Am Chem Soc 40: 1415–1425. doi: 10.1021/ja02242a008
[34]
Ruzzene M, Di Maira G, Tosoni K, Pinna LA (2010) Assessment of CK2 constitutive activity in cancer cells. Methods Enzymol 484: 495–514. doi: 10.1016/b978-0-12-381298-8.00024-1
[35]
Zanin S, Borgo C, Girardi C, O'Brien SE, Miyata Y, et al. (2012) Effects of the CK2 inhibitors CX-4945 and CX-5011 on drug-resistant cells. PLoS One 7: e49193. doi: 10.1371/journal.pone.0049193
[36]
Cozza G, Sarno S, Ruzzene M, Girardi C, Orzeszko A, et al. (2013) Exploiting the repertoire of CK2 inhibitors to target DYRK and PIM kinases. Biochim Biophys Acta 1834: 1402–1409. doi: 10.1016/j.bbapap.2013.01.018
[37]
Saul VV, de la Vega L, Milanovic M, Kruger M, Braun T, et al. (2013) HIPK2 kinase activity depends on cis-autophosphorylation of its activation loop. J Mol Cell Biol 5: 27–38. doi: 10.1093/jmcb/mjs053
[38]
Siepi F, Gatti V, Camerini S, Crescenzi M, Soddu S (2013) HIPK2 catalytic activity and subcellular localization are regulated by activation-loop Y354 autophosphorylation. Biochim Biophys Acta 1833: 1443–1453. doi: 10.1016/j.bbamcr.2013.02.018
[39]
Hofmann TG, Moller A, Sirma H, Zentgraf H, Taya Y, et al. (2002) Regulation of p53 activity by its interaction with homeodomain-interacting protein kinase-2. Nat Cell Biol 4: 1–10. doi: 10.1038/ncb715
[40]
Taira N, Nihira K, Yamaguchi T, Miki Y, Yoshida K (2007) DYRK2 is targeted to the nucleus and controls p53 via Ser46 phosphorylation in the apoptotic response to DNA damage. Mol Cell 25: 725–738. doi: 10.1016/j.molcel.2007.02.007
[41]
Yoshida K, Liu H, Miki Y (2006) Protein kinase C delta regulates Ser46 phosphorylation of p53 tumor suppressor in the apoptotic response to DNA damage. J Biol Chem 281: 5734–5740. doi: 10.1074/jbc.m512074200
[42]
Davies SP, Reddy H, Caivano M, Cohen P (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95–105. doi: 10.1042/0264-6021:3510095
[43]
Sarno S, Mazzorana M, Traynor R, Ruzzene M, Cozza G, et al. (2012) Structural features underlying the selectivity of the kinase inhibitors NBC and dNBC: role of a nitro group that discriminates between CK2 and DYRK1A. Cell Mol Life Sci 69: 449–460. doi: 10.1007/s00018-011-0758-7
