Differential phosphorylation determines the repressor and activator potencies of GLI1 proteins and their efficiency in modulating the HPV life cycle

Autoři: Alla Piirsoo aff001;  Anne Pink aff002;  Lagle Kasak aff002;  Martin Kala aff001;  Sergo Kasvandik aff001;  Mart Ustav aff001;  Marko Piirsoo aff001
Působiště autorů: Institute of Technology, University of Tartu, Tartu, Estonia aff001;  Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia aff002
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225775


The Sonic Hedgehog (Shh) signalling pathway plays multiple roles during embryonic development and under pathological conditions. Although the core components of the Shh pathway are conserved, the regulation of signal transduction varies significantly among species and cell types. Protein kinases Ulk3 and Pka are involved in the Shh pathway as modulators of the activities of Gli transcription factors, which are the nuclear mediators of the signal. Here, we investigate the regulation and activities of two GLI1 isoforms, full-length GLI1 (GLI1FL) and GLI1ΔN. The latter protein lacks the first 128 amino acids including the conserved phosphorylation cluster and the binding motif for SUFU, the key regulator of GLI activity. Both GLI1 isoforms are co-expressed in all human cell lines analysed and possess similar DNA binding activity. ULK3 potentiates the transcriptional activity of both GLI1 proteins, whereas PKA inhibits the activity of GLI1ΔN, but not GLI1FL. In addition to its well-established role as a transcriptional activator, GLI1FL acts as a repressor by inhibiting transcription from the early promoters of human papillomavirus type 18 (HPV18). Additionally, compared to GLI1ΔN, GLI1FL is a more potent suppressor of replication of several HPV types. Altogether, our data show that the N-terminal part of GLI1FL is crucial for the realization of its full potential as a transcriptional regulator.

Klíčová slova:

Gene expression – HPV-11 – Human papillomavirus – Phosphorylation – Transcription factors – Transcriptional control – Viral replication – Transcription activators


1. Kong JH, Siebold C, Rohatgi R. Biochemical mechanisms of vertebrate hedgehog signaling. Development. 2019; doi: 10.1242/dev.166892 31092502

2. Dennler S, André J, Alexaki I, Li A, Magnaldo T, Ten Dijke P, et al. Induction of sonic hedgehog mediators by transforming growth factor-β: Smad3-dependent activation of Gli2 and Gli1 expression in vitro and in vivo. Cancer Res. 2007; doi: 10.1158/0008-5472.CAN-07-0491 17638910

3. Dennler S, André J, Verrechia F, Mauviel A. Cloning of the human GLI2 promoter: Transcriptional activation by transforming growth factor-β via SMAD3/β-catenin cooperation. J Biol Chem. 2009; doi: 10.1074/jbc.M109.059964 19797115

4. Lo HW, Zhu H, Cao X, Aldrich A, Ali-Osman F. A novel splice variant of GLI1 that promotes glioblastoma cell migration and invasion. Cancer Res. 2009; doi: 10.1158/0008-5472.CAN-09-0886 19706761

5. Cao X, Geradts J, Dewhirst MW, Lo HW. Upregulation of VEGF-A and CD24 gene expression by the tGLI1 transcription factor contributes to the aggressive behavior of breast cancer cells. Oncogene. 2012; doi: 10.1038/onc.2011.219 21666711

6. Shimokawa T, Tostar U, Lauth M, Palaniswamy R, Kasper M, Toftgård R, et al. Novel human glioma-associated oncogene 1 (GLI1) splice variants reveal distinct mechanisms in the terminal transduction of the hedgehog signal. J Biol Chem. 2008; doi: 10.1074/jbc.M800299200 18378682

7. Niewiadomski P, Kong JH, Ahrends R, Ma Y, Humke EW, Khan S, et al. Gli protein activity is controlled by multisite phosphorylation in vertebrate hedgehog signaling. Cell Rep. 2014;6: 168–181. doi: 10.1016/j.celrep.2013.12.003 24373970

8. Niewiadomski P, Niedziółka SM, Markiewicz Ł, Uśpieński T, Baran B, Chojnowska K. Gli Proteins: Regulation in Development and Cancer. Cells. 2019; doi: 10.3390/cells8020147 30754706

9. Maloverjan A, Piirsoo M, Michelson P, Kogerman P, Østerlund T. Identification of a novel serine/threonine kinase ULK3 as a positive regulator of Hedgehog pathway. Exp Cell Res. 2010;316: 627–637. doi: 10.1016/j.yexcr.2009.10.018 19878745

10. Maloverjan A, Piirsoo M, Kasak L, Peil L, Østerlund T, Kogerman P. Dual function of UNC-51-like kinase 3 (Ulk3) in the Sonic hedgehog signaling pathway. J Biol Chem. 2010;285: 30079–90. doi: 10.1074/jbc.M110.133991 20643644

11. Reinson T, Toots M, Kadaja M, Pipitch R, Allik M, Ustav E, et al. Engagement of the ATR-Dependent DNA Damage Response at the Human Papillomavirus 18 Replication Centers during the Initial Amplification. J Virol. 2013; doi: 10.1128/JVI.01943-12 23135710

12. Orav M, Henno L, Isok-Paas H, Geimanen J, Ustav M, Ustav E. Recombination-Dependent Oligomerization of Human Papillomavirus Genomes upon Transient DNA Replication. J Virol. 2013; doi: 10.1128/JVI.01798-13 23986589

13. Sankovski E, Mannik A, Geimanen J, Ustav E, Ustav M. Mapping of Betapapillomavirus Human Papillomavirus 5 Transcription and Characterization of Viral-Genome Replication Function. J Virol. 2014; doi: 10.1128/JVI.01841-13 24198410

14. Piirsoo A, Piirsoo M, Kala M, Sankovski E, Lototskaja E, Levin V, et al. Activity of CK2α protein kinase is required for efficient replication of some HPV types. PLOS Pathog. 2019; doi: 10.1371/journal.ppat.1007788 31091289

15. Kogerman P, Grimm T, Kogerman L, Krause D, Undén AB, Sandstedt B, et al. Mammalian Suppressor-of-Fused modulates nuclear-cytoplasmic shuttling of GLI-1. Nat Cell Biol. 1999; doi: 10.1038/13031 10559945

16. Sasaki H, Hui C, Nakafuku M, Kondoh H. A binding site for Gli proteins is essential for HNF-3beta floor plate enhancer activity in transgenics and can respond to Shh in vitro. Development. 1997;

17. Geimanen J, Isok-Paas H, Pipitch R, Salk K, Laos T, Orav M, et al. Development of a Cellular Assay System To Study the Genome Replication of High- and Low-Risk Mucosal and Cutaneous Human Papillomaviruses. J Virol. 2011; doi: 10.1128/JVI.01985-10 21248030

18. Toots M, MU Jr., Männik A, Mumm K, Tämm K, Tamm T, et al. Identification of several high-risk HPV inhibitors and drug targets with a novel high-throughput screening assay. PLOS Pathog. 2017; doi: 10.1371/JOURNAL.PPAT.1006168 28182794

19. Meijer D, Graus A, Grosveld G. Mapping the transactivation domain of the Oct-6 POU transcription factor. Nucleic Acids Res. 1992; doi: 10.1093/nar/20.9.2241 1594443

20. Sheng T, Chi S, Zhang X, Xie J. Regulation of Gli1 localization by the cAMP/protein kinase A signaling axis through a site near the nuclear localization signal. J Biol Chem. 2006; doi: 10.1074/jbc.C500300200 16293631

21. Vishnoi K, Mahata S, Tyagi A, Pandey A, Verma G, Jadli M, et al. Cross-talk between Human Papillomavirus Oncoproteins and Hedgehog Signaling Synergistically Promotes Stemness in Cervical Cancer Cells. Sci Rep. 2016; doi: 10.1038/srep34377 27678330

22. Kinzler KW, Vogelstein B. The GLI gene encodes a nuclear protein which binds specific sequences in the human genome. Mol Cell Biol. 1990;

23. Winklmayr M, Schmid C, Laner-Plamberger S, Kaser A, Aberger F, Eichberger T, et al. Non-consensus GLI binding sites in Hedgehog target gene regulation. BMC Mol Biol. 2010; doi: 10.1186/1471-2199-11-2 20070907

24. Yoon JW, Kita Y, Frank DJ, Majewski RR, Konicek BA, Nobrega MA, et al. Gene expression profiling leads to identification of GLI1-binding elements in target genes and a role for multiple downstream pathways in GLI1-induced cell transformation. J Biol Chem. 2002; doi: 10.1074/jbc.M105708200 11719506

25. Vokes SA, Ji H, McCuine S, Tenzen T, Giles S, Zhong S, et al. Genomic characterization of Gli-activator targets in sonic hedgehog-mediated neural patterning. Development. 2007; doi: 10.1242/dev.001966 17442700

26. Toots M, Männik A, Kivi G, Ustav M, Ustav E, Ustav M. The transcription map of human papillomavirus type 18 during genome replication in U2OS cells. PLoS One. 2014; doi: 10.1371/journal.pone.0116151 25548925

27. Graham S V. Human Papillomavirus E2 Protein: Linking Replication, Transcription, and RNA Processing. J Virol. 2016; doi: 10.1128/jvi.00502-16 27412596

28. Kadaja M, Silla T, Ustav E, Ustav M. Papillomavirus DNA replication—From initiation to genomic instability. Virology. 2009. doi: 10.1016/j.virol.2008.11.032 19141359

29. Kaesler S, Luscher B, Ruther U. Transcriptional activity of GLI1 is negatively regulated by protein kinase A. Biol Chem. 2000; doi: 10.1515/BC.2000.070 10987360

30. Mazzone A, Gibbons SJ, Eisenman ST, Strege PR, Zheng T, D’Amato M, et al. Direct repression of anoctamin 1 (ANO1) gene transcription by Gli proteins. FASEB J. 2019; doi: 10.1096/fj.201802373R 30802137

31. Stecca B, Ruiz I Altaba A. A GLI1-p53 inhibitory loop controls neural stem cell and tumour cell numbers. EMBO J. 2009; doi: 10.1038/emboj.2009.16 19214186

Článek vyšel v časopise


2019 Číslo 11