RIPK4 activity in keratinocytes is controlled by the SCFβ-TrCP ubiquitin ligase to maintain cortical actin organization

  • Giel Tanghe
  • Corinne Urwyler-Rösselet
  • Philippe De Groote
  • Emmanuel Dejardin
  • Pieter-Jan De Bock
  • Kris Gevaert
  • Peter Vandenabeele
  • Wim Declercq
Original Article

Abstract

RIPK4 is a key player in epidermal differentiation and barrier formation. RIPK4 signaling pathways controlling keratinocyte proliferation and differentiation depend on its kinase activity leading to Dvl2, Pkp1 and IRF6 phosphorylation and NF-κB activation. However, the mechanism regulating RIPK4 activity levels remains elusive. We show that cultured keratinocytes display constitutive active phosphorylated RIPK4 while PKC signaling can trigger RIPK4 activation in various non-keratinocyte cell lines, in which RIPK4 is present in a non-phosphorylated state. Interestingly, we identified the SCFβ-TrCP ubiquitin E3 ligase complex responsible for regulating the active RIPK4 protein level. The SCFβ-TrCP complex binds to a conserved phosphodegron motif in the intermediate domain of RIPK4, subsequently leading to K48-linked ubiquitinylation and degradation. The recruitment of β-TrCP is dependent on RIPK4 activation and trans-autophosphorylation. β-TrCP knock-down resulted in RIPK4-dependent formation of actin stress fibers, cell scattering and increased cell motility, suggesting that tight control of RIPK4 activity levels is crucial to maintain cell shape and behavior in keratinocytes.

Keywords

RIPK4 β-TrCP Keratinocytes Proteasome Degradation PKC 

Notes

Acknowledgements

This research has been supported by the Flanders Institute for Biotechnology (VIB); Belgian Grants: Interuniversity Attraction Poles, IAP7/32; Stichting tegen Kanker (2010-162 and FAF-F/2016/868); Flemish Grants: FWO-Vlaanderen (G.0544.11) and a Methusalem Grant (BOF09/01M00709) from the Flemish Government to Peter Vandenabeele; a UGent Grant (GOA-01G01914). G.T. received a Ph.D. fellowship from FWO-Vlaanderen and C.U-R obtained a predoctoral Strategic Research fellowship from IWT-Vlaanderen. We also thank the VIB Bioimaging core facility for excellent assistance.

Compliance with ethical standards

Conflict of interest

The authors state no conflict of interest.

Supplementary material

18_2018_2763_MOESM1_ESM.mp4 (21.8 mb)
Inability to degrade RIPK4 in HaCaT keratinocytes results in increased cell motility. adHaCaT cells were reverse transfected with the following siRNAs (20nm): (a) non-targeting (control), (b)RIPK4, (c) β-TrCP1+2, (d) RIPK4 + β-TrCP1+2 and seeded on collagen-coated tissue culture plates.Imaging was started 24 h after transfection for the following 24 h with one image acquisition every 15 minwith an IncuCyte microscope
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Supplementary material 2 (MP4 18124 kb)
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Supplementary material 3 (MP4 20346 kb)
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Supplementary material 4 (MP4 21374 kb)
18_2018_2763_MOESM5_ESM.pdf (478 kb)
Supplementary Fig. 1 Tandem affinity purification of RIPK4 and identification of β-TrCP as binding partner. a-b HEK293T cells were transiently transfected with pCEMM N-TAP(GS)hRIPK4 or empty pCEMM N-TAP(GS) as a negative control. Purified TAP-tagged protein complexes were separated by gel electrophoresis followed by Coomassie staining (a), and subsequently gel fragments were excised for analysis by mass spectrometry (MS) analysis. For each gel fragment a corresponding gel fragment from the negative control purification was analyzed to allow the identification of false positives. Proteins that were identified by at least two peptides and which were absent from the negative control were retained as potential RIPK4 interactors (b). Raw data is available upon request. c Identified peptides in MS/MS analysis from samples in a corresponding to β-TrCP1 and β-TrCP2. Supplementary Fig. 2 Wnt3a stimulation does not induce RIPK4 hyper-phosphorylation. HEK293T cells were cultured for 16 h in growth medium containing 1% serum followed by stimulation with 200 ng/ml recombinant Wnt3a with or without MG132 (10 µM). Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. Simultaneously Wnt3a activity was measured using a Wnt reporter assay. HEK293T cells were transfected with the Wnt reporter one day before stimulation with 200 ng/ml Wnt3a. Lysates were made 24 h hours after stimulation and luciferase activity was measured. Supplementary Fig. 3 Inhibition of potential degron kinases does not affect PMA-induced RIPK4 phosphorylation and degradation. a HEK293T cells were cultured for 16 h in growth medium containing 1% serum followed by 1 h pretreatment with inhibitors for JNK (SP600125), p38 (SB202190), casein kinase I (D4476), casein kinase 2 (CX4945), IKKα and IKKβ (TPCA), GSK3β (CHIR99021) (at 10 µM final concentration) before stimulation with PMA (0.5 µM) with or without MG132 (10 µM). Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. b HEK293T cells were seeded and transfected with non-targeting (control) or with the indicated siRNA (20 nM). Next day the cells refreshed with 1% serum growth medium. 16 h later the cells were stimulated with 0.5 µM PMA with or without MG132 (10 µM) for the indicated time. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. Supplementary Fig. 4 β-TrCP knockdown does not induce RIPK4 mRNA expression levels. a HaCaT cells were transfected with non-targeting (control) or β-TrCP1+2 siRNA (20 nM). RIPK4, β-TrCP1 and β-TrCP2 mRNA levels were analyzed by qPCR analysis. Error bars represent standard error of the means (n = 2). Supplementary Fig. 5 RIPK4 mutagenesis and PKC isoform cloning primers used in this study. Online Resource 1: Inability to degrade RIPK4 in HaCaT keratinocytes results in increased cell motility. HaCaT cells were reverse transfected with non-targeting (control), RIPK4 and/or β-TrCP1+2 siRNA (20 nM) and seeded on collagen-coated tissue culture plates. Imaging was started 24 h after transfection for the following 24 h with one image acquisition every 15 min with a IncuCyte microscope. (PDF 478 kb)

References

  1. 1.
    Meylan E, Tschopp J (2005) The RIP kinases: crucial integrators of cellular stress. Trends Biochem Sci 30(3):151–159.  https://doi.org/10.1016/j.tibs.2005.01.003 CrossRefPubMedGoogle Scholar
  2. 2.
    Lippens S, Hoste E, Vandenabeele P, Agostinis P, Declercq W (2009) Cell death in the skin. Apoptosis Int J Progr Cell Death 14(4):549–569.  https://doi.org/10.1007/s10495-009-0324-z CrossRefGoogle Scholar
  3. 3.
    Kalay E, Sezgin O, Chellappa V, Mutlu M, Morsy H, Kayserili H, Kreiger E, Cansu A, Toraman B, Abdalla EM, Aslan Y, Pillai S, Akarsu NA (2012) Mutations in RIPK4 cause the autosomal-recessive form of popliteal pterygium syndrome. Am J Hum Genet 90(1):76–85.  https://doi.org/10.1016/j.ajhg.2011.11.014 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Mitchell K, O’Sullivan J, Missero C, Blair E, Richardson R, Anderson B, Antonini D, Murray JC, Shanske AL, Schutte BC, Romano RA, Sinha S, Bhaskar SS, Black GC, Dixon J, Dixon MJ (2012) Exome sequence identifies RIPK4 as the Bartsocas-Papas syndrome locus. Am J Hum Genet 90(1):69–75.  https://doi.org/10.1016/j.ajhg.2011.11.013 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    De Groote P, Tran HT, Fransen M, Tanghe G, Urwyler C, De Craene B, Leurs K, Gilbert B, Van Imschoot G, De Rycke R, Guerin CJ, Holland P, Berx G, Vandenabeele P, Lippens S, Vleminckx K, Declercq W (2015) A novel RIPK4–IRF6 connection is required to prevent epithelial fusions characteristic for popliteal pterygium syndromes. Cell Death Differ 22(6):1012–1024.  https://doi.org/10.1038/cdd.2014.191 CrossRefPubMedGoogle Scholar
  6. 6.
    Urwyler-Rosselet C, Tanghe G, Leurs K, Gilbert B, De Rycke R, De Bruyne M, Lippens S, Bartunkova S, De Groote P, Niessen C, Haftek M, Vandenabeele P, Declercq W (2018) Keratinocyte-specific ablation of RIPK4 allows epidermal cornification but impairs skin barrier formation. J Investig Dermatol.  https://doi.org/10.1016/j.jid.2017.12.031 PubMedGoogle Scholar
  7. 7.
    Holland P, Willis C, Kanaly S, Glaccum M, Warren A, Charrier K, Murison J, Derry J, Virca G, Bird T, Peschon J (2002) RIP4 is an ankyrin repeat-containing kinase essential for keratinocyte differentiation. Curr Biol 12(16):1424–1428CrossRefPubMedGoogle Scholar
  8. 8.
    Kwa MQ, Huynh J, Aw J, Zhang L, Nguyen T, Reynolds EC, Sweet MJ, Hamilton JA, Scholz GM (2014) Receptor-interacting protein kinase 4 and interferon regulatory factor 6 function as a signaling axis to regulate keratinocyte differentiation. J Biol Chem 289(45):31077–31087.  https://doi.org/10.1074/jbc.M114.589382 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Lee P, Jiang S, Li Y, Yue J, Gou X, Chen SY, Zhao Y, Schober M, Tan M, Wu X (2017) Phosphorylation of Pkp1 by RIPK4 regulates epidermal differentiation and skin tumorigenesis. EMBO J.  https://doi.org/10.15252/embj.201695679 Google Scholar
  10. 10.
    Bhr C, Rohwer A, Stempka L, Rincke G, Marks F, Gschwendt M (2000) DIK, a novel protein kinase that interacts with protein kinase Cdelta. Cloning, characterization, and gene analysis. J Biol Chem 275(46):36350–36357.  https://doi.org/10.1074/jbc.M004771200 CrossRefPubMedGoogle Scholar
  11. 11.
    Chen L, Haider K, Ponda M, Cariappa A, Rowitch D, Pillai S (2001) Protein kinase C-associated kinase (PKK), a novel membrane-associated, ankyrin repeat-containing protein kinase. J Biol Chem 276(24):21737–21744.  https://doi.org/10.1074/jbc.M008069200 CrossRefPubMedGoogle Scholar
  12. 12.
    Rosse C, Linch M, Kermorgant S, Cameron AJ, Boeckeler K, Parker PJ (2010) PKC and the control of localized signal dynamics. Nat Rev Mol Cell Biol 11(2):103–112.  https://doi.org/10.1038/nrm2847 CrossRefPubMedGoogle Scholar
  13. 13.
    Igumenova TI (2015) Dynamics and membrane interactions of protein kinase C. Biochemistry 54(32):4953–4968.  https://doi.org/10.1021/acs.biochem.5b00565 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Parkinson EK, Emmerson A (1984) Non-promoting hyperplasiogenic agents do not mimic the effects of phorbol, 12-myristate, 13-acetate on terminal differentiation of normal and transformed human keratinocytes. Carcinogenesis 5(5):687–690CrossRefPubMedGoogle Scholar
  15. 15.
    Muto A, Ruland J, McAllister-Lucas LM, Lucas PC, Yamaoka S, Chen FF, Lin A, Mak TW, Nunez G, Inohara N (2002) Protein kinase C-associated kinase (PKK) mediates Bcl10-independent NF-kappa B activation induced by phorbol ester. J Biol Chem 277(35):31871–31876.  https://doi.org/10.1074/jbc.M202222200 CrossRefPubMedGoogle Scholar
  16. 16.
    Kim SW, Oleksyn DW, Rossi RM, Jordan CT, Sanz I, Chen L, Zhao J (2008) Protein kinase C-associated kinase is required for NF-kappaB signaling and survival in diffuse large B-cell lymphoma cells. Blood 111(3):1644–1653.  https://doi.org/10.1182/blood-2007-05-088591 CrossRefPubMedGoogle Scholar
  17. 17.
    Komander D (2009) The emerging complexity of protein ubiquitination. Biochem Soc Trans 37(Pt 5):937–953.  https://doi.org/10.1042/BST0370937 CrossRefPubMedGoogle Scholar
  18. 18.
    Cohen-Kaplan V, Livneh I, Avni N, Cohen-Rosenzweig C, Ciechanover A (2016) The ubiquitin–proteasome system and autophagy: coordinated and independent activities. Int J Biochem Cell Biol 79:403–418.  https://doi.org/10.1016/j.biocel.2016.07.019 CrossRefPubMedGoogle Scholar
  19. 19.
    Kwon YT, Ciechanover A (2017) The ubiquitin code in the ubiquitin–proteasome system and autophagy. Trends Biochem Sci 42(11):873–886.  https://doi.org/10.1016/j.tibs.2017.09.002 CrossRefPubMedGoogle Scholar
  20. 20.
    Zheng N, Zhou Q, Wang Z, Wei W (2016) Recent advances in SCF ubiquitin ligase complex: clinical implications. Biochem Biophys Acta 1866 1:12–22.  https://doi.org/10.1016/j.bbcan.2016.05.001 Google Scholar
  21. 21.
    Skaar JR, Pagan JK, Pagano M (2013) Mechanisms and function of substrate recruitment by F-box proteins. Nat Rev Mol Cell Biol 14(6):369–381.  https://doi.org/10.1038/nrm3582 CrossRefPubMedGoogle Scholar
  22. 22.
    Heim D, Cornils K, Schulze K, Fehse B, Lohse AW, Brummendorf TH, Wege H (2015) Retroviral insertional mutagenesis in telomerase-immortalized hepatocytes identifies RIPK4 as novel tumor suppressor in human hepatocarcinogenesis. Oncogene 34(3):364–372.  https://doi.org/10.1038/onc.2013.551 CrossRefPubMedGoogle Scholar
  23. 23.
    Liu DQ, Li FF, Zhang JB, Zhou TJ, Xue WQ, Zheng XH, Chen YB, Liao XY, Zhang L, Zhang SD, Hu YZ, Jia WH (2015) Increased RIPK4 expression is associated with progression and poor prognosis in cervical squamous cell carcinoma patients. Sci Rep 5:11955.  https://doi.org/10.1038/srep11955 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Kopparam J, Chiffelle J, Angelino P, Piersigilli A, Zangger N, Delorenzi M, Meylan E (2017) RIP4 inhibits STAT3 signaling to sustain lung adenocarcinoma differentiation. Cell Death Differ.  https://doi.org/10.1038/cdd.2017.81 PubMedPubMedCentralGoogle Scholar
  25. 25.
    Rountree RB, Willis CR, Dinh H, Blumberg H, Bailey K, Dean C Jr, Peschon JJ, Holland PM (2010) RIP4 regulates epidermal differentiation and cutaneous inflammation. J Investig Dermatol 130(1):102–112.  https://doi.org/10.1038/jid.2009.223 CrossRefPubMedGoogle Scholar
  26. 26.
    Oleksyn D, Zhao J, Vosoughi A, Zhao JC, Misra R, Pentland AP, Ryan D, Anolik J, Ritchlin C, Looney J, Anandarajah AP, Schwartz G, Calvi LM, Georger M, Mohan C, Sanz I, Chen L (2017) PKK deficiency in B cells prevents lupus development in Sle lupus mice. Immunol Lett 185:1–11.  https://doi.org/10.1016/j.imlet.2017.03.002 CrossRefPubMedGoogle Scholar
  27. 27.
    Yang X, Boehm JS, Yang X, Salehi-Ashtiani K, Hao T, Shen Y, Lubonja R, Thomas SR, Alkan O, Bhimdi T, Green TM, Johannessen CM, Silver SJ, Nguyen C, Murray RR, Hieronymus H, Balcha D, Fan C, Lin C, Ghamsari L, Vidal M, Hahn WC, Hill DE, Root DE (2011) A public genome-scale lentiviral expression library of human ORFs. Nat Methods 8(8):659–661.  https://doi.org/10.1038/nmeth.1638 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):RESEARCH0034CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Staal FJ, van Noort M, Strous GJ, Clevers HC (2002) Wnt signals are transmitted through N-terminally dephosphorylated beta-catenin. EMBO Rep 3(1):63–68.  https://doi.org/10.1093/embo-reports/kvf002 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Low TY, Peng M, Magliozzi R, Mohammed S, Guardavaccaro D, Heck AJ (2014) A systems-wide screen identifies substrates of the SCFbetaTrCP ubiquitin ligase. Sci Signal 7(356):rs8.  https://doi.org/10.1126/scisignal.2005882 CrossRefPubMedGoogle Scholar
  31. 31.
    Wu X, Fukushima H, North BJ, Nagaoka Y, Nagashima K, Deng F, Okabe K, Inuzuka H, Wei W (2014) SCFbeta-TRCP regulates osteoclastogenesis via promoting CYLD ubiquitination. Oncotarget 5(12):4211–4221.  https://doi.org/10.18632/oncotarget.1971 PubMedPubMedCentralGoogle Scholar
  32. 32.
    Magliozzi R, Low TY, Weijts BG, Cheng T, Spanjaard E, Mohammed S, van Veen A, Ovaa H, de Rooij J, Zwartkruis FJ, Bos JL, de Bruin A, Heck AJ, Guardavaccaro D (2013) Control of epithelial cell migration and invasion by the IKKbeta- and CK1alpha-mediated degradation of RAPGEF2. Dev Cell 27(5):574–585.  https://doi.org/10.1016/j.devcel.2013.10.023 CrossRefPubMedGoogle Scholar
  33. 33.
    Hart M, Concordet JP, Lassot I, Albert I, del los Santos R, Durand H, Perret C, Rubinfeld B, Margottin F, Benarous R, Polakis P (1999) The F-box protein beta-TrCP associates with phosphorylated beta-catenin and regulates its activity in the cell. Curr Biol 9(4):207–210CrossRefPubMedGoogle Scholar
  34. 34.
    Zeke A, Misheva M, Remenyi A, Bogoyevitch MA (2016) JNK signaling: regulation and functions based on complex protein–protein partnerships. Microbiol Mol Biol Rev 80(3):793–835.  https://doi.org/10.1128/MMBR.00043-14 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Rojo AI, Medina-Campos ON, Rada P, Zuniga-Toala A, Lopez-Gazcon A, Espada S, Pedraza-Chaverri J, Cuadrado A (2012) Signaling pathways activated by the phytochemical nordihydroguaiaretic acid contribute to a Keap1-independent regulation of Nrf2 stability: role of glycogen synthase kinase-3. Free Radic Biol Med 52(2):473–487.  https://doi.org/10.1016/j.freeradbiomed.2011.11.003 CrossRefPubMedGoogle Scholar
  36. 36.
    Poligone B, Gilmore ES, Alexander CV, Oleksyn D, Gillespie K, Zhao J, Ibrahim SF, Pentland AP, Brown MD, Chen L (2015) PKK suppresses tumor growth and is decreased in squamous cell carcinoma of the skin. J Investig Dermatol 135(3):869–876.  https://doi.org/10.1038/jid.2014.428 CrossRefPubMedGoogle Scholar
  37. 37.
    Simpson CL, Patel DM, Green KJ (2011) Deconstructing the skin: cytoarchitectural determinants of epidermal morphogenesis. Nat Rev Mol Cell Biol 12(9):565–580.  https://doi.org/10.1038/nrm3175 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Meylan E, Martinon F, Thome M, Gschwendt M, Tschopp J (2002) RIP4 (DIK/PKK), a novel member of the RIP kinase family, activates NF-kappa B and is processed during apoptosis. EMBO Rep 3(12):1201–1208.  https://doi.org/10.1093/embo-reports/kvf236 CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Cotter TG, Lennon SV, Glynn JM, Green DR (1992) Microfilament-disrupting agents prevent the formation of apoptotic bodies in tumor cells undergoing apoptosis. Can Res 52(4):997–1005Google Scholar
  40. 40.
    Holland AJ, Lan W, Niessen S, Hoover H, Cleveland DW (2010) Polo-like kinase 4 kinase activity limits centrosome overduplication by autoregulating its own stability. J Cell Biol 188(2):191–198.  https://doi.org/10.1083/jcb.200911102 CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Hornbeck PV, Zhang B, Murray B, Kornhauser JM, Latham V, Skrzypek E (2015) PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res 43:D512–D520.  https://doi.org/10.1093/nar/gku1267 (database issue) CrossRefPubMedGoogle Scholar
  42. 42.
    Razani B, Zarnegar B, Ytterberg AJ, Shiba T, Dempsey PW, Ware CF, Loo JA, Cheng G (2010) Negative feedback in noncanonical NF-kappaB signaling modulates NIK stability through IKKalpha-mediated phosphorylation. Sci Signal 3(123):ra41.  https://doi.org/10.1126/scisignal.2000778 CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Bhatia N, Herter JR, Slaga TJ, Fuchs SY, Spiegelman VS (2002) Mouse homologue of HOS (mHOS) is overexpressed in skin tumors and implicated in constitutive activation of NF-kappaB. Oncogene 21(10):1501–1509.  https://doi.org/10.1038/sj.onc.1205311 CrossRefPubMedGoogle Scholar
  44. 44.
    Chen L, Hayden MS, Gilmore ES, Alexander-Savino C, Oleksyn D, Gillespie K, Zhao J, Poligone B (2017) PKK deletion in basal keratinocytes promotes tumorigenesis after chemical carcinogenesis. Carcinogenesis.  https://doi.org/10.1093/carcin/bgx120 Google Scholar
  45. 45.
    Forbes SA, Beare D, Boutselakis H, Bamford S, Bindal N, Tate J, Cole CG, Ward S, Dawson E, Ponting L, Stefancsik R, Harsha B, Kok CY, Jia M, Jubb H, Sondka Z, Thompson S, De T, Campbell PJ (2017) COSMIC: somatic cancer genetics at high-resolution. Nucleic Acids Res 45(D1):D777–D783.  https://doi.org/10.1093/nar/gkw1121 CrossRefPubMedGoogle Scholar

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© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Molecular Signaling and Cell Death UnitVIB-UGent Center for Inflammation ResearchGentBelgium
  2. 2.Department of Biomedical Molecular BiologyGhent UniversityGhentBelgium
  3. 3.Laboratory of Molecular Immunology and Signal Transduction, GIGA-InstituteUniversity of LiègeLiègeBelgium
  4. 4.VIB-UGent Center for Medical BiotechnologyGhentBelgium
  5. 5.Department of BiochemistryGhent UniversityGhentBelgium
  6. 6.Department of Biology, Institute of Molecular Health SciencesETH ZurichZurichSwitzerland

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