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

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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.

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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.

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18_2018_2763_MOESM1_ESM.mp4

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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18_2018_2763_MOESM5_ESM.pdf

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)

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Tanghe, G., Urwyler-Rösselet, C., De Groote, P. et al. RIPK4 activity in keratinocytes is controlled by the SCFβ-TrCP ubiquitin ligase to maintain cortical actin organization. Cell. Mol. Life Sci. 75, 2827–2841 (2018). https://doi.org/10.1007/s00018-018-2763-6

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