Wu KZL et al. (2019), Pathogenic FAM83G palmoplantar keratoderma muta...

XB-ART-56409

Wellcome Open Res 2019 Jan 01;4:133. doi: 10.12688/wellcomeopenres.15403.2.

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Pathogenic FAM83G palmoplantar keratoderma mutations inhibit the PAWS1:CK1α association and attenuate Wnt signalling.

Wu KZL , Jones RA , Tachie-Menson T , Macartney TJ , Wood NT , Varghese J , Gourlay R , Soares RF , Smith JC , Sapkota GP .

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Background: Two recessive mutations in the FAM83G gene, causing A34E and R52P amino acid substitutions in the DUF1669 domain of the PAWS1 protein, are associated with palmoplantar keratoderma (PPK) in humans and dogs respectively. We have previously reported that PAWS1 associates with the Ser/Thr protein kinase CK1α through the DUF1669 domain to mediate canonical Wnt signalling. Methods: Co-immunoprecipitation was used to investigate possible changes to PAWS1 interactors caused by the mutations. We also compared the stability of wild-type and mutant PAWS1 in cycloheximide-treated cells. Effects on Wnt signalling were determined using the TOPflash luciferase reporter assay in U2OS cells expressing PAWS1 mutant proteins. The ability of PAWS1 to induce axis duplication in Xenopus embryos was also tested. Finally, we knocked-in the A34E mutation at the native gene locus and measured Wnt-induced AXIN2 gene expression by RT-qPCR. Results: We show that these PAWS1 A34E and PAWS1 R52P mutants fail to interact with CK1α but, like the wild-type protein, do interact with CD2AP and SMAD1. Like cells carrying a PAWS1 F296A mutation, which also abolishes CK1α binding, cells carrying the A34E and R52P mutants respond poorly to Wnt signalling to an extent resembling that observed in FAM83G gene knockout cells. Consistent with this observation, these mutants, in contrast to the wild-type protein, fail to induce axis duplication in Xenopus embryos. We also found that the A34E and R52P mutant proteins are less abundant than the native protein and appear to be less stable, both when overexpressed in FAM83G-knockout cells and when knocked-in at the native FAM83G locus. Ala 34 of PAWS1 is conserved in all FAM83 proteins and mutating the equivalent residue in FAM83H (A31E) also abolishes interaction with CK1 isoforms. Conclusions: We propose that mutations in PAWS1 cause PPK pathogenesis through disruption of the CK1α interaction and attenuation of Wnt signalling.

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Species referenced: Xenopus
Genes referenced: axin2 cd2ap csnk1a1 gjb2 isyna1 myc smad1 wnt3a

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	Figure 1. . Pathogenic PPK point-mutations in PAWS1 disrupt its interaction with CK1α. A, B: HEK293 cells transiently expressing PAWS1-GFP and HA-SMAD1 ( A) or myc-CD2AP ( B) were subject to GFP immunoprecipitation (IP) and immunoblotting (IB) for the indicated proteins. C: Using retroviral transduction, PAWS1 or the indicated mutants were stably re-expressed in HaCaT PAWS1 −/− (KO) cells; GFP control, wildtype (WT), A34E (AE), R52P (RP), D262A (DA), F296A (FA). Cell extracts were analysed by IB. D: Immunoprecipitation of PAWS1 was performed from HaCaT cells described in ( C). E– G: PAWS1-GFP or the indicated mutants were stably expressed in PAWS1-KO HaCaT cells as described in ( C), and immunoprecipitated with GFP-Trap beads. GFP IP samples were separated by SDS-PAGE and Coomassie stained ( E). Each lane was cut into 6 pieces and subsequently processed for protein identification by mass spectrometry. Table showing total spectral counts for PAWS1 and CK1α ( F). Input and IP samples were analysed by IB with the indicated antibodies ( G).
	Figure 2. . PAWS1 A34E and PAWS1 R52P proteins have a shorter half-life in cells. A, B: U2OS PAWS1-KO cells transiently expressing PAWS1 WT, PAWS1 A34E, or PAWS1 R52P were treated with 100 µg/ml cycloheximide for the indicated times prior to sample collection. PAWS1 band intensities were measured, normalised to GAPDH loading control, and represented relative to the respective 0 h samples (n=3, error bars represent ± SD). Representative blots are shown in ( B). Two-way ANOVA. C, D: As in ( A, B), but indicated cells were treated with cycloheximide in the presence or absence of bortezomib (5 µM) for 6 h. Fold changes are shown relative to the respective untreated samples. Representative blots are shown in ( D). Multiple t-test. E, F: Cycloheximide chase performed as described in ( A) with PAWS1 WT and PAWS1 S614A. Representative blots are shown in ( F). Two-way ANOVA.
	Figure 3. . Pathogenic PPK PAWS1 point mutants impact canonical Wnt signalling. A, B: U2OS cells were transfected with PAWS1-GFP or the indicated mutants of PAWS1 or GFP alone. Levels of endogenous (e) or GFP-tagged (g) PAWS1 in protein extracts were analysed by immunoblotting ( A). TOPflash luciferase activity was measured after treatment with either control conditioned medium (L-CM) or Wnt3A conditioned medium (Wnt3a-CM) for 6 h ( B). Data are normalised to Renilla luciferase as internal control. Values shown relative to L-CM treated GFP control (n=3). Two-way ANOVA. C– E: 500 pg hPAWS1 mRNA was injected into Xenopus embryos at the four-cell stage. Protein levels were analysed by IB ( C). Representative images showing complete axis duplication and normal phenotypes at tadpole stage in injected embryos; scale bar = 1 mm ( D). % of embryos showing axis duplication phenotypes were quantified from three independent experiments. Error bars show SD; Two-way ANOVA ( E).
	Figure 4. . PAWS1 A34E knock-in reduces protein levels and impairs Wnt signalling. A: Schematic overview of CRISPR/Cas9 knock-in strategy. A GFP coding sequence, internal ribosome entry site (IRES), and mutations to the PAWS1 coding sequence in Exon2 were introduced by homology-directed repair with a plasmid donor. Further details are available in the Materials and methods section. L H, left homology arm; R H, right homology arm. B: U2OS, PAWS1-KO, and PAWS1 A34E KI cell extracts were analysed by IB with the indicated antibodies. C: PAWS1 transcript levels relative to GAPDH control in asynchronously growing cultures were assessed by RT-qPCR (n=3), and represented as fold-change relative to U2OS WT. One-way ANOVA. D: Cells were treated with L-CM or Wnt3a-CM for 3 h. Expression of AXIN2 was assessed by RT-qPCR relative to GAPDH, and represented as the fold-change over L-CM treated U2OS WT (n=3). Two-way ANOVA.
	Figure 5. . Conservation of the PAWS1 alanine 34 in FAM83A-H. A: Multiple sequence alignments were performed using Clustal Omega (EMBL-EBI) and visualised using BoxShade Server (EMBnet). Letters boxed in black indicate identical residues and letters shaded in grey indicate similar residues. Residues that are identical or similar in at least 50% of the FAM83 members are shaded. B: FLAG empty vector (-), FLAG-FAM83H WT (WT) and FLAG-FAM83H A31E (AE) were transiently expressed in FAM83H-KO U2OS cells. Cells were lysed, FLAG IPs were performed and IPs were analysed by immunoblotting for CK1 isoforms.

References [+] :

Amit, Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway. 2002, Pubmed