TY - JOUR
T1 - Pathogenic FAM83g palmoplantar keratoderma mutations inhibit the PAWS1:
T2 - Ck1α association and attenuate wnt signalling
AU - Wu, K. Z.L.
AU - Jones, Rebecca A.
AU - Tachie-Menson, Theresa
AU - Macartney, Thomas J.
AU - Wood, Nicola T.
AU - Varghese, Joby
AU - Gourlay, Robert
AU - Soares, Renata F.
AU - Smith, James C.
AU - Sapkota, Gopal P.
N1 - This work was supported by the Wellcome Trust through core funding to the Francis Crick Institute [FC001157]. RAJ and JCS
are supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK [FC001-157], the UK MRC [FC001-157],
and the Wellcome Trust [FC001-157]. KZLW is supported by the UK Medical Research Council Career Development Fellowship. TTM is supported
by the UK MRC PhD studentship. GPS is supported by the U.K. MRC [MC_UU_12016/3] and the pharmaceutical companies supporting the
Division of Signal Transduction Therapy (Boehringer-Ingelheim, GlaxoSmithKline, Merck-Serono).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PY - 2019/9/9
Y1 - 2019/9/9
N2 -
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.
AB -
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.
KW - Casein kinase
KW - Hereditary footpad hyperkeratosis
KW - Palmoplantar keratoderma
KW - Skin
KW - Wnt signalling
UR - http://www.scopus.com/inward/record.url?scp=85075354496&partnerID=8YFLogxK
U2 - 10.12688/wellcomeopenres.15403.1
DO - 10.12688/wellcomeopenres.15403.1
M3 - Article
C2 - 31656861
AN - SCOPUS:85075354496
VL - 4
SP - 1
EP - 18
JO - Wellcome Open Research
JF - Wellcome Open Research
SN - 2398-502X
M1 - 133
ER -