Characterisation of the biochemical and cellular roles of native and pathogenic amelogenesis imperfecta mutants of FAM83H

Abstract

The majority of mutations identified in patients with amelogenesis imperfecta have been mapped to FAM83H . As FAM83H expression is not limited to the enamel, how FAM83H contributes to amelogenesis is still largely unknown. We previously reported that members of the FAM83 family of proteins interact with and regulate the subcellular distribution of the promiscuous serine-threonine protein kinase CK1 family, through their shared N-terminal DUF1669 domains. FAM83H co-localises with CK1 isoforms to speckle-like structures in both the cytoplasm and nucleus. In this report, we show FAM83H, unlike other FAM83 proteins, interacts and colocalises with NCK1/2 tyrosine kinase adaptor proteins. This interaction is mediated by proline-rich motifs within the C-terminus of FAM83H, specifically interacting with the second and third SH3 domains of NCK1/2. Moreover, FAM83H pathogenic AI mutant proteins, which trigger C-terminal truncations of FAM83H, retain their interactions with CK1 isoforms but lose interaction with NCK1/2. These AI mutant FAM83H proteins acquire a nuclear localisation, and recruit CK1 isoforms to the nucleus where CK1 retains its kinase activity. As understanding the constituents of the FAM83H-localised speckles may hold the key to unravelling potential substrates of FAM83H-associated CK1 substrates, we employed a TurboID-based proximity labelling approach and uncovered several proteins including Iporin and BAG3 as potential constituents of the speckles.

Proximity‐dependent labeling methods for proteomic profiling in living cells: An update

“…Under extreme conditions (e.g., high expression levels, long labeling times), TurboID expression can be toxic in human cells, flies, and worms, suggesting that the evolution of this enzyme for increased activity may have effectively reached an upper limit. In addition to being useful in spatial proteomics (Branon et al, 2018), TurboID has also proven successful to discover new protein–protein interactions (Larochelle, Bergeron, Arcand, & Bachand, 2019; Mair, Xu, Branon, Ting, & Bergmann, 2019; Tachie‐Menson et al, 2020; Zhang et al, 2019). However, for some bait proteins, TurboID may increase the number of labeled background proteins relative to BioID (May, Scott, Campos, & Roux, 2020), perhaps due to its robust enzymatic activity.…”

Functions and regulation of the serine/threonine protein kinase CK1 family: moving beyond promiscuity

“…Both human cancer cells and Xenopus embryos harbouring these mutations, presented with dysfunctional Wnt signalling [64], suggesting that loss of the CK1α spatiotemporal regulation imparted by FAM83G may be a key event in the pathogenesis of the disease. Furthermore, mutations in FAM83H are associated with the dental disease amelogenesis imperfecta (AI) [137][138][139][140]. FAM83H interacts and co-localises with CK1α, δ and ɛ isoforms, on punctate cytoplasmic and nuclear speckles [56,139,[141][142][143].…”

“…Furthermore, mutations in FAM83H are associated with the dental disease amelogenesis imperfecta (AI) [137][138][139][140]. FAM83H interacts and co-localises with CK1α, δ and ɛ isoforms, on punctate cytoplasmic and nuclear speckles [56,139,[141][142][143]. AI-associated FAM83H mutations result in N-terminal truncations of the FAM83H protein, generating FAM83H species that maintain CK1-binding, but lack the C-terminus, which presumably regulates FAM83H localisation [56,57].…”

FAM83F regulates canonical Wnt signalling through an interaction with CK1α

“…Interestingly, two mutations within the DUF1669 domain of the FAM83G gene that cause palmoplantar keratoderma result in the loss of FAM83G-CK1α interaction and attenuation of Wnt signalling ( 7 ). FAM83D directs CK1α to the mitotic spindle to ensure proper spindle alignment and timely exit from mitosis ( 8 ), and FAM83H mutations that cause amelogenesis imperfecta retain interaction with CK1 isoforms but are mis-localised in cells ( 9 , 10 ). However, the biological and biochemical roles of FAM83F are poorly understood.…”