Members of the casein kinase 1 (CK1) family of serine-threonine protein kinases are implicated in the regulation of many cellular processes, including the cell cycle, circadian rhythms, and Wnt and Hedgehog signaling. Because these kinases exhibit constitutive activity in biochemical assays, it is likely that their activity in cells is controlled by subcellular localization, interactions with inhibitory proteins, targeted degradation, or combinations of these mechanisms. We identified members of the FAM83 family of proteins as partners of CK1 in cells. All eight members of the FAM83 family (FAM83A to FAM83H) interacted with the α and α-like isoforms of CK1; FAM83A, FAM83B, FAM83E, and FAM83H also interacted with the δ and ε isoforms of CK1. We detected no interaction between any FAM83 member and the related CK1γ1, CK1γ2, and CK1γ3 isoforms. Each FAM83 protein exhibited a distinct pattern of subcellular distribution and colocalized with the CK1 isoform(s) to which it bound. The interaction of FAM83 proteins with CK1 isoforms was mediated by the conserved domain of unknown function 1669 (DUF1669) that characterizes the FAM83 family. Mutations in FAM83 proteins that prevented them from binding to CK1 interfered with the proper subcellular localization and cellular functions of both the FAM83 proteins and their CK1 binding partners. On the basis of its function, we propose that DUF1669 be renamed the polypeptide anchor of CK1 domain.
The von Hippel–Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, aka FAM83G) loci, respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet-inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate.
The BMP and Wnt signalling pathways determine axis specification during embryonic development. Our previous work has shown that PAWS1 (also known as FAM83G) interacts with SMAD1 and modulates BMP signalling. Here, surprisingly, we show that overexpression of PAWS1 in Xenopus embryos activates Wnt signalling and causes complete axis duplication. Consistent with these observations in Xenopus, Wnt signalling is diminished in U2OS osteosarcoma cells lacking PAWS1, while BMP signalling is unaffected. We show that PAWS1 interacts and co‐localises with the α isoform of casein kinase 1 (CK1), and that PAWS1 mutations incapable of binding CK1 fail both to activate Wnt signalling and to elicit axis duplication in Xenopus embryos.
The eight members of the FAM83 (FAMily with sequence similarity 83) family of poorly characterised proteins are only present in vertebrates and are defined by the presence of the conserved DUF1669 domain of unknown function at their N-termini. The DUF1669 domain consists of a conserved phospholipase D (PLD)-like catalytic motif. However, the FAM83 proteins display no PLD catalytic (PLDc) activity, and the pseudo-PLDc motif present in each FAM83 member lacks the crucial elements of the native PLDc motif. In the absence of catalytic activity, it is likely that the DUF1669 domain has evolved to espouse novel function(s) in biology. Recent studies have indicated that the DUF1669 domain mediates the interaction with different isoforms of the CK1 (casein kinase 1) family of Ser/Thr protein kinases. In turn, different FAM83 proteins, which exhibit unique amino acid sequences outside the DUF1669 domain, deliver CK1 isoforms to unique subcellular compartments. One of the first protein kinases to be discovered, the CK1 isoforms are thought to be constitutively active and are known to control a plethora of biological processes. Yet, their regulation of kinase activity, substrate selectivity and subcellular localisation has remained a mystery. The emerging evidence now supports a central role for the DUF1669 domain, and the FAM83 proteins, in the regulation of CK1 biology.
Our previous studies of PAWS1 (protein associated with SMAD1; also known as FAM83G) have suggested that this molecule has roles beyond BMP signalling. To investigate these roles, we have used CRISPR/Cas9 to generate PAWS1-knockout U2OS osteosarcoma cells. Here, we show that PAWS1 plays a role in the regulation of the cytoskeletal machinery, including actin and focal adhesion dynamics, and cell migration. Confocal microscopy and live cell imaging of actin in U2OS cells indicate that PAWS1 is also involved in cytoskeletal dynamics and organization. Loss of PAWS1 causes severe defects in F-actin organization and distribution as well as in lamellipodial organization, resulting in impaired cell migration. PAWS1 interacts in a dynamic fashion with the actin/cytoskeletal regulator CD2AP at lamellae, suggesting that its association with CD2AP controls actin organization and cellular migration. Genetic ablation of CD2AP from U2OS cells instigates actin and cell migration defects reminiscent of those seen in PAWS1-knockout cells.This article has an associated First Person interview with the first authors of the paper.
Summary statement:PAWS1/FAM83G controls cell migration by influencing the organisation of F-actin and focal adhesions and the distribution of the actin stress fibre network through its association with CD2AP.. CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was . http://dx.doi.org/10.1101/106971 doi: bioRxiv preprint first posted online Feb. 8, 2017; . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/106971 doi: bioRxiv preprint first posted online Feb. 8, 2017; . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/106971 doi: bioRxiv preprint first posted online Feb. 8, 2017; . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was . http://dx.doi.org/10.1101/106971 doi: bioRxiv preprint first posted online Feb. 8, 2017; . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/106971 doi: bioRxiv preprint first posted online Feb. 8, 2017; . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was . http://dx.doi.org/10.1101/106971 doi: bioRxiv preprint first posted online Feb. 8, 2017; . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/106971 doi: bioRxiv preprint first posted online Feb. 8, 2017;. CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/106971 doi: bioRxiv preprint first posted online Feb. 8, 2017; . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/106971 doi: bioRxiv preprint first posted online Feb. 8, 2017; . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/106971 doi: bioRxiv preprint first posted online Feb. 8, 2017; . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/106971 doi: bioRxiv preprint first posted online Feb. 8, 2017; AbstractOur previous studies of PAWS1 (Protein Associated With SMAD1) have suggested t...
Transcriptional reporter systems allow researchers to investigate the function and regulation of transcription factors. Conventional systems employ artificial cDNA overexpression vectors containing either a promoter fragment or specific nucleotide sequence repeats upstream of firefly luciferase or fluorescent reporters, such as green fluorescence protein (GFP) cDNA (1). These systems suffer mainly from the lack of chromatin context. Here, we describe the rapid generation of endogenous transcriptional reporter cells for the Bone Morphogenetic Protein (BMP) pathway using CRISPR/Cas9 genome editing. In principle, our methodology can be applied to any cell line. The endogenous reporters will provide a robust system for the investigation of BMP transcriptional activity in the context of native chromatin landscape and facilitate chemical and genetic screens.
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