The DUF1669 domain of FAM83 family proteins anchor casein kinase 1 isoforms

Abstract

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.

Fig. 1. Generation of HEK 293 and U2OS cells for tetracycline-inducible expression of FAM83 proteins.

A. Schematic representation of the human FAM83 family of proteins and the conserved domain of unknown function, DUF1669, that characterises them. B. A single copy of each FAM83 gene (A–H) tagged with GFP at the N-terminus was stably inserted downstream of a tetracycline-inducible promoter in HEK 293 cells. Cells were treated with doxycycline and lysed at the indicated times after treatment. Extracts were resolved by SDS-PAGE and subjected to immunoblotting (IB) for GFP. ERK1 and ERK2 (ERK1/2) and GAPDH are loading controls. This blot is representative of 2 independent experiments. C. A single copy of each FAM83 gene (A–H) tagged with GFP at the C-terminus was stably inserted downstream of a tetracycline-inducible promoter in U2OS cells. Extracts of doxycycline-induced cells were immunoblotted for GFP and the loading control GAPDH. FAM83B is not included on the blot because we were unable to detect FAM83B-GFP expression in U2OS cells. This blot is representative of 2 independent experiments.

Fig. 2. FAM83 proteins interact with CK1 isoforms.

A. Mass fingerprinting of protein interactors of FAM83A–H proteins tagged N-terminally (HEK 293 cells) or C-terminally (U2OS cells) with GFP (fig. S2, A and B) identified one or more CK1 isoforms. These tables show the values for the top 3 precursor ion intensities of the indicated CK1 isoforms pulled down with each GFP-FAM83 protein (A–H) expressed in HEK 293 cells and each FAM83-GFP protein (A–H) expressed in U2OS cells. GFP expressed in each cell line was a negative control. Scaffold Q/Q+S V4.4.6 was used for analysis of the LC-MS/MS data from HEK 293 cells, and scaffold V4.3 was used for analysis of the LC-MS-MS data from U2OS cells. FAM83B-GFP did not express in U2OS cells. B. GFP Immunoprecipitates (IP) of GFP control or GFP-FAM83A–H proteins expressed in HEK 293 cells were immunoblotted (IB) with antibodies recognizing the indicated CK1 isoforms and other proteins known to interact with FAM83 family proteins. Short Exp., short exposure; Long Exp., long exposure. C. Extracts of wild-type (WT) or GFP-FAM83B knockin (^GFP/GFPFAM83B) HaCaT cells were immunoprecipitated with GFP-TRAP A beads and immunoblotted to detect the indicated CK1 isoforms. GAPDH was used as a loading control. FT, flow through. D. As in C., except that proteins were immunoprecipitated from WT and FAM83G-GFP knockin (FAM83G^GFP/GFP) U2OS cell extracts. E. U2OS extracts were immunoprecipitated using either pre-immune IgG or an antibody recognizing CK1α coupled to Protein-G sepharose beads and immunoblotted with antibodies recognizing the indicated FAM83 proteins and GAPDH. All blots are representative of 3 independent experiments.

Fig. 3. The DUF1669 domain is sufficient to mediate the interaction of FAM83 proteins with CK1.

A. The indicated fragments of Myc-tagged Xenopus laevis FAM83G (Myc-xFAM83G) were co-expressed with HA-CK1α in FAM83G^-/- U2OS cells, and then cell extracts or HA immunoprecipitates were subjected to immunoblotting (IB) with antibodies recognizing Myc or HA as indicated. This blot is representative of 3 independent experiments. B. A His-tagged fragment of FAM83A (amino acids122-304), which contains the DUF1669 and PLD-like domains, was mixed with recombinant CK1ε kinase domain (amino acids 1-294) in vitro. His-FAM83A(122-304) was then pulled down using Ni-sepharose (Ni²⁺) resin, which was washed twice before elution. The input, unbound flow-through (FT), wash solutions (W1 and W2), and eluate (E) were analysed by SDS-PAGE and stained with Coomassie blue. This gel is representative of 3 independent experiments. C. Empty Flag vector (ctrl) or the indicated FLAG-FAM83G mutant and wild-type (WT) proteins were overexpressed in FAM83G^-/- U2OS cells. Cell extracts (input) and FLAG immunoprecipitates (IP) were subjected to immunoblotting for FLAG, CK1α, or GAPDH as indicated. This blot is representative of 3 independent experiments. D. WT and Phe→Ala (FA) and Asp→Ala (DA) mutant forms of GFP-FAM83E–H were transiently expressed in U2OS cells, immunoprecipitated (IP) from cell extracts with a GFP-specific antibody, and immunoblotted for GFP, CK1α, and CK1ε as indicated. This blot is representative of 3 independent experiments.

Fig. 4. FAM83 proteins and CK1α colocalize in cells.

U2OS cells stably integrated with Tet-inducible expression of GFP-FAM83A–H were transfected with mCherry-CK1α. Cells were processed for fluorescence microscopy following 24h of doxycycline treatment. DNA was stained with DAPI. Images from one field of view representative of 3 independent experiments are shown. The number of cells that displayed staining patterns identical to the representative image were documented for each experiment: GFP-FAM83A (n=50); GFP-FAM83B (n=31); GFP-FAM83C (n=37); GFP-FAM83D (n=32); GFP-FAM83E (n=55); GFP-FAM83F (n=44); GFP-FAM83G (n=43); GFP-FAM83H (n=32). Fluorescence images for GFP-alone and mCherry-CK1α alone expressing cells are included in fig. S6. Scale bar, 20 µM.

Fig. 5. FAM83 proteins colocalize with endogenous CK1α in cells.

U2OS cells stably integrated with Tet-inducible expression of GFP, GFP-FAM83B, GFP-FAM83F, or GFP-FAM83H were treated with doxycycline for 16 h prior to processing cells for fluorescence microscopy to detect GFP and endogenous CK1α (anti- CK1α). DNA was stained with DAPI. Images from one field of view representative of three independent experiments are shown. The number of cells displaying staining patterns identical to the representative image were documented for each experiment: GFP-FAM83B (n=56); GFP-FAM83F (n=60); GFP-FAM83H (n=48); GFP only (n=38); no transgene (n=82). Scale bars, 20 µm.

Fig. 6. The association between FAM83 proteins and specific CK1 isoforms is selective in cells.

A. U2OS cells stably integrated with Tet-inducible expression of GFP-FAM83F or GFP-FAM83H were transfected with either mCherry-CK1α (α) or mCherry-CK1ε (ε). GFP-FAM83F and GFP-FAM83H expression was induced with doxycycline for 24 h prior to processing cells for fluorescence microscopy. DNA was stained with DAPI. Images from one field of view representative of three independent experiments are included. The number of cells that displayed staining patterns identical to the representative image were documented for each experiment: GFP-FAM83F + mCherry-CK1α (n=44); GFP-FAM83F + mCherry-CK1ε (n=40); GFP-FAM83H + mcherry-CK1α (n=32); GFP-FAM83H + mCherry-CK1ε (n=40). Scale bar, 20 µm. B. U2OS cells stably integrated with Tet-inducible expression of GFP-FAM83F or GFP-FAM83H were induced with doxycycline for 16 h prior to processing cells for fluorescence microscopy with CK1α (α) or CK1ε (ε) antibodies. Untransfected cells stained with CK1α or CK1ε antibodies were used as negative controls. Images from one field of view representative of three independent experiments are shown. The number of cells that displayed staining patterns identical to the representative image were documented for each experiment: GFP-FAM83F with CK1α (n=60); GFP-FAM83F with CK1ε (n=43); GFP-FAM83H with CK1α (n=48); GFP-FAM83H with CK1ε (n=35); no transgene with CK1α (n=82); no transgene with CK1ε (n=27). Scale bars, 20 µm.

Fig. 7. Association with CK1 determines the subcellular localization of FAM83C.

A. U2OS cells were cotransfected with plasmids encoding either GFP, GFP-FAM83C (WT), GFP-FAM83C(F293A) (FA), or GFP-FAM83C(D259A) (DA) plus a plasmid encoding HA-CK1α. Untransfected (UT) cells and cells transfected only with HA-CK1α were included as controls. Cell extracts (Input) or GFP-TRAP A Immunoprecipitates (IP) were immunoblotted (IB) with antibodies recognizing GFP and CK1α. α-Tubulin was used as a loading control. This blot is representative of 3 independent experiments. B. U2OS cells were transfected with plasmids encoding GFP-FAM83C, GFP-FAM83C(F293A), or GFP-FAM83C(D259A), together with mCherry-CK1α. Cells expressing GFP-FAM83C or mCherry-CK1α alone are negative controls. Cells were processed 24 h after transfection for fluorescence microscopy. DNA was stained with DAPI. Images from one field of view representative of three independent experiments are shown. The number of cells which displayed staining patterns identical to the representative image were documented for each experiment: GFP-FAM83C only (n=46); GFP-FAM83C + mCherry-CK1α (n=44); GFP-FAM83C (F293A) + mCherry-CK1α (n=41); GFP-FAM83C (D259A) + mCherry-CK1α (n=43); mCherry-CK1α only (n=45). Scale bar, 20 µm.

Fig. 8. FAM83H colocalizes with and contributes to the subcellular localization of endogenous CK1α.

A. FAM83H^-/- U2OS cells were transfected with plasmids encoding either GFP-FAM83H, GFP-FAM83H (D236A), or GFP-FAM83H (F270A). Untransfected knockout (FAM83H^-/-) cells were used as controls. Cells were processed for fluorescence microscopy with antibody recognizing CK1α. DNA was stained with DAPI. Images from one field of view representative of 3 independent experiments are included. Scale bar, 10 µm. B. The boxplot shows the range, mean, and lower and upper quartiles of the Pearson’s correlation coefficients of GFP-FAM83H and endogenous CK1α intensities within above-background pixels in the cytoplasm. C. GFP-FAM83H constructs were transfected into FAM83H^-/- U2OS cells, and extracts were immunoblotted (IB) with the indicated antibodies. Untransfected wild-type (WT) cells were used as controls. This blot is representative of three independent experiments.

Fig. 9. The intrinsic catalytic activity of CK1 is not affected by or required for the association of CK1 with FAM83 proteins.

A. An in vitro kinase assay was performed in the presence of [^γ32P]-ATP with recombinant GST-CK1α plus one of the following recombinant FAM83 fusion proteins: GST-FAM83A (A), MBP-FAM83B (B), GST-FAM83C (C), GST-FAM83D (D), GST-FAM83E (E), GST-FAM83F (F), GST-FAM83G-6His (G), or GST-FAM83H (H). After the reactions were stopped, samples were resolved by SDS-PAGE. The gel was stained with Instant blue, dried, and subjected to ³²P autoradiography for the indicated times. Instant blue–stained gel and autoradiograph representative of 3 independent experiments are shown. B. An in vitro kinase assay was set up with recombinant GST-CK1α, and either recombinant GST-FAM83G-6His or the GST-FAM83G (F296A, F300A) double mutant in the presence of increasing amounts of the optimized CK1 peptide substrate CK1tide. GST-CK1α, without FAM83G addition, was used as a control. Data points represent the average from three independent experiments, each including three replicates. Error bars, SEM. C. U2OS cells were transiently co-transfected with GFP-FAM83E, GFP-FAM83F, GFP-FAM83G, or GFP-FAM83H and either WT CK1α or a catalytically inactive (kinase dead, KD) form of CK1α. After 24 h cell extracts (Input) were immunoprecipitated (IP) with GFP-TRAP A beads and immunoblotted (IB) with the indicated antibodies. This blot is representative of 3 independent experiments. GAPDH is a loading control.