Research ArticleCell Biology

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

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Science Signaling  22 May 2018:
Vol. 11, Issue 531, eaao2341
DOI: 10.1126/scisignal.aao2341
  • Fig. 1 Generation of HEK 293 and U2OS cells for Tet-inducible expression of FAM83 proteins.

    (A) Schematic representation of the human FAM83 family of proteins and the conserved domain of unknown function DUF1669 that characterizes them. (B) A single copy of each FAM83 gene (FAM83A–H) tagged with GFP at the N terminus was stably inserted downstream of a Tet-inducible promoter in HEK 293 cells. Cells were treated with doxycycline and lysed at the indicated times after treatment. Extracts were resolved by SDS–polyacrylamide gel electrophoresis (PAGE) and subjected to immunoblotting (IB) for GFP. Extracellular signal–regulated kinase 1 (ERK1) and ERK2 (ERK1/2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are loading controls. This blot is representative of two independent experiments. (C) A single copy of each FAM83 gene (AH) tagged with GFP at the C terminus was stably inserted downstream of a Tet-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 two 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 three 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 (FAM83A–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 liquid chromatography tandem MS (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 knock-in (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 knock-in (FAM83GGFP/GFP) U2OS cell extracts. (E) U2OS extracts were immunoprecipitated using either preimmune 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 three 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 coexpressed 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 three 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 to 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 FT, wash solutions (W1 and W2), and eluate (E) were analyzed by SDS-PAGE and stained with Coomassie blue. This gel is representative of three independent experiments. (C) Empty Flag vector (ctrl) or the indicated FLAG-FAM83G mutant and 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 three 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 three 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 after 24 hours of doxycycline treatment. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI). 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 was 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), and 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 hours before 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 was documented for each experiment: GFP-FAM83B (n = 56), GFP-FAM83F (n = 60), GFP-FAM83H (n = 48), GFP only (n = 38), and 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 hours before 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 was documented for each experiment: GFP-FAM83F + mCherry-CK1α (n = 44), GFP-FAM83F + mCherry-CK1ε (n = 40), GFP-FAM83H + mcherry-CK1α (n = 32), and 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 hours before 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 was 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), and 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 three 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 hours 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 that displayed staining patterns identical to the representative image was 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), and 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 three independent experiments are included. Scale bar, 10 μm. (B) The boxplot shows the range, mean, and lower and upper quartiles of the PCCs 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 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 (adenosine 5′-triphosphate) with recombinant GST(glutathione S-transferase)–CK1α plus one of the following recombinant FAM83 fusion proteins: GST-FAM83A (A), MBP (myelin basic protein)–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 InstantBlue, dried, and subjected to 32P autoradiography for the indicated times. InstantBlue-stained gel and autoradiograph representative of three 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 represent SEM. (C) U2OS cells were transiently cotransfected 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 hours, cell extracts (input) were immunoprecipitated (IP) with GFP-Trap A beads and immunoblotted (IB) with the indicated antibodies. This blot is representative of three independent experiments. GAPDH is a loading control.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/531/eaao2341/DC1

    Fig. S1. Sequence alignment of the DUF1669 domain of the FAM83 proteins.

    Fig. S2. Coomassie images of GFP-Trap immunoprecipitations of FAM83A–H proteins used to identify interacting partners by MS.

    Fig. S3. Immunoblots of controls for Fig. 2.

    Fig. S4. FAM83G interacts with CK1α but not with CK1γ or TTBK1.

    Fig. S5. CK1-specificity switch with DUF1669 chimera.

    Fig. S6. Fluorescence images of GFP and mCherry-CK1α controls.

    Fig. S7. FAM83H colocalizes with and contributes to the subcellular localization of endogenous CK1ε.

    Fig. S8. Validation of CK1α and CK1ε antibodies for immunofluorescence applications.

    File S1. Supplemental ImageJ Macro for quantification of colocalization in cells.

  • Supplementary Materials for:

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

    Luke J. Fulcher, Polyxeni Bozatzi, Theresa Tachie-Menson, Kevin Z. L. Wu, Timothy D. Cummins, Joshua C. Bufton, Daniel M. Pinkas, Karen Dunbar, Sabin Shrestha, Nicola T. Wood, Simone Weidlich, Thomas J. Macartney, Joby Varghese, Robert Gourlay, David G. Campbell, Kevin S. Dingwell, James C. Smith, Alex N. Bullock, Gopal P. Sapkota*

    *Corresponding author. Email: g.sapkota{at}dundee.ac.uk

    This PDF file includes:

    • Fig. S1. Sequence alignment of the DUF1669 domain of the FAM83 proteins.
    • Fig. S2. Coomassie images of GFP-Trap immunoprecipitations of FAM83A–H proteins used to identify interacting partners by MS.
    • Fig. S3. Immunoblots of controls for Fig. 2.
    • Fig. S4. FAM83G interacts with CK1α but not with CK1γ or TTBK1.
    • Fig. S5. CK1-specificity switch with DUF1669 chimera.
    • Fig. S6. Fluorescence images of GFP and mCherry-CK1α controls.
    • Fig. S7. FAM83H colocalizes with and contributes to the subcellular localization of endogenous CK1ε.
    • Fig. S8. Validation of CK1α and CK1ε antibodies for immunofluorescence applications.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • File S1 (.txt format). Supplemental ImageJ Macro for quantification of colocalization in cells.

    © 2018 American Association for the Advancement of Science

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