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

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

6-OHDA-induced dopaminergic neurodegeneration in <i>Caenorhabditis elegans</i> is promoted by the engulfment pathway and inhibited by the transthyretin-related protein TTR-33

<div><p>Oxidative stress is linked to many pathological conditions including the loss of dopaminergic neurons in Parkinson’s disease. The vast majority of disease cases appear to be caused by a combination of genetic mutations and environmental factors. We screened for genes protecting <i>Caenorhabditis elegans</i> dopaminergic neurons from oxidative stress induced by the neurotoxin 6-hydroxydopamine (6-OHDA) and identified the <u>t</u>rans<u>t</u>hyretin-<u>r</u>elated gene <i>ttr-33</i>. The only described <i>C</i>. <i>elegans</i> transthyretin-related protein to date, TTR-52, has been shown to mediate corpse engulfment as well as axon repair. We demonstrate that TTR-52 and TTR-33 have distinct roles. TTR-33 is likely produced in the posterior arcade cells in the head of <i>C</i>. <i>elegans</i> larvae and is predicted to be a secreted protein. TTR-33 protects <i>C</i>. <i>elegans</i> from oxidative stress induced by paraquat or H₂O₂ at an organismal level. The increased oxidative stress sensitivity of <i>ttr-33</i> mutants is alleviated by mutations affecting the KGB-1 MAPK kinase pathway, whereas it is enhanced by mutation of the JNK-1 MAPK kinase. Finally, we provide genetic evidence that the <i>C</i>. <i>elegans</i> cell corpse engulfment pathway is required for the degeneration of dopaminergic neurons after exposure to 6-OHDA. In summary, we describe a new neuroprotective mechanism and demonstrate that TTR-33 normally functions to protect dopaminergic neurons from oxidative stress-induced degeneration, potentially by acting as a secreted sensor or scavenger of oxidative stress.</p></div

Mutations in the cell corpse engulfment pathway alleviate 6-OHDA-induced dopaminergic neurodegeneration.

<p>(A) Cartoon of the engulfment pathway (adapted from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007125#pgen.1007125.ref027" target="_blank">27</a>]). The recognition of the conserved ‘eat-me’ signal phosphatidylserine (PS) on the surface of apoptotic and necrotic cells triggers two partially redundant pathways in the engulfing cell [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007125#pgen.1007125.ref073" target="_blank">73</a>]. The genes tested in this study are highlighted in yellow and the part of the pathway that was shown to be required for axon regeneration [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007125#pgen.1007125.ref027" target="_blank">27</a>] is boxed in blue. <i>psr =</i> <u><i>p</i></u><i>hosphatidyl</i><u><i>s</i></u><i>erine</i> <u><i>r</i></u><i>eceptor family</i>, <i>ced =</i> <u><i>ce</i></u><i>ll</i> <u><i>d</i></u><i>eath abnormality</i>, <i>nrf =</i> <u><i>n</i></u><i>ose</i> <u><i>r</i></u><i>esistant to</i> <u><i>f</i></u><i>luoxetine</i> (lipid-binding protein). (B) Effect of mutations in engulfment pathway genes on dopaminergic neurodegeneration after treatment with 10 mM 6-OHDA. Error bars = SEM of 2 biological replicates, each with 70–120 animals per strain. Total number of animals per condition n = 175–230 (****p<0.0001, ***p<0.002, **p<0.01, n.s. p>0.05; G-Test comparing <i>ttr-33</i> mutant data to double mutant data). (C) Effect of mutations in engulfment pathway genes on dopaminergic neurodegeneration after treatment with 50 mM 6-OHDA. Error bars = SEM of 2 biological replicates, each with 85–140 animals per strain. Total number of animals per condition n = 225–235 (****p<0.0001, *p<0.05, n.s. p>0.05; G-Test).</p

Evidence that <i>ttr-33</i> is expressed in the posterior arcade cells and in the pharynx.

<p>(A) Head region of L1 stage larvae expressing the extrachromosomal transcriptional reporter Ex[P<i>ttr-33</i>::<i>gfp</i>]. Arrows indicate strongly expressing cells. (B) L1 stage larva expressing the transcriptional reporter Ex[P<i>ttr-33</i>::<i>gfp</i>]. (C) Pretzel stage embryo expression the transcriptional reporter Ex[P<i>ttr-33</i>::<i>gfp</i>]. The speckled signal is caused by intestinal autofluorescence.</p

<i>ttr-33</i> and <i>ttr-52</i> have independent functions.

<p>(A) Effect of <i>ttr-52</i> mutation on dopaminergic neurodegeneration after treatment with 2.5 mM 6-OHDA. Error bars = SEM of 2 biological replicates, each with 95–115 animals per strain. Total number of animals per condition n = 200–225 (n.s. p>0.05; G-Test). (B) Effect of <i>ttr-52</i> mutation on dopaminergic neurodegeneration after treatment with 25 mM 6-OHDA. Error bars = SEM of 4 biological replicates, each with 100–130 animals per strain. Total number of animals per condition n = 425–460 (n.s. p>0.05; G-Test). (C) Quantification of axonal regrowth, regeneration and fusion of the PLM axons 24 hours after UV-laser axotomy in <i>ttr-33(gt1983)</i> mutants and wild-type animals carrying the <i>zdIs5</i> transgene to visualise PLM. The percentage of successful reconnection is a proportion of the axons that showed regrowth. The percentage of successful axonal fusion is a proportion of the axons that successfully reconnected. Error bars = standard error of proportion. The number of animals tested is indicated at the bottom of each bar (*p<0.05, n.s. p>0.05; t-test). (D) Length of axonal regrowth in <i>ttr-33(gt1983)</i> mutants and wild-type animals (n.s. p>0.05; t-test).</p

Interference with the <i>jnk-1</i> MAPK pathway increases 6-OHDA sensitivity in <i>ttr-33</i> mutants.

<p>(A) Effect of p38 and JNK stress response pathway mutations on dopaminergic neurodegeneration after treatment with 2.5 mM 6-OHDA. Error bars = SEM of 2–4 biological replicates, each with 100–120 animals per strain and concentration. Total number of animals per condition n = 200–425 (*p<0.05, n.s. p>0.05; G-Test). (B) Effect of p38 and JNK stress response pathway mutations on dopaminergic neurodegeneration after treatment with 25 mM 6-OHDA. Error bars = SEM of 3 biological replicates, each with 100–110 animals per strain. Total number of animals per strain n = 310–330 (n.s. p>0.05; G-Test). (C) Effect of <i>pmk-1</i> mutation on dopaminergic neurodegeneration after treatment with 25 mM 6-OHDA. Error bars = SEM of 3 biological replicates, each with 100–135 animals per strain. Total number of animals per strain n = 350–360 (n.s. p>0.05; G-Test).</p

<i>ttr-33</i> mutants are sensitive to oxidative stress.

<p>(A) Percentage of larvae reaching the L3 larval stage 24 hours after an 1 hour incubation with indicated concentration of paraquat. Error bars = SEM of 3 biological replicates for 25 and 50 mM paraquat and 2 biological replicates for 0 mM paraquat, each with 60–245 animals per strain and concentration. Total number of animals per condition n = 275–550 (****p<0.0001, *p<0.05; two-tailed t-test comparing BY200 wild-type and mutant animal data). (B) Percentage of animals developed to L3 stage 48 hours after an 1 hour incubation with indicated concentration of H₂O₂. Error bars = SEM of 2 biological replicates, each with 165–547 animals per strain and concentration. Total number of animals per condition n = 311–942 (*p<0.05; two-tailed t-test comparing data of BY200 wild-type and mutant animal data at 12.5 and 25 mM H₂O₂). (C) Lifespan data for first biological replicate including 110–125 animals per strain. The inset shows the mean lifespan with the error bars depicting the standard error. Total number of animals for N2 wild-type (which was tested once) n = 110, and for all other strains n = 200–235 (****Bonferroni p<0.0001; Log-Rank Test).</p