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First published online 15 August 2007
doi: 10.1242/dev.005181

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GLH-1, the C. elegans P granule protein, is controlled by the JNK KGB-1 and by the COP9 subunit CSN-5

April M. Orsborn^1,*, Wensheng Li^1,, Tamara J. McEwen¹, Tomoaki Mizuno², Evgeny Kuzmin¹, Kunihiro Matsumoto² and Karen L. Bennett^1,

¹ Molecular Microbiology and Immunology Department, University of Missouri, Columbia, MO 65212, USA.
² Molecular Biology Department, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.

View larger version (34K): [in this window] [in a new window]   Fig. 1. GLH-1 levels are increased in C. elegans kgb-1 mutants. (A) N2 (wild-type) and kgb-1 worms grown at 20°C for 5 days beyond (d>) the L4 stage were analyzed by western blot (1000 worms in the wild-type lane and 800 worms in the kgb-1 lane), examining GLH-1 and GLH-4; ß-tubulin was a loading control. (B) Relative levels of GLH-1 in kgb-1 and N2 worms were compared for 1, 3 and 5 d>L4 at 20°C, with GLH-1 levels for N2 worms at 1d>L4 set to 1 and other levels determined relative to this. Values are the average of three experiments, with the standard error shown. *, differences in GLH-1 between kgb-1 and N2 are statistically significant (P<0.05). Beneath, a representative blot with a ß-tubulin loading control is shown. For B-D, 600 worms were loaded per lane. (C) N2 and kgb-1 worms grown at 26°C for 3.5d>L4 were analyzed by western blot to examine GLH-1 and GLH-4 levels. (D) GLH-1 levels were quantified for N2 worms and kgb-1 mutants grown at 26°C for 1, 2 and 3d>L4. *, GLH-1 differs significantly between N2 and kgb-1 animals (P<0.05). ß-tubulin was the loading control. (E) Northern blot analysis of RNA from N2 and kgb-1 adults grown at 20°C or 26°C shows relative abundance of glh-4, glh-1 and kgb-1 transcripts, with an eIF-4A loading control. The kgb-1 probe did not include the 5' end of the gene, for which RT-PCR detects a small product in kgb-1(um3) RNA (not shown).

View larger version (99K): [in this window] [in a new window]   Fig. 2. kgb-1 mutants have more GLH-1 with grossly expanded P granules. (A) Schematic of the adult C. elegans germline adapted from Navarro et al. (see Navarro et al., 2001; Smith et al., 2002). Both gonad arms are shown, containing mitotic nuclei, the transition zone from mitosis to meiosis, the pachytene region and the diakinetic region where oocytes cellularize. DTC, distal tip cell; D, distal gonad; E, embryo; U, uterus; S, sperm; P, proximal gonad; O, mature oocyte. (B) One arm of a splayed gonad from an N2 worm grown at 26°C for 2d>L4 (DAPI-stained). Germ cell nuclei in pachytene (arrowhead) and diakinesis (asterisk) are indicated. (C) A kgb-1 mutant treated as in B shows one gonad arm wrapped on itself, with the distal end at the top and the proximal end to the right (DAPI). Examples of EMO (endomitocially-replicating) nuclei are indicated with arrows. (D) Gonad in B with anti-GLH-1 antibody. (E) Gonad in C with anti-GLH-1 antibody. (F) Magnified view (1000x) of the pachytene region of the N2 gonad (the boxed region from D). (G) Magnified view (1000x) of the kgb-1 gonad (the boxed region from E).

View larger version (40K): [in this window] [in a new window]   Fig. 3. KGB-1 is uniformly cytoplasmic in the distal gonad, aggregating into dispersed particles when oocytes cellularize. (A) A splayed gonad of a young N2 adult C. elegans (DAPI-stained). (B) The gonad in A reacted with anti-GLH-1 (red). (C) The gonad in A, with anti-KGB-1 (green). (D) A merged image of B and C. (E) Magnified view (1000x) of maturing oocytes (the boxed region from D). (F) A splayed gonad from a young kgb-1(km21) worm (DAPI). (G) The gonad in F with anti-GLH-1. (H) The gonad in F with anti-KGB-1. The kgb-1(km21) strain (Mizuno et al., 2004) was used in F-H to establish the specificity of the N-terminal KGB-1 antibody as this region is deleted in kgb-1(km21), but not in kgb-1(um3).

View larger version (22K): [in this window] [in a new window]   Fig. 4. Interactions of KGB-1 and GLH-1. (A) Pull-down analysis. Lane 1, input kgb-1(um3) homogenate with anti-KGB-1 (C2-2) antibodies. Lane 2, input kgb-1(um3) homogenate, with anti-GLH-1. Lane 3, input N2 protein, with anti-KGB-1. Lane 4, N2 homogenate incubated with GST protein and GST beads, with anti-KGB-1. Lane 5, N2 homogenate incubated with GLH-1-GST and GST beads; reacted with anti-KGB-1. Lane 6, kgb-1(um3) homogenate incubated with GLH-1-GST and GST beads; reacted with anti-KGB-1. Lanes 4-6 are shown at a longer exposure than lanes 1-3, as the KGB-1 pulled down by GLH-1 was considerably less than the total KGB-1 in C. elegans, lane 3. (B) A schematic of 6-His-tagged GLH-1 constructs, with binding indicated (+). (C) Pull-down analysis. KGB-1-GST with GLH-1-6-His and three mutants of GLH-1, each tagged with 6-His. Lane 1, His-tagged GLH-1-K581N/L589W with KGB-1-GST. Lane 2, 6-His-tagged GLH-1-K581N/L588W with KGB-1-GST. Lane 3, 6-His-tagged GLH-1 lacking the D site (missing aa 581-588) with KGB-1-GST (on long exposures, GLH-1 is seen to weakly bind KGB-1, not shown). Lane 4, KGB-1-GST and full-length, wild-type GLH-1-6-His.

View larger version (44K): [in this window] [in a new window]   Fig. 5. KGB-1 can phosphorylate GLH-1; proteosomal or JNK inhibitors increase GLH-1. (A) In vitro kinase reactions were carried out with GLH-1-GST (first row) and c-Jun-GST (second row). Inputs of KGB-1 (third row) and MEK-1 (fourth row), both with HA tags, are shown. In lane 1, basal levels of GLH-1 or c-Jun phosphorylation occur when incubated with KGB-1 alone. Lane 2 shows phosphorylation levels of GLH-1 and c-Jun when KGB-1 is activated by MEK-1. A mutated inactive form of KGB-1 was used in lane 3. (B) HighFive cells were infected with GLH-1-6-His and KGB-1-GST (lanes 5 and 6), GLH-1-6-His and GST alone (lanes 1 and 2), GLH-1-6-His and CSN-5-GST (lanes 3 and 4) or 6-His-tagged GLH-1 alone (lanes 7 and 8) and grown for 18 hours. They were then treated (or not) with 1 µM MG132 and grown an additional 5 hours before lysing. GLH-1 levels were measured with anti-His antibody. A ß-tubulin loading control is shown. (C) Young N2 adult C. elegans were grown in liquid culture for 6 hours, with 1 µM M6132 added for 0-3 hours; GLH-1 levels were assayed by western blot with an -tubulin loading control. (D) Under the same conditions as in C, 50 µM SP600125 was added and GLH-1 tested by western blot, again with an -tubulin control.

View larger version (47K): [in this window] [in a new window]   Fig. 6. Biochemical and genetic interactions between C. elegans KGB-1 and CSN-5; Drosophila Vasa interacts with KGB-1 and CSN-5. (A) GST pull-downs using CSN-5-GST and KGB-1-6-His. Lane 1, CSN-5-GST with eGFP-6-His, negative control (the smaller band is likely to be a CSN-5 breakdown product). eGFP size indicated. Lane 2, CSN-5-GST with KGB-1-6-His. (B) After csn-5(RNAi) in kgb-1, GLH-1 was assayed by western blot using 30 worms/lane of: lane 1, uninjected N2 worms; lane 2, uninjected kgb-1 mutants; lane 3, fertile F1 progeny of csn-5(RNAi) into kgb-1. All worms assayed were 3d>L4 stage, 20°C. F1 worms were picked as adults 5-6 days after moving mothers to fresh plates to eliminate eggs formed before injection (purging). This experiment was repeated four times, with the decrease in GLH-1 for the fertile kgb-1; csn-5(RNAi) animals averaging 2.8-fold lower than their kgb-1 uninjected siblings (range 1.6-5.3) (lane 3 versus lane 2). This difference is statistically significant, P<0.03. ß-tubulin served as a loading control. (C) Baculoviral co-infections of insect cells treated as in Fig. 4B,C. Lane 1, Vasa-GST with eGFP-6-His; lane 2, Vasa-6-His with KGB-1-GST; lane 3, Vasa-6-His with CSN-5-GST. The His-tagged proteins pulled down are shown in the upper panel, with the GST proteins shown below. Input proteins for these pull-down assays are shown in Fig. S3 in the supplementary material.

View larger version (40K): [in this window] [in a new window]   Fig. 7. Model of possible interactions of KGB-1 and CSN-5 with GLH-1 in C. elegans. This model is based on data presented here, which suggest that CSN-5 protects GLH-1 from degradation. The MAPK docking site in GLH-1 is important for GLH-1/KGB-1 interactions. Kinase assays show that KGB-1 can phosphorylate GLH-1, perhaps at the conserved phosphodegron motif. In addition, GLH-1, CSN-5 and KGB-1 physically and functionally interact to allow cross-talk between CSN-5 and KGB-1, with CSN-5 antagonizing KGB-1 function and protecting GLH-1. While interacting with KGB-1 and GLH-1, CSN-5 might associate with the COP9 signalosome.

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