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First published online 14 January 2004
doi: 10.1242/dev.00987

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C. elegans SUR-6/PR55 cooperates with LET-92/protein phosphatase 2A and promotes Raf activity independently of inhibitory Akt phosphorylation sites

Gautam Kao^1,*, Simon Tuck², David Baillie³ and Meera V. Sundaram^1,

¹ Department of Genetics, University of Pennsylvania, Philadelphia, PA 19104, USA
² UCMP, Umea University, SE-901 87 Umeå, Sweden
³ Department of Biological Sciences, Simon Fraser University, Burnaby B.C. V5A 1S6, Canada

View larger version (17K): [in a new window]   Fig. 1. C. elegans vulval cell fate specification. (A) Six vulval precursor cells (VPCs) are competent to adopt vulval fates in response to cell signaling events, but only three VPCs (P5.p, P6.p and P7.p) normally do so (Moghal and Sternberg, 2003). Loss-of-function mutations in the Ras pathway cause fewer than three VPCs to adopt vulval fates, whereas gain-of-function mutations in the Ras pathway cause greater than three VPCs to adopt vulval fates. (B) Ras and Wnt pathways cooperate to specify vulval cell fates (Gleason et al., 2002). The Wnt pathway behaves genetically downstream of the Ras pathway as the pry-1 Muv phenotype is not suppressed by Ras pathway mutations (Gleason et al., 2002) but the activated MPK-1 phenotype is partly suppressed by bar-1 mutations (Eisenmann et al., 1998). Only pathway components relevant to this paper are depicted.

View larger version (79K): [in a new window]   Fig. 2. Abnormal cell divisions in sur-6(sv30) embryos. Cell division patterns (A-F), chromosome segregations during anaphase (G,H) and spindle orientation (I,J) were examined in wild-type embryos (A,C,E,G,I) or embryos produced by sur-6(sv30) mothers (B,D,F,H,J). (A-F) Nomarski images. (A) Wild-type embryo showing normal anaphase spindle in the AB cell. (B) sur-6(sv30) embryo showing an abnormal L-shaped spindle in the AB cell. (C) Wild-type embryo in which AB has divided before P1. (D) sur-6(sv30) embryo in which P1 has divided before AB. (E) Wild-type embryo showing the orthogonal orientation of the ABar and ABpr spindles. Doubleheaded arrows indicate the orientation of the spindle. (F) sur-6(sv30) embryo showing parallel orientation of the ABar and ABpr spindles. A similar defect has been reported in some Wnt pathway mutants (Rocheleau et al., 1997; Thorpe et al., 1997). (G,H) DAPI staining. (G) Wild-type one-cell embryo showing a normal anaphase figure. (H) sur-6(sv30) one cell embryo with anaphase bridging defects (arrow). (I,J) Anti-tubulin staining. (I) Wild-type multicellular embryo showing two centrosomes per dividing cell. (J) sur-6(sv30) multicellular embryo showing a dividing cell with supernumerary centrosomes (arrows).

View larger version (109K): [in a new window]   Fig. 3. sur-6(sv30) suppresses the lin-45S312A S453A Muv phenotype. Animals were observed by Nomarski optics at the L4 larval stage. Arrowheads indicate the real vulva, arrows indicate ectopic vulval invaginations (C,E,F) and asterisk in B indicates a non-vulval fate for P7.px in a partially vulvaless animal. (A) Wild type; (B) sur-6(sv30); (C) hs-torso4021-Draf; (D) sur-6(sv30); hs-torso4021-Draf. (E) hs-lin-45S312A S453A; (F) sur-6(sv30); hs-lin-45S312A S453A.

View larger version (36K): [in a new window]   Fig. 4. Activated MAP-kinase levels in wild-type and sur-6(sv30) mutant backgrounds. Western blots were performed using L4 animals. Membranes were probed with MAPK-YT antibody (Sigma) specific for dually phosphorylated MAP kinase, then stripped and reprobed with K23 antibody (Santa Cruz Biotechnology) which detects total MAP kinase for use as a loading control. Experiments were carried out in triplicate. (A) One representative western blot is shown. The mpk-1/map-kinase gene produces a 45 kDa soma-specific isoform in larvae (M. H. Lee and T. Schedl, personal communication). MAPK-YT reactive bands were absent in mek-2(h294lf) animals (Ohmachi et al., 2002). lin-45(ku112) animals were included as a positive control for reduced MPK-1 ERK phosphorylation. (B) Quantitation of three independent western blots was carried out using a BioRad GS670 imaging densitometer. Bands visualized by the K23 antibody were used as the loading control for the normalization of intensities of the bands visualized by the use of MAPK-YT.

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