spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online May 16, 2007
doi: 10.1242/10.1242/dev.02853

Development 134, 2061-2071 (2007)
Published by The Company of Biologists 2007
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zeng, Y. A.
Right arrow Articles by Verheyen, E. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zeng, Y. A.
Right arrow Articles by Verheyen, E. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Drosophila Nemo antagonizes BMP signaling by phosphorylation of Mad and inhibition of its nuclear accumulation

Yi Arial Zeng*,{dagger}, Maryam Rahnama*, Simon Wang, Worlanyo Sosu-Sedzorme and Esther M. Verheyen{ddagger}

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, V5A 1S6, Canada.


Figure 1
View larger version (89K):
[in this window]
[in a new window]

  		Fig. 1. Opposing effects of nmo and the Dpp pathway on Drosophila wing growth and patterning. (A) A wild-type adult wing. (B,C) Wings from transheterozygous combinations of dpp loss-of-function alleles (B, dppd5/dpphr56; C, dppd5/dpphr4) show reductions in vein spacing and loss of veins. (D-F) Ectopic Nemo decreases spacing of veins in a dose-sensitive manner. (D) omb-Gal4/+; UAS-nemo/+. (E,F) omb-Gal4/+; UAS-nmo/+; UAS-nmo/+. (G) UAS-Mad/+; vg-Gal4/+. (H) A nmoadk2/nmoadk2 loss-of-function wing. (I) Ectopic expression of the BMP antagonist Sog (UAS-sog/+; 69B-Gal4/+). (J) UAS-nmo/+; 69B-Gal4/+ phenocopies reduced BMP signaling.

 

Figure 2
View larger version (92K):
[in this window]
[in a new window]

  		Fig. 2. Modulation of Nemo affects the spacing of veins and wing cell density. (A-C) Comparison of intervein distances between wild-type (A) and nmoadk2/nmoadk2 (B) Drosophila wings. (C) Superimposition of the two wings, showing that nmo L2 and L5 veins (arrows) are spaced further apart than in the wild type. (D-F) Comparison of intervein distances between an omb-Gal4/+ control (D) and omb-Gal4/+; UAS-nmo/+ (E). (F) Superimposition of the two wings, showing that omb>nmo L2 and L5 veins (arrows) are closer together than in the omb-Gal4 control. (G) A wild-type wing with the numbering scheme depicted for the measured intervals, 1-4. (H) Measurement of intervein regions in the wild type, omb-Gal4/+, omb>nmo and nmoadk2. (I-L) Cell density within a given region in the wing blade of the wild type (I,J) and nmoadk2/nmoadk2 (K,L) was calculated by counting trichome density in the indicated squares (I,K; the location of the counted regions is indicated with an asterisk in J,L). The results are shown in Table 1.

 

Figure 3
View larger version (128K):
[in this window]
[in a new window]

  		Fig. 3. nmo antagonizes BMP signaling during Drosophila wing development. (A) dpp-Gal4>UAS-tkvQD results in a bifurcated wing blade. (B) dpp-Gal4>UAS-nmo has no visible wing defect. (C) Ectopic nmo is able to suppress the bifurcated phenotype in UAS-nmo/+; dppGal4/UAS-tkvQD wings. (D) ptc-Gal4>UAS-tkv causes loss of wing tissue and fusion of L3 and L4 veins. (E) ptc-Gal4>UAS-nmo shows no obvious phenotype. (F) ptc-Gal4/UAS-nmo; UAS-tkv/+ shows suppression of the ectopic tkv phenotype. (G) vg-Gal4>UAS-Mad showing both a widened wing blade and ectopic veins. (H) vg-Gal4>UAS-nmo shows no obvious phenotype. (I) UAS-Mad/+; vg-Gal4/UAS-nmo rescues the broad wing blade and ectopic wing veins phenotype caused by ectopic Mad. (J) The weak Dad mutant Dadj1E4 has no discernible wing phenotype. (K) nmoadk1 showing a mild ectopic vein phenotype. (L) Dadj1E4; nmoadk1 double-mutants have more severe ectopic vein phenotypes than nmoadk1 alone.

 

Figure 4
View larger version (68K):
[in this window]
[in a new window]

  		Fig. 4. nmo modulates Mad-dependent target gene expression and the pMad gradient. (A) vgQ-lacZ expression in the wild-type Drosophila third instar wing imaginal disc. (B) vgQ expression is abolished in the dorsal wing pouch when UAS-nmo is expressed using the dorsal-specific driver ap-Gal4. (C,D) nmoDB24 somatic clones (marked by the absence of GFP, green). (E) Expression of vgQ-lacZ is increased in the clone abutting the A/P boundary (arrow) but shows no detectable change in the clone further away from the levels of highest Dpp signaling, in which nmo expression is normally low (arrowhead). (F-H) Salm expression in wild-type, nmoDB24/nmoadk2 and omb>2x nmo third instar wing discs. The width of Salm expression along the D/V boundary is indicated by a white line. (I-K) nmolacZ expression in late third instar stage wing discs (green) co-localizes in the L3 and L4 vein primordia flanking the A/P boundary with highest levels of pMad staining (red in J,K). (L-N) nmoDB24 somatic clones (marked by the absence of GFP, green). (M,N) pMad staining is unchanged in nmo clones. (O-Q) pMad staining in wild-type (O), nmoDB24/nmoadk2 (P) and omb>1x nmo (Q) discs. Arrowheads indicate the position of peaks of pMad staining.

 

Figure 5
View larger version (61K):
[in this window]
[in a new window]

  		Fig. 5. The inhibition of Mad is specific to Nemo and not Erk MAPK. (A) A wild-type adult Drosophila wing. (B) The extra vein phenotype induced by vg>Mad. (C) A wing from a rlSem/+ hypermorphic fly. (D) Co-expression of UAS-nmo suppresses the vg>Mad phenotype. (E) Co-expression of UAS-rlSem (indicated as Sem) enhances the vg>Mad phenotype (F) Heterozygosity for the rlSem mutant enhances the vg>Mad phenotype.

 

Figure 6
View larger version (40K):
[in this window]
[in a new window]

  		Fig. 6. Drosophila Nemo binds to and phosphorylates serine 25 in the MH1 domain of Mad. (A) pXJ-Flag-nemo and pCMV-T7-mad were co-transfected into HEK293 cells. Cell lysates were immunoprecipitated with anti-Flag, anti-T7 or IgG (control). Immunoblotting was performed with anti-Flag and anti-T7 antibodies. (B) Nemo phosphorylates Mad and autophosphorylates. HEK293 cells were transfected with expression vectors as indicated. Immunoprecipitated complexes with indicated antibodies were subjected to in vitro kinase assays and analyzed by autoradiography. The immunoprecipitates were also immunoblotted with the indicated antibodies to confirm loading. (C) Schematic of the full-length Mad protein showing the MH1, MH2 and linker domains, as well as the site of the nuclear localization sequence (NLS). Potential Nemo phosphorylation sites are each indicated directly above the protein structure as a numbered S residue, followed by proline (P). The constructs shown beneath were generated to identify residues that are phosphorylated by Nemo. (D) In vitro kinase assays performed with wild-type Mad, Mad 4SA, Mad AAVA, Mad-{Delta}MH1 and MadS25A demonstrate that Nemo specifically targets serine 25, and that Nemo autophosphorylates. (E) Immunoblot of cell extracts used in kinase assays showing relative expression levels of these proteins.

 

Figure 7
View larger version (57K):
[in this window]
[in a new window]

  		Fig. 7. Drosophila Nemo-mediated phosphorylation inhibits the nuclear accumulation of Mad and MadS25A shows receptor-independent nuclear localization. COS-7 cells were transfected with (A) T7-Mad; (B) T7-Mad and HA-TkvQD (constitutively active form); (C) T7-Mad, HA-TkvQD and Flag-Nemo; (D) T7-Mad, HA-TkvQD and Flag-NemoK69M (kinase-dead); (E) T7-MadS25A. Immunostaining was preformed using anti-T7 and anti-HA antibodies to indicate the localization of T7-Mad (left-hand column) and expression of HA-TkvQD (center column). DAPI staining was also performed prior to mounting (right-hand column). Expression of Nemo (C) can inhibit the Mad nuclear accumulation that occurs upon Tkv signaling (B). Expression of kinase-dead Nemo does not affect Mad localization (D). (E) Mutation of the Nemo target site renders MadS25A constitutively nuclear even in the absence of receptor activation. (F,G) In vivo consequences of en-Gal4 expressing UAS-MadS25A (G) are very mild compared with wild-type UAS-Mad (F).

 

Figure 8
View larger version (66K):
[in this window]
[in a new window]

  		Fig. 8. Drosophila Nemo phosphorylation promotes the nuclear export of Mad. COS-7 cells were transfected with the constructs indicated and stained for the localization of Mad (green, upper panel of each pair) and with DAPI to indicate nuclei (blue, lower panel of each pair). MadS25A is primarily nuclear (A), whereas MadS25D is heavily enriched in the cytoplasm (B). Percentages indicate the number of cells displaying a primarily nuclear localization. (C,D) The localization of Mad is influenced by Tkv receptor activation. (E,F) Co-transfection of Nemo inhibits the Tkv-induced nuclear accumulation in the absence of leptomycin B (LMB) (E), but does not block nuclear retention in the presence of LMB (F).

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2007