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Involvement of a proline-rich motif and RING-H2 finger of Deltex in the regulation of Notch signaling

Kenji Matsuno1,*,{dagger}, Mikiko Ito2,*, Kazuya Hori1, Fumiyasu Miyashita1, Satoshi Suzuki3, Noriyuki Kishi3, Spyros Artavanis-Tsakonas4 and Hideyuki Okano3,5,6

1 Department of Biological Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
2 Department of Nutrition, School of Medicine, University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503, Japan
3 Department of Neuroanatomy, Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
4 Department of Cell Biology, MGH Cancer Center, 149-7309 Harvard Medical School, Building 149, 13th Street, Charlestown, MA 02129, USA
5 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology corporation, 2-6-15 Shibakoen, Minato-ku, Tokyo 105-0011, Japan
6 Department of Physiology, Keio University, School of medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
* These two authors contributed equally to this work



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  		Fig. 1. deltex and Notch are required for the normal development of the wing margin. (A) Wild-type adult wing. The wing veins III and IV are indicated by arrowheads (see D). (B) deltex/Y wing. (C) Notch54l9/+ wing. (D) Protein expression from a UAS responder line under the control of the ptc-GAL4 driver. UAS-GFP was crossed to ptc-GAL4, and wing discs of the third-instar larvae were stained with mouse anti-Delta antibody. GFP and Delta are shown in green and red, respectively. Presumptive cells of wing veins III and IV expressing Delta are indicated by arrowheads.

 


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  		Fig. 2. Wild-type and mutant Deltex proteins used in this study. The Deltex protein is arbitrarily divided into three regions (domains I, II and III), based on the position of two OPA repeats, which separate the regions. The region binding to the Notch CDC10/Ankyrin repeats (blue box), a proline-rich motif that is a putative SH3-binding site (red box) and a RING-H2 finger motif (green box) are shown. The full-length Deltex protein consists of 737 amino acids (Busseau et al., 1994Go). All the cDNAs encoding the Deltex derivatives shown were inserted into the pUAST vector, and transgenic flies carrying these constructs were generated. (A) Deltex derivatives are shown schematically. Dx{Delta}NBS lacks the domain capable of mediating Notch and Deltex interactions (amino acids 46-204). Dx{Delta}PRM lacks the proline-rich motif (amino acids 475-483). DxmRZF has point mutations in the RING-H2 finger motif: two histidine residues (amino acids 570 and 573) are replaced by alanine residues. Dx{Delta}NBS-{Delta}PRM lacks both the binding sites for Notch and the proline-rich motif. Dx{Delta}NBS-mRZF is a double mutation that lacks the binding site for Notch and has point mutations in the RING-H2 finger motif. Amino acid numbers are according to Busseau et al. (Busseau et al., 1994Go). (B) The Deltex derivatives listed in A were also made as fusion proteins with GST (yellow box), and are shown schematically. GST is wild-type GST used as a control. (C,D) Western blot analysis of the Deltex mutant derivatives shown in A,B. Flies carrying UAS constructs capable of expressing the Deltex derivatives (A,B) were crossed to the hs-GAL4 line. Samples isolated before (shown by –) and after (shown by +) heat shock. (C) Lanes 1 and 2, Canton-S; lanes 3 and 4, Dxfull; lanes 5 and 6, Dx{Delta}NBS; lanes 7 and 8, Dx{Delta}PRM; lanes 9 and 10, DxmRZF; lanes 11 and 12, Dx{Delta}NBS-{Delta}PRM; lanes 13 and 14, Dx{Delta}NBS-mRZF. The protein blot was probed with the anti-Deltex antibody. Molecular weight markers are shown in kDa. (D) Lanes1 and 2, Dxfull+GST; lanes 3 and 4, Dx{Delta}NBS+GST; lane 5 and 6, Dx{Delta}PRM+GST; lanes 7 and 8, DxmRZF+GST; lanes 9 and 10, Dx{Delta}NBS-mRZF+GST; lanes 11 and 12, GST alone. The protein blot was probed with anti-GST antibody. Molecular weight markers are shown in kDa.

 


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  		Fig. 3. Effects of overexpression of Deltex derivatives under the control of the ptc-GAL4 driver. Transgenic flies carrying the various UAS constructs expressing the Deltex derivatives were crossed to ptc-GAL4 or ptc-GAL4/CyO; A101/TM6B. (A-L) Adult wings and high magnifications of regions of wings. (M-R) The SOP cells in the third-instar wing discs are shown in green, and the Deltex derivatives are shown in red. An enhancer trap line, A101, was used to visualize the SOP cells. (A,G,M) Wild-type wing and disc. (B,H,N) Overexpression of Dxfull. Note a secondary wing margin-like structure (black arrowheads) and ectopic SOPs (white arrowhead). (C,I,O) Overexpression of Dx{Delta}NBS. Occasionally, a missing crossvein (white arrow) and a few extra bristles (white arrowhead) are observed. (D,J,P) Overexpression of Dx{Delta}PRM. Note the wing-notch phenotype. (E,K,Q) Overexpression of DxmRZF. (F,L,R) Overexpression of Dx{Delta}NBS-mRZF.

 


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  		Fig. 4. Dominant-negative behavior of Dx{Delta}PRM. (A-D) Adult wings. (E,F) Wing discs of third-instar larvae. SOPs are shown in green, and Deltex derivatives are shown in red. (G-I) Wing discs of third-instar larvae. Wg and Deltex proteins are shown in green and red, respectively. (A,C,E) Co-expression of Dxfull and Dx{Delta}PRM. Note that phenotypes induced by either Dxfull or Dx{Delta}PRM were suppressed by co-expression of both proteins (see Fig. 3B,D,H,J,N,P). (B,D,F) Overexpression of Dx{Delta}NBS-{Delta}PRM with the ptc-Gal4 driver. Note that overexpression of Dx{Delta}NBS-{Delta}PRM did not result in the wing-notch phenotype. (G) Endogenous expression of Wg (green) was detected along the boundary of the dorsal/ventral compartments in the wild-type wing discs of third-instar larvae. (H) Overexpression of Dx{Delta}PRM. Endogenous expression of Wg (green) was suppressed in the cells expressing Dx{Delta}PRM (red). A high-magnification photograph is shown at the top right. (I) Overexpression of Dx{Delta}NBS-{Delta}PRM. Note that Dx{Delta}NBS-{Delta}PRM (red) did not suppress the Wg (green) expression. A high-magnification photograph is shown at the top right.

 


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  		Fig. 5. The dominant-negative form of Deltex (Dx{Delta}PRM) suppressed Notch signaling downstream of the full-length Notch and upstream of an activated form of Notch. (A) Notch and its derivative. Protein motifs in Notch: SP, a signal peptide; EGF, 36 EGF-like repeats; N, 3 Notch/Lin-12 repeats; TM, the transmembrane domain; NLS, two nuclear localization signals; ANK, 6 CDC10/Ankyrin repeats; opa, polyglutamine repeat. The full-length Notch and an activated form of Notch are shown at the top and bottom of A, respectively. Nact is a truncated form that lacks the entire extracellular domain and the transmembrane domain. It functions as a constitutively active form of Notch. (B-G) UAS-Nfull or UAS-Nact was expressed alone or co-expressed with UAS-Dx{Delta}PRM under the control of the ptc-GAL4 driver. Wing discs of third-instar larvae are shown. (B) Overexpression of Nact. SOPs are shown in green. Note that a row of ectopic SOP cells was formed (arrowhead). (C) Co-expression of Nact and Dx{Delta}PRM (red). Ectopic formation of SOPs (green) was not suppressed. (D) Overexpression of Nact induced the ectopic expression of Wg (green) (Couso et al., 1994Go; Williams et al., 1994Go). (E) Co-expression of Nact and Dx{Delta}PRM (red). Note that the ectopic Wg expression (green) remained essentially the same. (F) Overexpression of Nfull (red) induced the ectopic Wg expression (green). (G) Co-expression of Nfull and Dx{Delta}PRM (red). Note that the ectopic expression of Wg (green) was suppressed.

 


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  		Fig. 6. Mutations in the RING-H2 finger motif of Deltex disrupted its homotypic interaction. UAS-Dxfull, -DxmRZF, -Dxfull+GST, and -DxmRZF+GST (see Fig. 2A,B) were expressed with pWA-GAL4 in Drosophila S2 cells. Deltex derivatives were detected by western blot with an anti-Deltex antibody. Lanes 1-7, the total cell extract that was added to the resin; lanes 8-14, eluants from the Glutathione-Sepharose 4B resin. Dxfull and DxmRZF are indicated by an arrowhead (~90 kDa). Dxfull+GST and DxmRZF+GST are indicated by an arrow (~120 kDa). Molecular weight markers are shown in kDa.

 


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  		Fig. 7. GST-mediated dimerization substituted for the function of the Deltex RING-H2 finger motif. Transgenic flies carrying UAS constructs expressing Deltex derivatives fused to GST were crossed to ptc-GAL4 or ptc-GAL4/CyO;A101/TM6B. (A-L) Adult wings and high-magnification photographs. (M-R) The SOP cells in the third-instar wing discs are shown in green, and Deltex derivatives are shown in red. An enhancer trap line, A101, was used to visualize SOP cells. (A,G,M) Overexpression of Dxfull+GST did not produce a noticeable effect. (B,H,N) Overexpression of Dx{Delta}NBS+GST resulted in wild-type wings and discs. (C,I,O) Overexpression of Dx{Delta}PRM+GST gave wild-type wings and discs. (D,J,P) Overexpression of DxmRZF+GST resulted in the induction of a secondary wing margin-like structure and ectopic SOP formation (indicated by an arrow), which resembled the effect of Dxfull overexpression (see Fig. 3B,H,N). Note that DxmRZF (non-GST form) did not show a substantial effect under the same conditions (see Fig. 3E,K,Q). (E,K,Q) Overexpression of Dx{Delta}NBS-mRZF+GST resulted in wild-type wings and discs. (F,L,R) Overexpression of GST alone did not show a substantial effect.

 


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  		Fig. 8. Loss-of-function deltex phenotype was rescued by the overexpression of DxmRZF+GST. Adult wings from flies of the following genotypes. (C,F) deltex24 / Y;; hs-GAL4/DxmRZF+GST. Wing phenotype of deltex was rescued (27% showed complete rescue). (A-C) Wings without heat-shock treatment and (D-F) wings from flies heat-shocked at the early pupal stage. (A,D) deltex24 / Y;; hs-GAL4. Small deltas of extra vein material are visible where the veins reach the wing margin. (B,E) deltex24/Y;; hs-GAL4/UAS-Dxful. Wing phenotype of deltex was rescued (18% showed complete rescue).

 




© The Company of Biologists Ltd 2002