The activity of the Drosophila morphogenetic protein Bicoid is inhibited by a domain located outside its homeodomain
Chen Zhao1,
Allen York1,
Fan Yang1,*,
David J. Forsthoefel2,
Vrushank Dave1,
Dechen Fu1,
Dongyi Zhang1,
Maria S. Corado3,
Stephen Small3,
Mark A. Seeger2 and
Jun Ma1,
1 Division of Developmental Biology, Children’s Hospital Research Foundation, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229, USA
2 Department of Molecular Genetics and the Neurobiotechnology Center, The Ohio State University, 125 Rightmire Hall, 1060 Carmack Road, Columbus, OH 43210, USA
3 Department of Biology, New York University, 100 Washington Square East, New York, NY 10003, USA*Present address: Department of Molecular Genetics, University of Cincinnati College of Medicine, 231 Sabin Drive, Cincinnati, OH 45267, USA
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Fig. 1. The N-terminal domain of Bcd inhibits its own activity. (A) A truncated Bcd derivative [Bcd(92-489)] exhibits an activity higher than that of the full-length protein. CAT assay results from Drosophila S2 cells that were transfected with the hb-CAT reporter plasmid and effector plasmids that express Bcd(1-489) or Bcd(92-489). The CAT activity for wild-type Bcd (expressed from 1 µg transfected effector DNA) on this reporter is arbitrarily assigned as 100 throughout this report. The standard error (s.e.m.) for the activity of Bcd(92-489) on hb-CAT was 23%. While the representative CAT assay results shown here were obtained with the same length of enzymatic reaction time (30 minutes), accurate CAT activities (as measured numbers) were obtained with different lengths of reaction time to keep the assays in the linear range. (B) Delineating the self-inhibitory domain of Bcd. Shown are CAT activities for the N-terminal deletion derivatives of Bcd in transient transfection assays on the hb-CAT reporter. The homeodomain (HD) of Bcd (residues 92-151) is marked.
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Fig. 2. Alanine mutations at residues 52-56 of full-length Bcd increase its activity. (A) Wild-type Bcd and Bcd(A52-56), which contains five alanines at positions 52-56 of full-length Bcd. HD, homeodomain. (B) CAT assay results from S2 cells that were transfected with increasing amounts of effector plasmids expressing wild-type Bcd or Bcd(A52-56): 0.2 µg (lanes 2, 6), 0.5 µg (lanes 3, 7), 1.0 µg (lanes 4, 8), 2.0 µg (lanes 5, 9). Lane 1 shows the result from cells transfected with an empty vector expressing no Bcd. All CAT reactions shown here were carried out in 30 minutes. (C) A plot of CAT activities against the amount of the transfected effector DNA. To measure accurately the activity difference between the proteins, a shorter length of the CAT reaction time was used for the mutant protein (also see legend to Fig. 1). (D) Western blot analysis showing the total amount of Bcd proteins in transfected cells. The amount of transfected DNA is the same as in B.
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Fig. 3. Neither subcellular localization nor DNA binding is affected by mutations in the self-inhibitory domain of Bcd. (A) Western blot results for wild-type Bcd and Bcd(A52-56) in nuclear (N) and cytoplasmic (C) fractions of the transfected cells. Lanes 1 and 2 represent results from cells that were transfected with an empty effector plasmid expressing no Bcd. (B) DNA-binding assay using Bcd proteins synthesized in an in vitro transcription/translation system. The left panel shows the proteins (labeled with 35S) and the right panel shows the gel shift results using a 32P-labeled DNA probe containing a Bcd binding site. In both panels, lanes 1 to 3 represent no Bcd, wild-type Bcd and Bcd(A52-56), respectively. The Bcd-DNA complex is marked with a solid arrowhead. (C) Gel shift assays for a Scatchard analysis to determine the dissociation constants (KD) for wild type Bcd (left panel) and Bcd(A52-56) (right panel) expressed in S2 cells. In this assay, the nuclear extracts generated from transfected S2 cells were used in gel shift assays with increasing concentrations of the radioactively labeled DNA probe: 5x10–10 M, 1x10–9 M, 2x10–9 M, 5x10–9 M, 1x10–8 M and 3.3x10–8 M for lanes 1 to 6, respectively. The solid arrowhead indicates the full-length Bcd-DNA complex, which was not formed using nuclear extracts made from non-transfected cells (not shown); smaller bands seen on the gel are presumably complexes containing breakdown products of Bcd. Quantitation was based on the amount of the full-length Bcd-DNA complex. (D) Scatchard plots for wild-type Bcd and Bcd(A52-56) expressed in S2 cells. Three independent assays yielded an estimated KD value (–1/KD=slope) of 3.0±0.9 nM and 4.0±0.5 nM for wild-type Bcd and Bcd(A52-56), respectively.
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Fig. 4. The self-inhibitory domain of Bcd works as an independent module. (A) Deletion derivatives of Bcd, either wild-type or Bcd(A52-56), were used in transient transfection assays. The derivative shown at the bottom (6) contains the DNA-binding domain of GAL4 (residues 2-94) in place of the homeodomain of Bcd. The activities, shown in the table, of the wild-type and mutant forms of this hybrid protein were obtained from the GAL4-CAT reporter gene, which contains five GAL4 sites upstream of the CAT gene. All other derivatives were assayed on the hb-CAT reporter gene. *The activity of the wild type Bcd-GAL4(2-94) hybrid protein on the GAL4-CAT reporter is assigned as 100, a standard for the relative CAT activity shown in B. (B) The self-inhibitory domain of Bcd can repress the activity of heterologous activators. Activities of hybrid activators that contain the DNA-binding domain of GAL4 (residues 1-147) fused to bacterially derived activation sequences (B6 and B42) are shown in the table. In addition, the first 91 amino acids of Bcd, either wild type or Bcd(A52-56), were attached to the N termini of these activators. The column designated –Bcd shows results of an activator lacking any Bcd sequence.
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Fig. 5. Bcd(A52-56) causes severe patterning defects in Drosophila embryos. (A-H) Representative cuticle phenotypes of embryos from bcd(A52-56) transgenic females (A-D) and higher magnification showing their corresponding head regions (E-H). Both moderate (B,F) and severe (C-H) phenotypes are shown for embryos from bcd+ females carrying one copy of bcd(A52-56)18A. A and E represent a completely normal embryo from bcd+ transgenic females carrying two copies of bcd(A52-56) for transgenic line 3-4 (see Table 3). Cuticles are orientated with anterior towards the left and (except C,G) dorsal upwards.
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Fig. 6. Bcd(A52-56) severely affects the expression patterns of target genes in Drosophila embryos. Shown are embryos from bcd+ females carrying either no (A,C,E,G,I) or one copy (B,D,F,H,J) of bcd(A52-56)18A, hybridized with digoxigenin-labeled hb (A-F) or otd (G-J) antisense RNA probes. Different developmental stages are shown: pre-cellular (A,B), cellularizing (C,D,G,H) and cellularized embryos (E,F,I,J). Embryos are oriented with anterior towards the left and dorsal upwards.
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Fig. 7. Bcd-dependent activation of hb and otd is unaffected in embryos from P1590 GLCs. Cellularizing (A,B,E,F) and cellularized (C,D,G,H) embryos were hybridized with digoxigenin-labeled hb (A-D) or otd (E-H) antisense RNA probes. Embryos are oriented with anterior towards the left and dorsal upwards. Wild-type embryos (left column) are compared with embryos from P1590 GLCs (right column), which have greatly reduced maternal CtBP function. No change is detected in the expression pattern of either gene with the exception of the later hb pattern (D), which shows a posterior expansion of the PS4 stripe (*), and an anterior expansion of the posterior hb domain. This perturbation is only detected in a small percentage ( 10%) of embryos from P1590 GLCs. See text for further details.
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Fig. 8. Interaction between Bcd and dSAP18. Shown are results of GST pull-down experiments in which bacterially expressed GST-dSAP18 (lanes 2, 4, 6 and 8) or GST alone (lanes 1, 3, 5 and 7) were used to pull down in vitro translated and radioactive labeled Bcd proteins. Note that the Bcd derivatives are pulled down by GST-dSAP18 above the background levels for GST alone.
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© The Company of Biologists Ltd 2002
