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Separable and redundant regulatory determinants in Cactus mediate its dorsal group dependent degradation

Nielsen Q. Fernandez¹, Jörg Grosshans^2,*, Jason S. Goltz^1,3 and David Stein^1,3,

¹ Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA
² Max-Planck-Institut fur Entwicklungsbiologie, Tubingen, Germany
³ Section of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA
^* Present address: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA

View larger version (25K): [in a new window]   Fig. 1. Structural comparison between Cactus and IB. Schematic diagrams of Cactus and IB are shown with hatched yellow regions indicating the positions of the six complete ankyrin repeats in each protein. The position and sequence of the identified IKK target motif is shown above IB, as are the position of the two Cactus regulatory elements discussed in this study. The position of serine (red) and lysine (blue) residues located N-terminal to the first ankyrin repeat are shown. Lysine and serine residues present in other parts of the proteins are not shown.

View larger version (64K): [in a new window]   Fig. 2. Maternally expressed Cactus-lacZ fusion protein rescues Cactus function and exhibits dorsal group-dependent polar degradation. The Cactus-lacZ fusion protein was expressed in the female germline under the transcriptional control of the alpha-tubulin 67C promoter in cact011/ cact011 females, resulting in hatching embryos with normal DV polarity (B). cact011/ cact011 females normally produce ventralized embryos (A). Cactus-lacZ protein (visualized by enzymatic activity (C) or with anti-ß-galactosidase antibody (E)) exhibited a dorsal enrichment in the progeny of wild-type females (pipe/+, C) similar to that seen for endogenous Cactus protein (Bergmann et al., 1996). Cactus-lacZ protein exhibited a uniform distribution in the progeny of dorsal group mutant females (pipe/pipe, D). In this and all other figures embryos are positioned with dorsal side upwards, anterior towards the left and maternal genotypes are shown at top right. Similarly, in all panels, arrowheads identify ventral denticle bands, while FK indicates the position of Filzkörper.

View larger version (101K): [in a new window]   Fig. 3. Amino acid determinants present in the N-terminal 125 residues of Cactus are required for polarized degradation. The distribution of full-length Cactus-lacZ (A), or deleted derivatives lacking the first 51(B), 76(C), 101(D) or 125(E) amino acids, expressed under the control of the alpha-tubulin 67C promoter in cactD13/+ females, visualized by ß-galactosidase activity. Deletion of the first 125 amino acid residues resulted in loss of regulated degradation (E) and the production of dominantly dorsalized embryos, as assessed in the cuticular pattern (F). While Cactus-lacZ deletion derivatives that lacked as many as 101 amino acids gave rise to a normal Dorsal protein nuclear gradient (G), the deletion of 125 amino acids resulted in a Cactus-lacZ protein that retains Dorsal in the cytoplasm (H).

View larger version (89K): [in a new window]   Fig. 4. Polarized degradation of the Cactus-lacZ protein requires Dorsal. Maternally expressed full-length Cactus-lacZ visualized by lacZ activity exhibits a normal polarized distribution in the progeny of females heterozygous for dl1 (A), while ventral degradation is not observed the progeny of females homozygous for dl1 (B). Similarly, a deletion derivative Cactus(Nterm)lacZ in which the N-terminal 161 amino acids of Cactus are fused to lacZ, and which lacks the Dorsal-binding ankyrin repeat region, did not exhibit spatially regulated degradation (C).

View larger version (106K): [in a new window]   Fig. 5. Polarized degradation of Cactus-lacZ fusion proteins does not rely on endogenous Cactus. Enzymatic activity was used to determine Cactus-lacZ distributions in the progeny of cactBQ/cactE10 females, expressing endogenous mutant Cactus that does not undergo regulated degradation (Bergmann et al., 1996). While the 101Cactus-lacZ variant undergoes polarized degradation (A), the 125Cactus-lacZ variant does not (B). Similarly, 101Cactus-lacZ restored ventral and lateral elements (D) to the cuticles of embryonic progeny of cactBQ/cactE10 females (C), while 125Cactus-lacZ did not (E). Two-hybrid prey constructs containing full-length, N terminally deleted and C terminally deleted segments of Cactus fused to a bacterial activator segment in plasmid JG4-5 (Gyuris et al., 1993) were constructed (for extent of Cactus present in each construct, see G) and tested for their interaction with a lexA-Cactus bait (F). An interaction between Pelle and Pellino is shown as a positive control, while empty vector (JG4-5) and Tube and Pellino fusions to the activation domain fail to interact with lexA-Cactus and represent negative controls.

View larger version (97K): [in a new window]   Fig. 6. The putative IB Kinase (IKK) target motif is dispensable for the spatially regulated degradation of Cactus-lacZ. Mutated derivatives of Cactus-lacZ in which serines 74 and 78 (A) or serines 74, 78, 82 and 83 (B) had been converted to alanine residues were expressed under maternal transcriptional control and their distributions determined by lacZ activity. Both proteins exhibited normal polarized distributions and were capable of restoring ventral pattern elements (D) to the otherwise dorsalized progeny of cactBQ/cactE10 females (C). Similarly, the mutated constructs were capable of restoring lateral and dorsal pattern elements (F) and the ability to hatch to the otherwise ventralized progeny of cact011/cact011 females (E).

View larger version (74K): [in a new window]   Fig. 7. Two separable regions in the Cactus N terminus direct polarized degradation. (A) Cactus-lacZ derivative lacking the N terminal 101 amino acid residues was subjected to site-directed mutagenesis to assess regulatory roles of serines 104 and 116 (A-D). The unmutated (A) and S104A (C) derivatives exhibited polarized degradation, as visualized by lacZ activity, while the S104,116A double mutant (B) or the S116A single mutant (D) led to a loss of degradation. Conversion of lysine108 to arginine did not perturb the Cactus-lacZ degradation gradient (I). To assess the regulatory roles of the putative IKK target motif in the absence of the influence of the S116 residue, the first 100 amino acids of Cactus were fused in frame to 144Cactus-lacZ and its distribution (E) compared with that of a mutant derivative in which serines 74, 78, 82 and 83 had been converted to alanine residues (F). Full-length Cactus-lacZ bearing the S74A, S78A and S116A mutants substitutions failed to undergo polarized degradation (G) and acted as a dominant dorsalizing mutant (H), demonstrating that the mutagenized residues are key regulatory determinants in the N terminus of Cactus.

View larger version (70K): [in a new window]   Fig. 8. The N terminus of IB can substitute for the N terminus of Cactus to direct regulated degradation. A Cactus-lacZ fusion derivative in which the first 71 amino acids of human IB were fused to the N-terminus of 144Cactus-lacZ was expressed under alpha-tubulin 67C transcriptional control and its distribution determined by lacZ activity. This construct exhibited polarized degradation (A) and the ability to rescue DV polarity in the otherwise ventralized progeny of cact011/ cact011 mutant females (C). In contrast, a derivative in which the IKK target site serines 32 and 36 had been converted to alanine exhibited a uniform distribution (B) and led to the dominant dorsalization of what would have otherwise been normal hatching progeny from cact011/+ females (D).