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.