FIG. 4.

Conjugation of Smt3 to Dorsal occurs at lysine 382. (A) Dorsal^K382R is resistant to Smt3 conjugation. 529SU cells were left untransfected (lane 1) or were transiently transfected with 10 μg of an expression vector for wild-type (WT) Dorsal (lanes 2 and 3) or Dorsal^K382R (lanes 4 and 5). Cells were either untreated (lanes 1, 2, and 4) or treated with Cu²⁺ to induce Ubc9 and Smt3 expression (lanes 3 and 5). (B) Disruption of the Smt3 conjugation site enhances transcriptional activation. S2 cells were transfected with the DE5 reporter and a Twist expression vector along with a Dorsal, Dorsal^K382R, or Dorsal^K382A expression vector. Data were analyzed as described in the legend to Fig. 2A. The inset displays the expression levels of the three Dorsal variants corresponding to the bar graph revealed by anti-Dorsal immunoblotting of whole-cell extracts. (C) Dorsal^K382R is unresponsive to Ubc9/Smt3. The DE5 reporter was introduced into S2 cells with expression vectors for mutant or wild-type Dorsal and Twist with or without Ubc9 and Smt3. Either 8 ng (+) or 64 ng (+++) of Dorsal expression vector was employed in this study. Data were analyzed as described in the legend to Fig. 2A. (D) Dorsal^K382R is not responsive to Ulp1. S2 cells were transfected with the DE5 reporter alone or in combination with expression vectors encoding Dorsal and Twist with or without either 1 or 10 μg of an expression vector for Ulp1.