Conjugation of Smt3 to dorsal may potentiate the Drosophila immune response

Abstract

A variety of transcription factors are targets for conjugation to the ubiquitin-like protein Smt3 (also called SUMO). While many such factors exhibit enhanced activity under conditions that favor conjugation, the mechanisms behind this enhancement are largely unknown. We previously showed that the Drosophila melanogaster rel family factor, Dorsal, is a substrate for Smt3 conjugation. The conjugation machinery was found to enhance Dorsal activity at least in part by counteracting the Cactus-mediated inhibition of Dorsal nuclear localization. In this report, we show that Smt3 conjugation occurs at a single site in Dorsal (lysine 382), requires just the Smt3-activating and -conjugating enzymes, and is reversed by the deconjugating enzyme Ulp1. Mutagenesis of the acceptor lysine eliminates the response of Dorsal to the conjugation machinery and results in enhanced levels of synergistic transcriptional activation. Thus, in addition to controlling Dorsal localization, Smt3 also appears to regulate Dorsal-mediated activation, perhaps by modulating an interaction with a negatively acting nuclear factor. Finally, since Dorsal contributes to innate immunity, we examined the role of Smt3 conjugation in the immune response. We find that the conjugation machinery is required for lipopolysaccharide-induced expression of antimicrobial peptides in cultured cells and larvae, suggesting that Smt3 regulates Dorsal function in vivo.

FIG. 1.

Conjugation of Smt3 to Dorsal. (A) Detection of the Dorsal-Smt3 conjugate. 529SU cells (S2 cells stably transformed with expression vectors encoding HA-Ubc9 and FLAG-Smt3 under the control of a Cu²⁺ inducible promoter) were left untransfected (lane 1) or were transiently transfected with 10 μg of Dorsal expression vector with (lane 2) or without (lane 3) subsequent Cu²⁺ induction. Cells were lysed 48 h later, lysates were fractionated by SDS-10% PAGE and analyzed by immunoblotting with anti-Dorsal antibody. An arrow indicates Dorsal-Smt3 (lane 3); the bands with apparent molecular masses of approximately 63 and 46 kDa are unknown proteins that cross-react with the anti-Dorsal antibody nonspecifically. (B) Conjugation of Smt3 to Dorsal in vitro. Epitope-tagged forms of Dorsal and components of the Smt3 conjugation machinery were expressed in E. coli (Smt3^FL, Smt3^GG, Ubc9) or Sf9 cells (Dorsal, SAE1/2) and purified to homogeneity. These proteins were then used in an in vitro conjugation assay (described in Materials and Methods) in the indicated combinations. Reactions were fractionated by SDS-PAGE, blotted onto polyvinylidene difluoride membranes, and probed with anti-Dorsal antibodies. An arrow indicates Dorsal-Smt3 (lane 8).

FIG. 2.

Stimulation of Dorsal-mediated activation by the Smt3 conjugation machinery in the absence of Cactus. (A) Transfection assays. S2 cells were transfected with a Dorsal-responsive luciferase gene reporter construct (DE5) alone or in combination with expression vectors encoding Dorsal and Twist, with or without Ubc9 and Smt3. A Renilla luciferase internal control reporter was also included in each transfection. (B) Smt3 and Ubc9 influence Dorsal in the absence of Twist. S2 cells were transfected with the DE5 reporter alone or in combination with expression vectors encoding Dorsal (without Twist) with or without Ubc9 and Smt3. (C) Catalytically defective Ubc9. The DE5 reporter was introduced into S2 cells with expression vectors for Dorsal and Twist and with (25 ng [+] or 500 ng [+++]) or without a mutant or wild-type expression vector for HA-tagged Ubc9. The inset shows equivalent expression levels of mutant and wild-type Ubc9, at both levels indicated in the bar graph, detected by anti-HA immunoblotting of whole-cell extracts. Each bar represents the average (+ standard deviation [error bar]) of duplicate experiments. Relative luminescence was first calculated by dividing the firefly luciferase activity (from the Dorsal-responsive reporter) by the Renilla luciferase activity (from the internal control). Fold activation values were then obtained by dividing each relative luminescence value by the relative luminescence value obtained for the reporters alone.

FIG. 3.

Conjugation of Smt3 to Dorsal is reversible. (A) Alignment of yeast and human Ulp1 with Drosophila Ulp1. Only the C-terminal 181 amino acids are shown. The conserved catalytic triad residues (arrowheads) and oxyanion hole Gln residue (asterisk) are indicated. (B) Deconjugation by Ulp1. 529SU cells were untransfected (lane 1) or transiently transfected with 10 μg of Dorsal and 0 (lane 2), 0.5 (lane 3), 1 (lane 4), or 2 (lane 5) μg of a Ulp1 expression vector. Whole-cell lysates were analyzed as described for Fig. 1A. (C) Processing of Smt3-GFP fusion by Ulp1. The contents of each lane were incubated in the presence of 150 mM NaCl, 1 mM dithiothreitol, 10 mM Tris (pH 8.0), and 0.2% Triton X-100. The reactions were stopped at the times indicated above the lanes (in minutes). Lane 1, HA-Smt3; lane 2, HA-Smt3-GFP; lanes 3 to 8, HA-Smt3-GFP and crude GST-Ulp1 (or GST). In lane 8, the GST-Ulp1 was preincubated with 20 mM NEM at room temperature for 15 min prior to its addition to the reaction mixture. Lane 9 contained the same ingredients as lane 7 except that GST-Ulp1 was replaced with GST. The reactions were stopped by boiling in SDS loading buffer, subjected to SDS-12% PAGE, and analyzed by anti-HA immunoblotting. A similar blot was also probed with an anti-GFP antibody, demonstrating that the appearance of HA-Smt3 was accompanied by the parallel appearance of GFP (data not shown). (D) Biphasic response of Dorsal to Ulp1. The DE5 reporter was cotransfected without Dorsal and Twist expression vectors or with Dorsal and Twist expression vectors plus 0, 1, 2, 5, or 10 μg of a Ulp1 expression vector. Data were analyzed as described for Fig. 2A.

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.

FIG. 5.

Lysine 382 is part of a potential SC motif. (A) Alignment of putative steroid receptor and rel family SC motifs. Many rel family transcription factors contain motifs that resemble possible Smt3 conjugation sites and/or SC motifs found in steroid receptors. The conserved residues in the (I/L/V)KXE consensus sequence are highlighted, and the target lysine is underlined. (B) Reporters used in this study. Both reporters used in this work contain a module consisting of tandem Dorsal and Twist binding sites driving expression of the firefly luciferase gene. The DE1 and DE5 reporters contain one or five tandem copies of this module, respectively, and are described in further detail in Materials and Methods. (C) The DE1 reporter is synergistically activated by Dorsal and Twist. S2 cells were transfected with the DE1 reporter alone or in combination with expression vectors encoding Dorsal and/or Twist. (D) Dorsal^K382R does not superactivate a reporter containing a single Dorsal binding site. S2 cells were transfected with the DE5 or DE1 reporter along with expression vectors for mutant or wild-type (WT) Dorsal, Twist, and/or Ubc9/Smt3 (2.5 μg), as indicated. (E) Superactivation by Dorsal^K382R does not require Twist. The DE5 reporters were transfected into S2 cells along with expression vectors for mutant or wild-type Dorsal with or without Twist, as indicated.

FIG. 6.

Ubc9 and Smt3 are required for the induction of antimicrobial peptides. (A) Conjugation of Smt3 to Dorsal correlates with increased LPS induction of CecA1. 529SU cells were induced with Cu²⁺ for 24 h (lanes 1 and 2) and/or challenged with LPS for 6 h (lanes 2 and 4). Total RNA was then isolated and subjected to quantitative RT-PCR with primers specific to the CecA1 mRNA (top panel). Whole-cell lysates from these cells were fractionated by SDS-PAGE (15 or 5% acrylamide) and analyzed by immunoblotting to confirm Ubc9 and Smt3 expression (middle panel, lanes 3 and 4) and Smt3-Dorsal production (bottom panel, lanes 3 and 4). (B) Smt3 and Ubc9 are required for the induction of CecA1 and Drs in S2 cells. mRNA corresponding to the genes indicated at the top were disrupted using dsRNAi (36 h). Total RNA was isolated following 6 h of LPS challenge. RT-PCR was then performed using primers specific to the genes indicated to the right of the gels.

FIG. 7.

Smt3 and Ubc9 are required for the induction of CecA1 in first instar larvae. Larvae homozygous for a null allele of dl (dl¹) or for hypomorphic P-element disruptions of either ubc9 or smt3 were isolated and challenged with LPS for 6 h. Total RNA was isolated and subjected to quantitative RT-PCR using primers corresponding to either CecA1 or β-actin. WT, wild type.

FIG. 8.

Potential mechanism for synergy control via Smt3 conjugation. Twist and Dorsal bind to the multiple binding sites to bring about reporter activation. In its wild-type, unconjugated form, Dorsal recruits a putative SCF that attenuates Dorsal activity, giving rise to moderate levels of transcriptional activation. However, disruption of the SC motif by mutagenesis or Smt3 conjugation disallows binding of the SCF. As a result, Dorsal brings about the much higher levels of transcriptional activation observed experimentally.