Volume 1, Issue 6, Pages 817-827 (December 2001) Regulation of Drosophila Tracheal System Development by Protein Kinase B  Jing Jin, Norman Anthopoulos, Benjamin Wetsch, Richard C. Binari, Daniel D. Isaac, Deborah J. Andrew, James R. Woodgett, Armen S. Manoukian  Developmental Cell  Volume 1, Issue 6, Pages 817-827 (December 2001) DOI: 10.1016/S1534-5807(01)00090-9

Figure 1 Genetic Interactions between Dakt1 and trh (A) Graphic representation of the percent lethality of embryos observed in Dakt1q transheterozygous crosses. Genotypes of the crosses are depicted on the left. The bar graph is a diagrammatic representation of the lethality observed in control embryos and in Dakt1q/mutant transheterozygous embryos. The number (n) of total embryos assayed from each cross is also indicated. (B–D) Cuticle preparations of wild-type (B), p60A/+; Dakt1q/+ (C), and Dakt1q/trhP1747 (D) larvae. Developmental Cell 2001 1, 817-827DOI: (10.1016/S1534-5807(01)00090-9)

Figure 2 Dakt1 Signaling Positively Regulates trachealess Function Wild-type embryos showing the expression of Trh at stage 11 (A), btl transcripts at stage 11 (B), and tracheal structure visualized with MAb2A12 antibody at stage 16 (C). At stage 11, the tracheal placodes express both Trh and btl and are ten in number. The first placode is anterior-ventral and the tenth placode is anterior-dorsal at this stage (and in our panels). In trh mutant (trh7J83) embryos, the expression of Trh at stage 11 (D), btl at stage 11 (E), and MAb2A12 antigen at stage 16 (F) is lost. Zygotic stage 11 Dakt1 mutant (Dakt1q/Df(3R)sbd45) embryo (G), showing lower Trh levels as compared to wild-type (A). The expression of btl is also reduced in Dakt1 embryo at stage 11 (H). The first four tracheal placodes show even lower expression of btl. (I) MAb2A12 staining of stage 16 Dakt1 embryo showing only slight tracheal branching defects. (J) PI3′K regulatory subunit, p60 (p60A) zygotic mutants at stage 11 also show lower Trh levels. (K) In p60 zygotic mutant (p60A/p60B) embryos, lower levels of btl transcripts are also observed. (L) Transcription of btl in embryos expressing ectopic dominant-negative PI3′K (Dp110D954) is almost completely repressed. (M) The expression pattern of btl transcripts in a stage 11 dPTEN GLC embryo shows expansion of the btl transcription domain in all placodes. Note the induction of an ectopic pit (arrow) in this embryo. Also, the first placode shows the most dramatic expansion of the btl transcription domain. (N) Expression of btl transcripts in stage 11 embryos overexpressing dPTEN (da GAL4, UAS dPTEN). The expression of btl in the first seven placodes is repressed. (O) The expression pattern of zygotic Dakt1 at stage 11 as assayed by detection of β-gal protein in Dakt1P1627/+ embryo. Developmental Cell 2001 1, 817-827DOI: (10.1016/S1534-5807(01)00090-9)

Figure 3 PKB Phosphorylates Trh at S665 In Vitro (A) Schematic representation of Trh structure showing various protein domains as well as S571 and S665. (B) Sequence comparison of the two putative PKB phosphorylation sites (S571 and S665) in Trh. (C) Phosphorylation of recombinant Trh by PKB. HA-tagged wild-type or kinase-dead (KD: K179A) PKB proteins were immunoprecipitated from 293 cells with (+) or without (−) activation and incubated with wild-type or mutant (S571A or S665A; the double mutant S571A; S665A is denoted as AA) recombinant Trh in the presence of [32P]ATP. Upper panel is an autoradiographic image of 32P incorporation into Trh on SDS-PAGE. Lower panel is the Coomassie staining of the recombinant Trh proteins. (D) Substrate specificity of Trh in comparison to other PKB targets, such as Crosstide and GST-GSK-3β in PKB kinase assay. The bottom panel is the anti-HA immunoblot of HA-tagged recombinant PKB used in each experiment. (E) In vitro kinase assay with immunoprecipitated Dakt1 using either Trh or TrhS665A as substrate and measuring the incorporation of 32P by autoradiography. Note the reduced phosphorylation of the TrhS665A protein compared to wild-type Trh. (F) Kinase assay with 10 mU recombinant Akt1/PKBα (active or heat-inactivated, denoted as “+” or “−,” respectively) measuring phosphate incorporation by either 32P autoradiography (top panel) or immunoblotting with Akt substrate phospho-specific antibodies (second panel). The bottom two panels are loading controls for Trh and PKB proteins from the kinase reactions. Developmental Cell 2001 1, 817-827DOI: (10.1016/S1534-5807(01)00090-9)

Figure 4 PKB Phosphorylates Trh at S665 In Vivo (A) Two-dimensional (2D) electrophoretic analysis (TLC at pH 1.9 and chromatography) and autoradiography of tryptic digests of phosphorylated Trh. Upper panels: Drosophila S2 cells were transfected with either wild-type or S665A Flag-tagged Trh and metabolically labeled with 32P-phosphate. Trh proteins isolated using anti-Flag immunoprecipitation followed by SDS-PAGE purification were subjected to tryptic digestion. Lower panels: tryptic mapping of in vitro-labeled recombinant Trh from the PKB kinase assays depicted in Figure 3C. The origin is marked by an arrowhead. (B) Phospho-amino acid mapping of phosphorylated Trh from S2 cells. The phosphopeptide (marked) from (A) (upper left panel) was partially digested in acid and subsequently electrophoretically separated in 2D (pH 1.9 and pH 3.5, respectively). Phospho-amino acids were visualized by autoradiography. (C) Phospho-specific Western blot analysis of S665 phosphorylation of Trh. S2 cells were transfected with Flag-tagged Trh or TrhS665A and either treated with pervanadate (+) or left untreated (−). Anti-Flag immunoprecipitations were analyzed by Western blotting with either phospho-Akt substrate antibody (upper panel) or Flag antibody (lower panel). Developmental Cell 2001 1, 817-827DOI: (10.1016/S1534-5807(01)00090-9)

Figure 5 PI3′K/PKB Signaling Activates Trh Reporter Gene Transcription via Phosphorylation of S665 of Trh (A) Regulation of Trh activity in tissue culture cells. S2 cells were transiently cotransfected with control β-gal and pGL3-B-123 reporter vectors with the addition of either control or Trh constructs. β-Gal vector is a control and the pGL3-B-123 vector (containing the luciferase gene) provides the reporter for the assay. Cell lysates were measured for luciferase activity 48 hr after transfection and the results are tabulated in the bar graphs indicated. Activities tabulated in these bar graphs represent the ratio of luciferase/β-gal activities to normalize for transfection. Therefore, the units of activity are arbitrary. The bottom panel is an anti-Flag immunoblot showing expression levels of wild-type and mutant Trh protein in each experiment. (B) Same experiment as in (A) except with the addition of plasmid directing the expression of Tgo. (C) Transfection experiments performed in cells overexpressing either bovine PKB or Drosophila Dakt. The presence or absence of PKB or Dakt1 is indicated by “+” or “−.” These results are denoted as bar graphs of relative luciferase activity. (D) Similar assays were conducted in S2 cell lines with stable expression of various Myc-tagged versions of the catalytic subunit of Drosophila PI3′K (Dp110). The Dp110-CAAX mutation results in constitutive activation of Dp110, whereas the kinase-dead Dp110 (Dp110-KD) provides a negative control for the experiment. The presence or absence of Dp110 induction in each experiment is indicated by “+” or “−.” The inserted panel is the anti-Myc immunoblot showing relative levels of Dp110 proteins in the experiment. Developmental Cell 2001 1, 817-827DOI: (10.1016/S1534-5807(01)00090-9)

Figure 6 Effects of Ectopic Expression of Wild-Type and Mutant Trh on btl Transcription Expression of btl mRNA in stage 11 embryos. (A) Expression of ectopic Trh using arm GAL4 (arm GAL4-UAS Trh) induces two ectopic domains of btl expression marked by arrows (as compared with wild-type in Figure 2B). (B) Expression of ectopic TrhS665A does not induce ectopic btl transcription domains. (C) Expression of Trh in trh7J83 mutant background showing rescue of all btl expression including the ectopic domains indicated by arrows. (D) Expression of TrhS665A in a trh7J83 mutant background does not restore btl transcription. (E and F) Expression of ectopic Trh in a Dakt1q mutant background fails to induce btl transcription in ectopic domains ([F]; as compared with [A]) indicated by arrows, whereas ectopic TrhS665D is able to induce some ectopic btl transcription (F). Developmental Cell 2001 1, 817-827DOI: (10.1016/S1534-5807(01)00090-9)

Figure 7 Expression and Cellular Localization of Wild-Type and Mutant Trh Expression of Trh wild-type and S665A mutant proteins in embryos. Protein extracts from embryos collected from wild-type (lane 1), arm GAL4 x UAS Trh-Flag (lane 2), and arm GAL4 x UAS trhS665A-Flag (lane 3) flies were immunoblotted with anti-Flag M2 antibodies (A). Fluorescent-tagged confocal imaging of optical sections of embryos expressing Trh-Flag (B) or TrhS665A-Flag (C) using anti-Flag M2 antibodies. While Trh-Flag is localized to the nucleus (B), TrhS665A-Flag is excluded from the nucleus (C). Confocal imaging of Trh expression from arm GAL4 x UAS trh (arm-trh) in wild-type (D) or Dakt1q/Df(3R)sbd45 embryos (E). Ectopic Trh from arm-trh is nuclear in all cells of the wild-type embryo (D). In the absence of zygotic Dakt1, ectopic Trh from arm-trh is largely cytoplasmic (E). Note that the endogenous Trh expression in arm-trh; Dakt1 embryos shows some nuclear localization in vicinity of the tracheal placodes (E). In (B–E), tracheal pits are marked with arrowheads. Developmental Cell 2001 1, 817-827DOI: (10.1016/S1534-5807(01)00090-9)