kuzbanian-mediated cleavage of Drosophila Notch

Abstract

Loss of Kuzbanian, a member of the ADAM family of metalloproteases, produces neurogenic phenotypes in Drosophila. It has been suggested that this results from a requirement for kuzbanian-mediated cleavage of the Notch ligand Delta. Using transgenic Drosophila expressing transmembrane Notch proteins, we show that kuzbanian, independent of any role in Delta processing, is required for the cleavage of Notch. We show that Kuzbanian can physically associate with Notch and that removal of kuzbanian activity by RNA-mediated interference in Drosophila tissue culture cells eliminates processing of ligand-independent transmembrane Notch molecules. Our data suggest that in Drosophila, kuzbanian can mediate S2 cleavage of Notch.

Figure 1

Diagram of N constructs and localization of epitopes recognized by antibodies. (A) The antibodies used in this work, above the regions of N used to generate them, and the region of N fused to GST (BD) used in the pull-down assays in Figure 2B. The top molecule in B is wild-type N tagged with the DNA-binding domain of LexA, N^LexA. (S) Signal sequence; (EGF) epidermal growth factor-like repeats; (LNG) Lin-12, N, Glp-1 repeats; (S2) location of TACE cleavage site in mammals; (S3) location of Psn-dependent cleavage site; (TM) transmembrane domain; (nls1, nls2) nuclear localization signals; (CDC10) cdc10 or ankyrin repeats; (polyQ) polymeric glutamines; (PEST) PEST sequence thought to be involved in protein stability. Shown beneath N^LexA are the various deletions used in this work.

Figure 2

Kuz can associate with N. (A) S2 cells transfected with actin-driven N alone (lanes 1,4) or with actin N plus actin-driven myc-tagged kuz (lane 2) or kuz^DN (lane 3) were immunoprecipitated with anti-myc (lanes 1–3) or anti-NI (lane 4) antibodies, and the Western probed with anti-NPCR antisera. See Figure 1A for location of the epitopes recognized by the antibodies. (B) Bacterially produced GST–N fusion protein encoding amino acids 1623–1893 of N (from the end of the LNG repeats to the start of the ankyrin repeats, BD in Fig. 1A) was used in pull-down assays of in vitro translated Su(H) (lane 3), Kuz (lane 6) and human insulin receptor (hIR, lane 9) labeled with ³⁵S methionine. (I) 1% of the input in vitro translation product; (V) GST alone; (N) GST–N fusion.

Figure 3

A comparison of the activities of LN^LexA and LNR^LexA. (A–E) The effect that expressing N^LexA (A), LN^LexA (B,C), or LNR^LexA (D,E) under control of daughterless (da) GAL4 (a ubiquitous driver) has on the nervous system of wild-type embryos. (A,B,D) Ventral views; (C,E) lateral views. The embryos were stained with an anti-horseradish peroxidase (HRP) antibody that reacts with the nervous system. (F) Western blot showing increased production of N^pp114LexA in embryos expressing LNR^LexA. Extracts of embryos expressing N^LexA (lanes 1,2), LN^LexA (lanes 3,4), or LNR^LexA (lanes 5,6) under the control of daGAL4 were immunoprecipitated with anti-Su(H) antisera, and the Western was reacted with anti-LexA antisera. The immunoprecipitates in lanes 2, 4, and 6 were treated with phosphatase prior to electrophoresis. N^LexA is the full-length N protein that coimmunoprecipitates with Su(H); N^pp114LexA is the phosphorylated cleaved cytoplasmic domain that associates with Su(H); and N^p100LexA is the phosphatased cytoplasmic domain that associates with Su(H) (Kidd et al. 1998). (G–I) The increase in nuclear entry of the cytoplasmic domain derived from LNR^LexA. S2 cells were transfected with UAS constructs encoding N^LexA, LN^LexA, or LNR^LexA, and a plasmid encoding heat shock (hs) GAL4 at a ratio of 1:10. Cells were heat-shocked for 30 min and allowed to recover for 2 h prior to fixation. They were then reacted with the NT antibody, which reacts with the extracellular domain; the NPCR antibody, which reacts with the intracellular domain; and sytox green to label the DNA. See Figure 1A for location of the epitopes recognized by the antibodies. (J,K) embryos expressing N^LexA (J) or LNR^LexA (K) under control of daGal4 were reacted with anti-LexA antibody to detect the N protein derived from the transgene and propidium iodide, which reacts with the DNA.

Figure 4

Signaling from LN^LexA and LNR^LexA is decreased in the absence of Dl. (A–D) Embryos expressing LN^LexA (A), LNR^LexA (B), and N^IntraLexA (C,D) under control of daGAL4, reacted with anti-HRP antibody. The embryo in C is Dl⁺, the other three are zygotic Dl nulls. The same transgenic lines were used in wild-type (Fig. 3B–E; Fig. 4C) and Dl backgrounds. Of the three antineurogenic N proteins, N^IntraLexA produces the strongest antineurogenic phenotype in a wild-type background. (E) Extracts of embryos expressing N^LexA (lanes 1,2), LN^LexA (lanes 3,4), and LNR^LexA (lanes 5,6) under control of daGAL4, in either wild-type (lanes 1,3,5) or zygotic Dl backgrounds (lanes 2,4,6), were immunoprecipitated with anti-Su(H) antibody and the Western reacted with anti-LexA antibody. To ensure that the protein being characterized is derived from Dl embryos, both the N transgenes and daGAL4 were recombined onto Dl chromosomes.

Figure 5

Signaling from LN^LexA and LNR^LexA is decreased in the absence of Kuz. All the embryos (A–E) were reacted with anti-HRP antibody. (A) Ventral view of an embryo that is maternally and zygotically kuz null. (B–E) The effect of expressing in a maternal and zygotic kuz background N^LexA (B), LN^LexA (C), LNR^LexA (D), and N^IntraLexA (E). All the N proteins were expressed under control of daGAL4. The white stars in E point out the remnants of the nervous system in kuz; N^IntraLexA embryos. The same transgenic lines were used in wild-type (Fig. 3A–E; Fig. 4C) and kuz backgrounds. (F) Extracts of embryos expressing N^LexA (lanes 1,2), LN^LexA (lanes 3,4), and LNR^LexA (lanes 5,6) under control of daGAL4, in either wild-type (lanes 1,3,5) or maternal and zygotic kuz backgrounds (lanes 2,4,6) were immunoprecipitated with anti-Su(H) antibody and the Western reacted with anti-LexA antibody. To ensure that the protein being characterized is derived from kuz embryos, the N transgenes were recombined onto kuz chromosomes.

Figure 6

Anti-N immunoprecipitates of extracts from Dl and kuz embryos differ. (A) Extracts of embryos expressing N^LexA (lanes 2,3), LN^LexA (lanes 4,5), and LNR^LexA (lanes 6,7), under the control of daGAL4 were immunoprecipitated with anti-NI antibody (see Fig. 1A) and the Western reacted with anti-LexA antibody. Extracts of embryos expressing N^LexA were also immunoprecipitated with anti-Su(H) antibody (lane 1). The immunoprecipitates in lanes 1, 3, 5, and 7 were treated with phosphatase prior to electrophoresis. N^p100LexA is the phosphatased cytoplasmic domain that interacts with Su(H) (Kidd et al. 1998). (S2 ●) Migration of S2-cleaved N; (S3 *) migration of S3-cleaved N; (S) Su(H); (▴ in lanes 2,3) an ∼97-kD cleavage product present in NI immunoprecipitates of extracts expressing N^LexA that does not associate with Su(H), but is dependent on Psn (see text); (○ in lanes 5,6) an ∼108-kD cleavage product found in NI immunoprecipitates of LN^LexA. (B) Extracts of embryos expressing N^LexA (lanes 1–3), LN^LexA (lanes 4–6), and LNR^LexA (lanes 7–9), under the control of daGAL4, which had been fractionated into membrane and soluble fractions, were immunoprecipitated with anti-NI antibody and the Western reacted with anti-LexA antibody. (T) Unfractionated extract; (M) membrane fraction; (So) soluble fraction. All immunoprecipitates were phosphatased prior to electrophoresis. (C) Extracts of embryos expressing LNR^LexA under the control of daGAL4 were fractionated into membrane, cytoplasmic, and nuclear fractions prior to immunoprecipitation with anti-NI antibody. The immunoprecipitates in lanes 2, 4, and 6 were phosphatased. The Western was reacted with anti-LexA antibody. (M) Membrane; (C) cytoplasm; (N) nuclear. The smear representing phosphorylated S3 in the nuclear fraction (lane 5) is more easily visible on longer exposures. (D,E) Extracts of embryos expressing N^LexA were immunoprecipitated with anti-NI antibody (D, lanes 1–5; E, lanes 2–7) and the Western reacted with anti-LexA antibody. The extracts in lanes 1 and 5 of D and lanes 2 and 5 of E were from wild-type embryos (WT). The extracts in lane 2 of D and lanes 3 and 6 of E were from zygotic Dl embryos. The extracts in lane 3 of D and lanes 4 and 7 of E were from maternal and zygotic kuz embryos. The extract in lane 4 of D was from maternal and zygotic Psn embryos. (D, lane 6; E, lane 1) Extracts of N^LexA embryos were also immunoprecipitated with anti-Su(H) antibody (S). The immunoprecipitates in lanes 4 and 5 of D were derived from embryos expressing N^LexA under the control of armadillo (arm) GAL4. All the other immunoprecipitates were derived from embryos expressing N^LexA under the control of daGAL4. (▴ in D, lanes 1,2,5; E, lanes 2,3,5,6) The N^LexA derived protein that is dependent on Psn but does not associate with Su(H). (E, lanes 5–7) A longer exposure of lanes 2–4. (S2* in E) The N cleavage product the size of S2-cleaved N that is found in kuz extracts but is not processed further (see text). (F,G) Comparisons of the anti-NI immunoprecipitates of WT (lane 3), Dl (lane 4), kuz (lane 5), and Psn (lane 6) embryos expressing LN^LexA (F) and LNR^LexA (G). In lanes 1 and 2 extracts of embryos expressing N^LexA were immunoprecipitated with anti-Su(H) antibody (S) and anti-NI antibody respectively. (G, lane 7) extracts of Psn embryos expressing N^LexA were immunoprecipitated with anti-NI antibody. (G*) S3-cleaved N. All the immunoprecipitates in D–G were phosphatased prior to electrophoresis. To ensure that the protein being characterized is derived from Dl embryos, both the N transgenes and daGAL4 were recombined onto Dl chromosomes and to ensure that the protein being characterized is derived from kuz and Psn embryos, the N transgenes were recombined onto kuz and Psn chromosomes, respectively. All the immunoprecipitates within each panel (A–G) were electrophoresed on the same gel. In some of the panels different exposures of the lanes were used to generate the figure. Only the regions where the S2 and S3 cleavage products migrate are shown.

Figure 6

Anti-N immunoprecipitates of extracts from Dl and kuz embryos differ. (A) Extracts of embryos expressing N^LexA (lanes 2,3), LN^LexA (lanes 4,5), and LNR^LexA (lanes 6,7), under the control of daGAL4 were immunoprecipitated with anti-NI antibody (see Fig. 1A) and the Western reacted with anti-LexA antibody. Extracts of embryos expressing N^LexA were also immunoprecipitated with anti-Su(H) antibody (lane 1). The immunoprecipitates in lanes 1, 3, 5, and 7 were treated with phosphatase prior to electrophoresis. N^p100LexA is the phosphatased cytoplasmic domain that interacts with Su(H) (Kidd et al. 1998). (S2 ●) Migration of S2-cleaved N; (S3 *) migration of S3-cleaved N; (S) Su(H); (▴ in lanes 2,3) an ∼97-kD cleavage product present in NI immunoprecipitates of extracts expressing N^LexA that does not associate with Su(H), but is dependent on Psn (see text); (○ in lanes 5,6) an ∼108-kD cleavage product found in NI immunoprecipitates of LN^LexA. (B) Extracts of embryos expressing N^LexA (lanes 1–3), LN^LexA (lanes 4–6), and LNR^LexA (lanes 7–9), under the control of daGAL4, which had been fractionated into membrane and soluble fractions, were immunoprecipitated with anti-NI antibody and the Western reacted with anti-LexA antibody. (T) Unfractionated extract; (M) membrane fraction; (So) soluble fraction. All immunoprecipitates were phosphatased prior to electrophoresis. (C) Extracts of embryos expressing LNR^LexA under the control of daGAL4 were fractionated into membrane, cytoplasmic, and nuclear fractions prior to immunoprecipitation with anti-NI antibody. The immunoprecipitates in lanes 2, 4, and 6 were phosphatased. The Western was reacted with anti-LexA antibody. (M) Membrane; (C) cytoplasm; (N) nuclear. The smear representing phosphorylated S3 in the nuclear fraction (lane 5) is more easily visible on longer exposures. (D,E) Extracts of embryos expressing N^LexA were immunoprecipitated with anti-NI antibody (D, lanes 1–5; E, lanes 2–7) and the Western reacted with anti-LexA antibody. The extracts in lanes 1 and 5 of D and lanes 2 and 5 of E were from wild-type embryos (WT). The extracts in lane 2 of D and lanes 3 and 6 of E were from zygotic Dl embryos. The extracts in lane 3 of D and lanes 4 and 7 of E were from maternal and zygotic kuz embryos. The extract in lane 4 of D was from maternal and zygotic Psn embryos. (D, lane 6; E, lane 1) Extracts of N^LexA embryos were also immunoprecipitated with anti-Su(H) antibody (S). The immunoprecipitates in lanes 4 and 5 of D were derived from embryos expressing N^LexA under the control of armadillo (arm) GAL4. All the other immunoprecipitates were derived from embryos expressing N^LexA under the control of daGAL4. (▴ in D, lanes 1,2,5; E, lanes 2,3,5,6) The N^LexA derived protein that is dependent on Psn but does not associate with Su(H). (E, lanes 5–7) A longer exposure of lanes 2–4. (S2* in E) The N cleavage product the size of S2-cleaved N that is found in kuz extracts but is not processed further (see text). (F,G) Comparisons of the anti-NI immunoprecipitates of WT (lane 3), Dl (lane 4), kuz (lane 5), and Psn (lane 6) embryos expressing LN^LexA (F) and LNR^LexA (G). In lanes 1 and 2 extracts of embryos expressing N^LexA were immunoprecipitated with anti-Su(H) antibody (S) and anti-NI antibody respectively. (G, lane 7) extracts of Psn embryos expressing N^LexA were immunoprecipitated with anti-NI antibody. (G*) S3-cleaved N. All the immunoprecipitates in D–G were phosphatased prior to electrophoresis. To ensure that the protein being characterized is derived from Dl embryos, both the N transgenes and daGAL4 were recombined onto Dl chromosomes and to ensure that the protein being characterized is derived from kuz and Psn embryos, the N transgenes were recombined onto kuz and Psn chromosomes, respectively. All the immunoprecipitates within each panel (A–G) were electrophoresed on the same gel. In some of the panels different exposures of the lanes were used to generate the figure. Only the regions where the S2 and S3 cleavage products migrate are shown.

Figure 7

A Dl-independent N protein requires kuz for activity. (A–C) Ventral views of anti-HRP stains of embryos expressing ΔEGF 1–18 LNRLexA under control of daGAL4, in wild-type (A), Dl (B), or kuz (C) backgrounds. (D) Extracts of embryos expressing ΔEGF 1–18 LNRLexA under control of daGAL4 were immunoprecipitated with anti-NI antibody (lanes 2,3) or anti-Su(H) antibody (lanes 6,7). The extracts in lanes 2 and 6 are from wild-type embryos, and the extracts in lanes 3 and 7 are from Dl embryos. (Lane 1) An anti-NI immunoprecipitate of wild-type embryos expressing N^LexA; (lanes 4,5) anti-Su(H) immunoprecipitates of embryos expressing N^LexA. The immunoprecipitates in lanes 1–4 were treated with phosphatase. (*, lanes 2,3) S3-cleaved N; (●, lane 1) S2-cleaved N. (E) Extracts of embryos expressing ΔEGF 1–18 LNRLexA under control of daGAL4 were immunoprecipitated with anti-NI antibody (lanes 3,4) or anti-Su(H) antibody (lanes 6,7). The extracts in lanes 3 and 6 are from wild-type embryos, and the extracts in lanes 4 and 7 are from kuz embryos. (Lane 2) An anti-NI immunoprecipitate of wild-type embryos expressing N^LexA; (lanes 1,5) anti-Su(H) immunoprecipitates of embryos expressing N^LexA. The immunoprecipitates in lanes 1–4 were treated with phosphatase. (*, lane 3) S3-cleaved N. The Westerns in both D and E were reacted with anti-LexA antisera.

Figure 8

Kuzbanian promotes the cleavage of Notch. (A, lanes 3–12) are anti-NI immunoprecipitates of S2 cells expressing heat-shock-induced LNR^KasLexA (see Fig. 1B) that have been treated with the indicated double-stranded (ds) RNAs. (Lane 2) An anti-NI immunoprecipitate of S2 cells expressing heat-shock-induced N^KasLexA (see Fig. 1B); (lanes 1,13) anti-Su(H) immunoprecipitates of S2 cells expressing LNR^KasLexA. (B, lanes 3–15) anti-NI immunoprecipitates of S2 cells expressing heat-shock-induced LNR^KasLexA (lanes 3–11) or ΔEGF 1–18 LNR^KasLexA (lanes 12–15; see Fig. 1B) that have been treated with the indicated double-stranded (ds) RNAs. The cells in lanes 7–10 and 14 were cotransfected with an actin-driven TACE construct. (Lane 1) An anti-NI immunoprecipitate of S2 cells expressing heat-shock-induced N^KasLexA; (lane 2) an anti-Su(H) immunoprecipitate of S2 cells expressing LNR^KasLexA. All the immunoprecipitates were phosphatased prior to electrophoresis, and the Westerns were reacted with anti-LexA antisera. (S2, S3) Locations of S2 and S3 cleavage products.