Ligand-induced cleavage and regulation of nuclear entry of Notch in Drosophila melanogaster embryos

Abstract

Notch, a transmembrane protein found in a wide range of organisms, is a component of a pathway that mediates cell-fate decisions that involve intercellular communication. In this paper, we show that in Drosophila melanogaster, Notch (N) is processed in a ligand-dependent fashion to generate phosphorylated, soluble intracellular derivatives. Suppressor of Hairless [Su(H)] is predominantly associated with soluble intracellular N. It has been demonstrated by others that N has access to the nucleus, and we show that when tethered directly to DNA, the cytoplasmic domain of N can activate transcription. Conversely, a viral activator fused to Su(H) can substitute for at least some N functions during embryogenesis. We suggest that one function of soluble forms of N is to bind to Su(H), and in the nucleus, to act directly as a transcriptional transactivator of the latter protein. Although N has functional nuclear localization signals, the N/Su(H) complex accumulates in the cytoplasm and on membranes suggesting that its nuclear entry is regulated. Localization studies in cultured cells and embryos suggest that Su(H) plays a role in this regulation, with the relative levels of Delta, N and Su(H) determining whether a N/Su(H) complex enters the nucleus.

Figure 1

Constructs and antibodies. (A) A diagram of the N protein (N). (EGF) Epidermal growth factor-like repeats; (LNG) lin-12, Notch, Glp-1 repeats; (cc) evolutionary conserved cysteine residues; (CDC10) CDC10 or ankyrin repeats; (nls1, nls2) putative nuclear localization signals; (polyQ) polymeric glutamines; (PEST) PEST sequence thought to be involved in protein stability (Rogers et al. 1986). The bars above the diagram of N indicate the various regions used as antigens to create anti-N antibodies. Beneath the diagram of N are the N constructs used in this paper. (B) Su(H) constructs used in this paper.

Figure 2

Su(H) is associated with phosphorylated N proteins. (A,B) Western blot analysis of anti-Su(H) immunoprecipitates from Drosophila embryos probed with anti-N (NPCR) antibody. For A, an overnight collection of embryos was used and in B, staged embryos (age indicated in hours at top of lanes; H denotes hatching) were used for detergent extracts. Prior to electrophoresis, some of the samples were treated with alkaline phosphatase with or without inhibitor. (+) Presence of phosphatase and/or inhibitor. (A, Lane 4 and B, lane 9) In vitro-translated N^Intra1768, the size of which is not affected by phosphatase treatment; (A, lane 5) Antibody but no extract; (A, lane 6) Embryo extract recovered on protein A–Sepharose beads that were not conjugated to antibody. The locations of N (N), N^Intra1768, N^pp114, N^p100A, N^p100B, and N^p100C proteins are shown along side each blot, and also by dots on the blot. The × in A indicates a cross-reacting protein nonspecifically bound to the protein A–Sepharose beads; the asterisk (*) in B indicates a hypophosphorylated N protein that comigrates with N^p100B. (A, all lanes with extract; B, lanes 3–6) 500 μg of protein was used for each immunoprecipitation; the remaining lanes in B contain 2.5 mg(lanes 1,2) and 1.75 mg (lanes 7,8). (C) Western blot analysis of anti-N (NI) immunoprecipitates from Drosophila embryos probed with anti-N (NPCR) antibody. (Lanes 1, 2) Immunoprecipitates from yw embryos; (lanes 3,4) immunoprecipitates of embryos resulting from a cross of Su(H) hsN^intra1790/CyO males to Su(H)^SF8 FRT40A/CyO virgins; (lanes 5,6) immunoprecipitates of embryos resulting from a cross of Su(H) hsN^intra1790/CyO males to hsflp12/yw; Su(H)^SF8 FRT40A/ovo^D FRT40A virgins. All N^Intra expressing embryos are both zygotically and maternally Su(H) null. For all genotypes, 3- to 5 hr-old embryos were subjected to a 30 min heat shock at 37°C and allowed to recover for 15–30 min prior to collection. (+) Phosphatase treatment. The locations of N (N) and N^Intra are indicated next to the blot.

Figure 2

Su(H) is associated with phosphorylated N proteins. (A,B) Western blot analysis of anti-Su(H) immunoprecipitates from Drosophila embryos probed with anti-N (NPCR) antibody. For A, an overnight collection of embryos was used and in B, staged embryos (age indicated in hours at top of lanes; H denotes hatching) were used for detergent extracts. Prior to electrophoresis, some of the samples were treated with alkaline phosphatase with or without inhibitor. (+) Presence of phosphatase and/or inhibitor. (A, Lane 4 and B, lane 9) In vitro-translated N^Intra1768, the size of which is not affected by phosphatase treatment; (A, lane 5) Antibody but no extract; (A, lane 6) Embryo extract recovered on protein A–Sepharose beads that were not conjugated to antibody. The locations of N (N), N^Intra1768, N^pp114, N^p100A, N^p100B, and N^p100C proteins are shown along side each blot, and also by dots on the blot. The × in A indicates a cross-reacting protein nonspecifically bound to the protein A–Sepharose beads; the asterisk (*) in B indicates a hypophosphorylated N protein that comigrates with N^p100B. (A, all lanes with extract; B, lanes 3–6) 500 μg of protein was used for each immunoprecipitation; the remaining lanes in B contain 2.5 mg(lanes 1,2) and 1.75 mg (lanes 7,8). (C) Western blot analysis of anti-N (NI) immunoprecipitates from Drosophila embryos probed with anti-N (NPCR) antibody. (Lanes 1, 2) Immunoprecipitates from yw embryos; (lanes 3,4) immunoprecipitates of embryos resulting from a cross of Su(H) hsN^intra1790/CyO males to Su(H)^SF8 FRT40A/CyO virgins; (lanes 5,6) immunoprecipitates of embryos resulting from a cross of Su(H) hsN^intra1790/CyO males to hsflp12/yw; Su(H)^SF8 FRT40A/ovo^D FRT40A virgins. All N^Intra expressing embryos are both zygotically and maternally Su(H) null. For all genotypes, 3- to 5 hr-old embryos were subjected to a 30 min heat shock at 37°C and allowed to recover for 15–30 min prior to collection. (+) Phosphatase treatment. The locations of N (N) and N^Intra are indicated next to the blot.

Figure 2

Su(H) is associated with phosphorylated N proteins. (A,B) Western blot analysis of anti-Su(H) immunoprecipitates from Drosophila embryos probed with anti-N (NPCR) antibody. For A, an overnight collection of embryos was used and in B, staged embryos (age indicated in hours at top of lanes; H denotes hatching) were used for detergent extracts. Prior to electrophoresis, some of the samples were treated with alkaline phosphatase with or without inhibitor. (+) Presence of phosphatase and/or inhibitor. (A, Lane 4 and B, lane 9) In vitro-translated N^Intra1768, the size of which is not affected by phosphatase treatment; (A, lane 5) Antibody but no extract; (A, lane 6) Embryo extract recovered on protein A–Sepharose beads that were not conjugated to antibody. The locations of N (N), N^Intra1768, N^pp114, N^p100A, N^p100B, and N^p100C proteins are shown along side each blot, and also by dots on the blot. The × in A indicates a cross-reacting protein nonspecifically bound to the protein A–Sepharose beads; the asterisk (*) in B indicates a hypophosphorylated N protein that comigrates with N^p100B. (A, all lanes with extract; B, lanes 3–6) 500 μg of protein was used for each immunoprecipitation; the remaining lanes in B contain 2.5 mg(lanes 1,2) and 1.75 mg (lanes 7,8). (C) Western blot analysis of anti-N (NI) immunoprecipitates from Drosophila embryos probed with anti-N (NPCR) antibody. (Lanes 1, 2) Immunoprecipitates from yw embryos; (lanes 3,4) immunoprecipitates of embryos resulting from a cross of Su(H) hsN^intra1790/CyO males to Su(H)^SF8 FRT40A/CyO virgins; (lanes 5,6) immunoprecipitates of embryos resulting from a cross of Su(H) hsN^intra1790/CyO males to hsflp12/yw; Su(H)^SF8 FRT40A/ovo^D FRT40A virgins. All N^Intra expressing embryos are both zygotically and maternally Su(H) null. For all genotypes, 3- to 5 hr-old embryos were subjected to a 30 min heat shock at 37°C and allowed to recover for 15–30 min prior to collection. (+) Phosphatase treatment. The locations of N (N) and N^Intra are indicated next to the blot.

Figure 3

The presence of Notch ligand and the extracellular domain of N are required for the isolation of N^p100 bound to Su(H). (A) Detergent extracts from embryos expressing either N^LexA (lanes 1,3) or N^{ΔEGF1-36–LexA} [lanes 2,4 (see Fig. 1 for structures of N^LexA and N^{ΔEGF1-36–LexA}] were immunoprecipitated with antibodies against either N (NI) (lanes 1,2) or Su(H) (lanes 3,4) treated with phosphatase, and then probed with antibodies against LexA. Antibodies against N immunoprecipitate N^LexA (lane 1) and N^{ΔEGF1-36–LexA} (lane 2); the smaller proteins seen in lane 2 are missing from lane 1 and, are most likely nonspecific breakdown products visible because of massive overexpression of N^{ΔEGF1-36–LexA} compared with N^LexA. N^LexA and N^{ΔEGF1-36–LexA} are coimmunoprecipitated by anti-Su(H) antibodies as is a smaller, processed form of N^LexA (N^p100–LEXA, lane 3, *). No such processed protein is seen when the extracellular ligand binding domain of N is deleted (lane 4). (B) Dl temperature-sensitive mutants reduce the level of processed N protein. yw (wt), Dl^6B/TM6 (a strong) or Dl^RF/TM6 (a weak temperature-sensitive Dl allele) males were mated to Dl^x/TM6 (an amorphic Dl allele) females. All the embryos resulting from the above crosses were incubated at either room temperature (lanes 1,3,5) or at the nonpermissive temperature, 29°C (lanes 2,4,6). N proteins coimmunoprecipitated from detergent extracts by anti-Su(H) antibody were detected with the NPCR anti-N antibody. Beneath each lane the ratio of processed N to N (termed N^pp114/N) is shown. The strong temperature-sensitive Dl allele (lanes 3,4) has a considerably greater effect on the level of processed N than the weaker allele (lanes 5,6)

Figure 4

Ligand-induced cleavage of N. (A) Anti-N antibodies immunoprecipitate a N protein, the size of N^p100B (*) from embryos exposed to ectopic Dl (lane 5). Embryos aged from 2 to 4 hr were heat-shocked at 37°C for 30 min and allowed to recover at 30°C for 2 hr prior to detergent extraction. Extracts from either heat-shocked yw (lanes 1,2) or heat-shocked GAL4; UAS Dl with (lanes 5,6), or without heat shock (lanes 3,4) were immunoprecipitated with anti-N NI antibody. Lane 7 lacks embryo extract; lane 8 contains N^Intra1768 in vitro translation products. After immunoprecipitation, lanes marked with a + were treated with alkaline phosphatase prior to electrophoresis. N proteins were detected with the anti-N antibody NPCR. (Labels) Locations of N (N) and N^Intra1768(N^Intra). (B) Coexpression of N^LexA and Dl results in an increase in the level of N^pp114–LexA. Detergent extracts from 4–8 hr UASN^lexA × hGAL4 embryos (lanes 1,2,5,6) or UAS Dl30B; UAS N^lexA × hGAL4 embryos (lanes 3,4,7,8) were immunoprecipitated with either anti-N antibody (NI) (lanes 1–4) or anti-Su(H) antibody (lanes 5–8). Samples in lanes 2, 4, 6, and 8 were treated with alkaline phosphatase prior to electrophoresis. After blotting, proteins were detected with an anti-LexA monoclonal antibody. Coexpression of Dl and N^LexA causes the appearance of a novel hypophosphorylated N^LexA protein in the anti-N immunoprecipitations (*, cf. lanes 1 and 3). In this experiment, there was an ∼40%–50% increase in the amount of N^pp114–LexA bound to Su(H) (cf. the ratios of N^pp114LexA to N^LexA in lane 5 with lane 7, and in lane 6 with lane 8). (C) A histogram comparing the relative amounts of processed vs. full-length N associated with Su(H) in the absence or presence of ectopic Dl. The average of the results of four immunoprecipitations are plotted. The fold increase in the amount of processed N compared with full-length N in the presence of UAS Dl for each of the four experiments was as follows: 1.7, 1.5, 2.0, and 1.8.

Figure 5

N^pp114 is a soluble protein. (A) Subcellular distribution of N^pp114. Subcellular fractions were prepared from Drosophila embryos that had been lysed under hypotonic conditions (10 mm KCl). (Nuc) The nuclear fraction; (P100) the membrane fraction; (S100) the soluble fraction. The position of N^pp114 is indicated by a square bracket, N^p100B by an arrowhead, and N^p86 by a dot. Equal proportions of each fraction were immunoprecipitated with antibodies against either N (NI) (lanes 1–6) or Su(H) (lanes 7–12) and then detected with the anti-NPCR antibody. The autoradiograph has been overexposed to show the presence of processed N in the soluble fraction. Because the amount of N in the membrane fraction is so high, lanes 1 and 2 are from a shorter exposure. (B) Evidence for additional processing of the cytoplasmic domain of N. Soluble proteins extracted in 0.4 m KCl were immunoprecipitated with anti-NI antibody and detected with anti-NPCR antibody. As well as immunoprecipitating the same N proteins as those coimmunoprecipitated by anti-Su(H) antibodies, anti-N antibodies immunoprecipitate two novel proteins of ∼99 kD (N^pp99) and 86 kD (N^p86) (lane 1), treatment with alkaline phosphatase (lane 2) reduces the amount of N^pp99 and increases the amount of N^p86, suggesting that N^pp99 is a phosphorylated form of N^p86. (C) Some N^pp114 is associated, but not stably, with membranes. Postnuclear supernatants were incubated with increasing amounts of KCl prior to fractionation by centrifugation into membrane bound (lanes marked M) or soluble proteins (lanes marked S). Fractionated proteins were then immunoprecipitated with anti-Su(H) antibody and detected with the anti-N NPCR antibody, a longer exposure of the relevant region of the resulting autoradiograph is shown at bottom. Some N^pp114 can be seen to be associated with membranes when extracted at 0.1 m KCl but is removed at higher salt concentrations. The protein that comigrates with N^p100B and appears to be stably associated with membranes is marked by an asterisk.

Figure 6

N^Intra is retained in the cytoplasm in embryos. Confocal images showing the localization of N^Intra in embryos and Drosophila S2 cells as detected by immunofluorescence. With the exception of the left panel in C, N^Intra protein is represented in green and the nuclei in red. This panel is a pseudocolored representation to illustrate the relative amounts of N^Intra1768. The correspondence of the colors with the intensity of the signal is indicated by the pseudocolor bar, with the more intense signals being depicted by colors higher up the bar. (A) Heat shock-induced N^Intra1768 appears to be totally nuclear in S2 cells. UAS N^Intra1768 was cotransfected along with HS GAL4 and detected with a rabbit anti-N (NI) antibody. The nucleus was detected with SYTOX Green. (B,C) In embryos (hGAL4; UAS N^intra1768) there is retention of N^Intra1768 in the cytoplasm. (B,C, right) The merged images of the N signal in green and the nuclei in red. (C, left) N^Intra1768 is primarily nuclear in those cells in which it is most highly expressed. Mouse anti-NPCR antibody was used to detect N^Intra1768. (B) Mouse anti-Flag antibody was used to detect Flag-tagged N^Intra1768. The nuclei were detected by propidium iodide. (D) N^Intra1768 is still retained in the cytoplasm in embryos that are maternally and zygotically N null (N^264–47 FRT/ovo^D FRT; hGAL4/hsflp X FM7/Y; N^intra1768. (D, left) Mouse NPCR antibody was used to detect N^Intra1768; (D, right) a merged image of N (green) and nuclei (red). (E) N^Intra1790 is predominantly nuclear in embryos with reduced levels of Su(H). Anti- myc antibody was used to detect myc-tagged N^intra1790 in embryos that are maternally Su(H)⁻ (hsflp/yw;Su(H)^SF8 FRT/ovo^D FRT;hGAL4 X Su(H)/CyO; UASN^Intra1790). In embryos that are maternally Su(H)⁺ (hGAL4 X Su(H)/CyO;UASN^Intra1790) there is retention of N^Intra1790 in the cytoplasm (data not shown).

Figure 7

Su(H) can retain N^Intra in the cytoplasm. Confocal images showing the localization of Su(H) and N^Intra1768. With the exception of F, which is a pseudocolored image, Su(H) is represented in blue, N^Intra in green, and the nuclei in red. (A) Su(H) is expressed in both the cytoplasm and nuclei of transfected S2 cells. S2 cells were transfected with a construct expressing Su(H) under the control of an actin promoter. Su(H) is detected with rat anti-Su(H) antibody and the DNA with SYTOX Green. (B,C) Coexpression of high levels of N^Intra1768 along with Su(H) results in both being found in nuclei of S2 cells. (B) Two cells are depicted showing Su(H) expression in blue and N expression in green. In the lower cell, no N^Intra1768 is present and Su(H) is localized ubiquitously. In contrast, the upper cell expresses high levels of N^Intra1768 along with Su(H) and both are localized in the nucleus. A merged image of the first two panels is shown at right. The cell shown in C was probed with a DNA marker as well as with anti-N and Su(H) antibodies. Actin driven Su(H) was cotransfected with UAS N^Intra1768 and HS GAL4. Su(H) protein was detected by rat anti-Su(H) antibody, N protein was detected by a mouse anti-Flag antibody, and the DNA with SYTOX Green. (D,E,G) The difference in localization of N^Intra1768 promoted by expression of an excess of Su(H). N^Intra is represented in green and the nuclei in red. (D) UAS N^Intra1768 and HS GAL4 were transfected into S2 cells. (E,G) A 20× mass excess of actin Su(H) was cotransfected along with UAS N^Intra1768 and HS GAL4. (D,E) Localization of N^Intra1768 in nuclei is indicated by dots and retention in the cytoplasm by asterisks. Two cells from those shown in E are depicted at higher magnification in G. As cytoplasmic localization of N^Intra1768 is never seen in cells lacking ectopic Su(H) (D), the upper cell in G must have received more Su(H) relative to N^Intra1768 than the lower one, resulting in N^Intra1768 being retained in the cytoplasm. N^Intra1768 was detected with a rabbit anti-N (NI) antibody and the nuclei with SYTOX Green. (F) When low levels of N^Intra1768 are expressed along with Su(H), N^Intra1768 is retained in the cytoplasm. A pseudocolored confocal image showing the relative levels on N at right and Su(H) at left is portrayed. The intensity of staining is depicted by the pseudocolor bar with the colors representing the more intensely stained regions being higher up the bar. In the lower cell, N^Intra1768 is expressed at relatively high levels and both N^Intra1768 and Su(H) are found in the nucleus. In the upper cell, N^Intra1768 is expressed at relatively low levels and both N^Intra1768 and Su(H) are found in the cytoplasm. When cells are doubly stained for DNA and N^Intra1768, the two stains converge only when levels of N^Intra1768 are high compared with Su(H) (data not shown). Su(H) was detected with a rat anti-Su(H) antibody and N^Intra1768 with a mouse anti-Flag antibody.

Figure 8

The cytoplasmic domain of N can behave as a transcriptional activator. (A) The cytoplasmic domain of N is a transcriptional activator in yeast. The transcriptional activation activity of various N constructs fused to the DNA-binding domain of LexA (Fig. 1) was determined by their ability to drive expression from a LexA–β-galactosidase reporter. The bar marked LexA is the β-galactosidase activity of yeast expressing the vector alone, LexA–Bicoid^2-160 is a negative control. The height of the bar is the average of three samples, standard deviations are shown by the error bars. (B) N^Intra1790 cannot activate transcription from LexA–β-galactosidase reporter. A lexA–β-gal; UAS N^intra1790; HS GAL4 embryo is stained with anti β-galactosidase antibody. There is anti β-galactosidase reactivity in the secretory cells and the anal pads that results from leakiness of the reporter. (C) N^{Intra1790–LexA} accumulates to its highest levels in the salivary glands, amnioserosa, and midgut. A lexA–β-gal; UAS N^{intra1790–lexA}; HS GAL4 embryo is stained with anti-NPCR antibody. (D) N^{Intra1790–LexA} induces expression of β-gal from the LexA–β-gal reporter. A lexA–β-gal; UAS N^{intra1790–LexA}; HS GAL4 embryo is stained with anti β-galactosidase antibody. β-Galactosidase accumulates in the salivary glands, amnioserosa and midgut, which correspond with regions that accumulate the highest levels of N^{Intra1790–LexA} in C. (B,C,D) Embryos were fixed 2 hr after a 30-min heat shock. (E) A heterologous activator fused to Su(H) can substitute for N function and activate transcription of m8. N^264-47 FRT/ovo^D FRT; h GAL4/hsflp × FM7 lac-Z/Y; myc Su(H)–VP16 embryos are stained with an m8 probe. m8 expression is induced in seven stripes, in which h is expressed. Overexpression of Su(H) alone does not result in induction of m8 expression (data not shown). Anti β-galactosidase antibody was used to distinguish the N null embryos.