A family of Rhomboid intramembrane proteases activates all Drosophila membrane-tethered EGF ligands

Abstract

Drosophila has three membrane-tethered epidermal growth factor (EGF)-like proteins: Spitz, Gurken and Keren. Spitz and Gurken have been genetically confirmed to activate the EGF receptor, but Keren is uncharacterized. Spitz is activated by regulated intracellular translocation and cleavage by the transmembrane proteins Star and the protease Rhomboid-1, respectively. Rhomboid-1 is a member of a family of seven similar proteins in Drosophila. We have analysed four of these: all are proteases that can cleave Spitz, Gurken and Keren, and all activate only EGF receptor signalling in vivo. Star acts as an endoplasmic reticulum (ER) export factor for all three. The importance of this translocation is highlighted by the fact that when Spitz is cleaved by Rhomboids in the ER it cannot be secreted. Keren activates the EGF receptor in vivo, providing strong evidence that it is a true ligand. Our data demonstrate that all membrane-tethered EGF ligands in Drosophila are activated by the same strategy of cleavage by Rhomboids, which are ancient and widespread intramembrane proteases. This is distinct from the metalloprotease-induced activation of mammalian EGF-like ligands.

Fig. 1. Drosophila Rhomboids 1–4 are proteases that can cleave Spitz. (A) A dendrogram illustrating the sequence relationship between the seven Drosophila Rhomboids. Rhomboids 1–4 are most similar to each other. (B) GFP-tagged Spitz was cleaved by Rhomboids 1–4 in a Star-dependent manner when transiently expressed in COS cells and analysed by western blotting; the cleaved N-terminus of Spitz accumulated in the medium in all four cases. Note that Rhomboids 2, 3 and 4 could cleave Spitz intracellularly in the absence of Star (small arrowhead, full-length Spitz indicated by large arrowhead: compare the relative levels of full-length and cleaved Spitz in the presence of each Rhomboid), but this cleaved product was not secreted. The white arrowhead shows the hyperglycosylated form of Spitz caused by Star expression in the absence of Rhomboid (Lee et al., 2001). (C) Rhomboid levels in the Spitz cleavage assay were reduced by decreasing the amount of transfected rhomboid DNA (shown in ng) (Urban et al., 2001). All four Rhomboids cleaved and secreted Spitz at equivalent levels, even when they became limiting.

Fig. 2. Analysis of Rhomboid 1–4 activity in Drosophila. Rhomboids 1–4 were ectopically expressed in wings using the MS1096-Gal4 driver and caused EGF receptor hyperactivation phenotypes, including thick veins and blisters. Multiple transgenic lines were isolated in each case: Rhomboids 2 and 4 produced a spectrum of phenotypes (each indicated with three panels), which included phenotypes identical to Rhomboids 1 and 3, but their average phenotypes were significantly weaker. While the expression of Star and the weakest Rhomboid-4 transgene (which was X-linked and examined in females) yielded only subtle effects, if any, their co-expression resulted in strongly synergistic phenotypes (bottom row).

Fig. 3. Keren processing by Star and Rhomboids 1–4. (A) A GCG pileup alignment of Keren, Gurken and Spitz. A dashed line indicates the locations of the EGF domains. Transmembrane domains predicted by TMHMM (Krogh et al., 2001) are indicated with a solid line. (B) Western blots of conditioned medium and lysates from COS cells transfected with Keren with or without Star and/or Rhomboids 1–4. GFP–Keren was expressed alone (–) or in the presence (+S) or absence (–S) of Star with each of the four Rhomboids (R1–R4). Keren was cleaved and secreted efficiently by all four Rhomboids in the presence of Star; it was also cleaved and secreted at a lower level by Rhomboids 3 and 4 in the absence of Star. An intracellular cleaved product (arrowhead) was produced by all four Rhomboids. (C) Processing of Keren by Star and Rhomboid-1 in S2 cells: compared with Spitz, there is a higher level of cleavage and secretion triggered by Star or Rhomboid-1 alone, but the presence of Rhomboid-1 and Star together enhanced processing. (D and E) Intracellular localization of Keren. Cells were transfected with GFP–Keren with or without Myc-Star. The localization of each protein was detected by immunofluorescence. (D) GFP–Keren co-localized with an antibody against an endogenous ER marker (αPDI). (E) Co-expression of Star with GFP–Keren caused Keren to be exported from the ER and accumulate in the Golgi apparatus and at the cell surface.

Fig. 4. Ectopic expression of Keren in wings and the ventral epidermis. (A) Wild-type wing. (B and C) Full-length UAS-Keren expressed in wings with the MS1096-Gal4 driver produced a range of EGF receptor hyperactivity phenotypes: thickened wing veins and extra wing vein material. (D–F) Expression of secreted Spitz in the embryo with the arm-Gal4 driver produced typical EGF receptor overactivation phenotypes in the ventral epidermis (Szüts et al., 1997; Payre et al., 1999). Wild-type denticle belts have a characteristic arrangement of six rows (D); overexpression of sSpi (E) or sKer (F) caused the formation of extra denticles.

Fig. 5. Gurken processing by Star and Rhomboids 1–4. (A) Western blots of conditioned medium and lysates from COS cells transfected with Gurken with or without Star and/or Rhomboids 1–4. GFP–Gurken was expressed alone (–) or in the presence (+S) or absence (–S) of Star with each of the four Rhomboids (R1–R4). In COS cells, Gurken was cleaved and secreted by all four Rhomboids, independently of Star; all four also caused intracellular cleavage. (B) Processing of Gurken in S2 cells was similar to that in COS cells. (C and D) Intracellular localization of Gurken. Cells were transfected with GFP–Gurken and/or Myc-Star. The localization of each protein was detected by immunofluorescence. (C) GFP-tagged Gurken co-localized with an antibody against an endogenous ER marker (αPDI). (D) Co-expression of Star with GFP–Gurken caused Gurken to be exported from the ER and accumulate in the Golgi apparatus but, in contrast to Spitz and Keren, not at the cell surface.

Fig. 6. Intracellular localization of HA-tagged Rhomboids 1–4 in COS cells. Rhomboids 2 and 3 could only be detected in the Golgi apparatus like Rhomboid-1, and co-localized with the known Golgi marker p115. Rhomboid-4 displayed a pronounced cell surface distribution, although it was also detected in the Golgi apparatus, but not in the ER.

Fig. 7. The subcellular site of Spitz cleavage determines whether Spitz is secreted. (A) A limiting dilution series of Rhomboid-4 resulted in efficient intracellular Spitz cleavage in the absence of Star (arrowhead), but this form was not secreted (as detected by western blotting). Note that in the presence of Star, full-length Spitz became hyperglycosylated (arrow) when it passed through the Golgi apparatus without being cleaved (Lee et al., 2001) as a result of limiting Rhomboid-4 expression. (B) Rhomboid-1 targeted to the ER by a KDEL signal in its C-terminus cleaved Spitz in the absence of Star, but this cleaved product was not secreted, even in the presence of Star. Rhomboid-1 carrying the mutated ER retention signal KDAS behaved as wild-type Rhomboid-1. (C) GFP–Spitz (green) was confined to the ER even in the presence of Rhomboid-1–KDEL, when ∼50% was cleaved, see (B). Note that there was no co-localization of GFP–Spitz with a Golgi marker (red). (D) When the N-terminus of Spitz was replaced by TGF-α (junction at the last cysteine of the EGF repeat), this chimeric protein (GFP–TGF–Spi) was cleaved by Rhomboid-1–KDEL, and the cleaved form (of ∼45 kDa) was secreted from cells efficiently.