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Activation of the Aspergillus PacC zinc finger transcription factor requires two proteolytic steps

2002; Springer Nature; Volume: 21; Issue: 6 Linguagem: Inglês

10.1093/emboj/21.6.1350

1460-2075

Eliecer Dı́ez, Josué Álvaro, Eduardo A. Espeso, Lynne Rainbow, Teresa Suárez, Joan Tilburn, Herbert N. Arst, Miguel Á. Peñalva,

NF-κB Signaling Pathways

Article15 March 2002free access Activation of the Aspergillus PacC zinc finger transcription factor requires two proteolytic steps Eliecer Díez Eliecer Díez Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas del CSIC, Velázquez 144, Madrid, 28006 Spain Search for more papers by this author Josué Álvaro Josué Álvaro Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas del CSIC, Velázquez 144, Madrid, 28006 Spain Search for more papers by this author Eduardo A. Espeso Eduardo A. Espeso Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas del CSIC, Velázquez 144, Madrid, 28006 Spain Search for more papers by this author Lynne Rainbow Lynne Rainbow Department of Infectious Diseases, Faculty of Medicine, Imperial College of Science, Technology & Medicine, Hammersmith Hospital, Du Cane Road, London, W12 0NN UK Present address: Division of Reproductive and Child Health, Medical and Molecular Genetics, The Medical School, Edgbaston, Birmingham, B15 2TT UK Search for more papers by this author Teresa Suárez Teresa Suárez Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas del CSIC, Velázquez 144, Madrid, 28006 Spain Search for more papers by this author Joan Tilburn Joan Tilburn Department of Infectious Diseases, Faculty of Medicine, Imperial College of Science, Technology & Medicine, Hammersmith Hospital, Du Cane Road, London, W12 0NN UK Search for more papers by this author Herbert N. Arst Jr Herbert N. Arst Jr Department of Infectious Diseases, Faculty of Medicine, Imperial College of Science, Technology & Medicine, Hammersmith Hospital, Du Cane Road, London, W12 0NN UK Search for more papers by this author Miguel Á. Peñalva Corresponding Author Miguel Á. Peñalva Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas del CSIC, Velázquez 144, Madrid, 28006 Spain Search for more papers by this author Eliecer Díez Eliecer Díez Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas del CSIC, Velázquez 144, Madrid, 28006 Spain Search for more papers by this author Josué Álvaro Josué Álvaro Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas del CSIC, Velázquez 144, Madrid, 28006 Spain Search for more papers by this author Eduardo A. Espeso Eduardo A. Espeso Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas del CSIC, Velázquez 144, Madrid, 28006 Spain Search for more papers by this author Lynne Rainbow Lynne Rainbow Department of Infectious Diseases, Faculty of Medicine, Imperial College of Science, Technology & Medicine, Hammersmith Hospital, Du Cane Road, London, W12 0NN UK Present address: Division of Reproductive and Child Health, Medical and Molecular Genetics, The Medical School, Edgbaston, Birmingham, B15 2TT UK Search for more papers by this author Teresa Suárez Teresa Suárez Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas del CSIC, Velázquez 144, Madrid, 28006 Spain Search for more papers by this author Joan Tilburn Joan Tilburn Department of Infectious Diseases, Faculty of Medicine, Imperial College of Science, Technology & Medicine, Hammersmith Hospital, Du Cane Road, London, W12 0NN UK Search for more papers by this author Herbert N. Arst Jr Herbert N. Arst Jr Department of Infectious Diseases, Faculty of Medicine, Imperial College of Science, Technology & Medicine, Hammersmith Hospital, Du Cane Road, London, W12 0NN UK Search for more papers by this author Miguel Á. Peñalva Corresponding Author Miguel Á. Peñalva Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas del CSIC, Velázquez 144, Madrid, 28006 Spain Search for more papers by this author Author Information Eliecer Díez1, Josué Álvaro1, Eduardo A. Espeso1, Lynne Rainbow2,3, Teresa Suárez1, Joan Tilburn2, Herbert N. Arst2 and Miguel Á. Peñalva 1 1Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas del CSIC, Velázquez 144, Madrid, 28006 Spain 2Department of Infectious Diseases, Faculty of Medicine, Imperial College of Science, Technology & Medicine, Hammersmith Hospital, Du Cane Road, London, W12 0NN UK 3Present address: Division of Reproductive and Child Health, Medical and Molecular Genetics, The Medical School, Edgbaston, Birmingham, B15 2TT UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:1350-1359https://doi.org/10.1093/emboj/21.6.1350 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Aspergillus PacC transcription factor undergoes proteolytic activation in response to alkaline ambient pH. In acidic environments, the 674 residue translation product adopts a 'closed' conformation, protected from activation through intramolecular interactions involving the ≤150 residue C-terminal domain. pH signalling converts PacC to an accessible conformation enabling processing cleavage within residues 252–254. We demonstrate that activation of PacC requires two sequential proteolytic steps. First, the 'closed' translation product is converted to an accessible, committed intermediate by proteolytic elimination of the C-terminus. This ambient pH-regulated cleavage is required for the final, pH-independent processing reaction and is mediated by a distinct signalling protease (possibly PalB). The signalling protease cleaves PacC between residues 493 and 500, within a conserved 24 residue 'signalling protease box'. Precise deletion or Leu498Ser substitution prevents formation of the committed and processed forms, demonstrating that signalling cleavage is essential for final processing. In contrast, signalling cleavage is not required for processing of the Leu340Ser protein, which lacks interactions preventing processing. In its two-step mechanism, PacC processing can be compared with regulated intramembrane proteolysis. Introduction A number of transcription factors are synthesized as inactive precursors, which are activated by proteolytic processing in response to appropriate environmental signals. A subgroup, typified by NF-κB p105 (Ghosh et al., 1998) or yeast SPT23 and MGA2 (Hoppe et al., 2000), includes proteins which are substrates of limited proteolysis mediated by the proteasome. A second subgroup, typified by SREBP, undergoes regulated intra membrane proteolysis (Rip) (Brown and Goldstein, 1999; Brown et al., 2000). In Rip, a regulated primary cleavage that takes place outside the lipid bilayer is a prerequisite for a second intramembrane cleavage of a transmembrane protein. A third subgroup includes Drosophila Cubitus interruptus (Ci, the transducer of the Hedgehog signal; Ingham, 1998) and Aspergillus PacC (mediating pH regulation of gene expression; Tilburn et al., 1995). Members of this third subgroup share features of their zinc finger DNA-binding domains, and their respective processing proteases have unusual specificities in that their action does not appear to require the region encompassing the processing site (Methot and Basler, 1999; Mingot et al., 1999). This subgroup also includes the GLI metazoan homologues of Ci, which are key regulatory factors in development (Ruiz i Altaba, 1999). We address here the mechanisms underlying the proteolytic activation of PacC by exploiting the ease with which the lower eukaryote Aspergillus nidulans can be manipulated genetically. The 674 residue zinc finger protein PacC (Figure 1A) is activated by limited proteolysis in response to the alkaline ambient pH signal, which is transmitted to the transcription factor via the six pal gene signal transduction pathway (Orejas et al., 1995; Mingot et al., 1999). The processed factor (the 248–250 N-terminal residues) localizes to the nucleus (Mingot et al., 2001) where it activates transcription of genes preferentially expressed under alkaline conditions and represses transcription of genes preferentially expressed under acidic conditions, in both cases through 5′-GCCARG-3′ sites in the promoters of the target genes (Tilburn et al., 1995; Espeso and Peñalva, 1996; Espeso et al., 1997; Espeso and Arst, 2000). Mutational inactivation of genes in the pal pathway (palA, B, C, F, H and I) or the pacC gene itself (pacC− and pacC+/− mutations) leads to an acidity-mimicking phenotype; gain-of-function pacCc mutations bypassing the need for the ambient pH signal lead to alkalinity mimicry (Caddick et al., 1986; Arst et al., 1994; Denison et al., 1995, 1998; Orejas et al., 1995; Tilburn et al., 1995; Negrete-Urtasun et al., 1997, 1999). Figure 1.(A) Schematic representation of PacC deletion mutations used in this work. To avoid confusion with earlier publications, numbering indicates amino acid positions on the basis of 678 residues, although translation starts at codon 5 (Mingot et al., 1999). (B) A processing intermediate is detected with PacCΔ10. EMSA shows PacC–DNA complexes corresponding to the different PacC forms. Strains and growth conditions are indicated. Here, and in all other panels, an asterisk indicates the processing intermediate; FL and P indicate full-length and processed form complexes, respectively. (C) Western blot analysis of protein extracts used in (B). Proteins were detected with the indicated antisera. The anti-PacC(529–678) antiserum was raised against the C-terminal PacC domain. (D) Deducing the approximate C-terminal residue of the processing intermediate. Protein extracts corresponding to the indicated mutant proteins were analysed by western blot with α-PacC(5–265) antiserum. The deduced molecular weight of the intermediates was used to estimate the approximate position of their C-terminal truncations. The pacCc75 translation product (Mr 45 478) was used as internal standard. (E) pH and pal dependence of the formation of the processing intermediate in pacCΔ8 strains. Protein extracts from acidic (H+) and neutral (N) growth conditions were analysed by EMSA. Neutral (pH 6.8) conditions leading to pH signalling were used as strains carrying palA1 or palB7 loss-of-function mutations do not grow at alkaline pH, and palI30 mutants grow poorly. (F) The translation product of PacCΔ10 and the processing intermediate show a precursor–product relationship. The relevant portion of an EMSA gel showing conversion of the PacCΔ10 translation product to the processing intermediate. Cells were grown under acidic conditions, incubated in the presence of cycloheximide for 30 min and shifted to neutral conditions also in the presence of cycloheximide. Numbers indicate hours after the pH shift. Download figure Download PowerPoint The ambient pH signal regulates the accessibility of PacC to the processing protease. Upon translation, PacC adopts a 'closed' conformation, held together by the interaction of a C-terminal domain (interacting domain C, located within residues 529–678) with two domains upstream, which prevents processing under inappropriate circumstances (i.e. at acidic pH, resulting in the absence of pal signalling) (Espeso et al., 2000). When the pH signal is received (i.e. at alkaline ambient pH), the protein shifts to an 'open' (protease-accessible) conformation (Espeso et al., 2000). The mutant pacC+/−20205 product is deficient in the pH signal response and behaves as if locked in the 'closed' conformation. Single residue mutations, typified by pacCc69 (L340S), or pacCc truncating mutations removing the C-terminal 492 residues and <501 residues. Download figure Download PowerPoint We used a similar pH shift western analysis to show that the appearance, apparently at the expense of the translation product, of the wild-type intermediate is completely prevented by the palA1 mutation, even after long exposure to alkaline pH (Figure 4A and C), demonstrating, in agreement with Figure 1E for PacCΔ8, that the formation of the wild-type intermediate requires an intact pal signalling pathway. Figure 4.Sequential conversion of the translation product into the intermediate and the processed form in a pH- and pal-dependent manner. (A–C) Wild-type or palA1 cells grown under acidic conditions (H+) were shifted to alkaline conditions (OH−). Where indicated (+ CHX), cycloheximide was added 30 min before the pH shift and maintained in the alkaline media. Protein extracts taken at the indicated time points after the pH shift were analysed by western blot with α-PacC(5–265) antiserum. FL, translation product; IT, intermediate; P, processed form. Download figure Download PowerPoint Figure 4A and B shows pH shift experiments in the presence or absence of cycloheximide. The relatively high PacC levels occurring in the absence of cycloheximide after 30 min exposure to alkaline pH are probably due to the positive transcriptional autoregulation of pacC (an alkaline expressed gene; Tilburn et al., 1995) resulting in de novo PacC synthesis, as evidenced by the appearance of the full-length form after 120 min. The relatively low levels of PacC and the complete absence of the full-length form in the presence of cycloheximide after 30 min are consistent with its prevention of de novo protein synthesis. In mycelia shifted to alkaline pH in the presence of cycloheximide, ∼50% of the wild-type translation product is converted to the intermediate within 15 min after the shift and, within 30 min the intermediate largely predominates (Figure 4B). These and the above data are fully consistent with a precursor–product relationship between the translation product and the intermediate, in agreement with data for PacCΔ10 (Figure 1F). The decrease in levels of the intermediate visible after 30 min (the minor mobility shift results from phosphorylation; J.Álvaro and T.Suárez, unpublished) correlated with an increase in the relative levels of the processed form (Figure 4B). As no translation product remains at 30 min, these data are fully consistent with a precursor–product relationship between the intermediate and the processed form. Moreover, the fact that pal− mutations (Figure 4C), two different pacC deletions removing the signalling protease target sequence and two missense mutations within this sequence (Figures 5, 6 and 7, see below) precluding pacC function block the signalling protease cleavage leading to the intermediate and, simultaneously, the formation of the processed form, establishes that, under normal circumstances, the translation product is converted to the processed form through the intermediate. Figure 5.The pacC+/−20205 product, permanently locked in the 'closed' conformation, is not converted to the processing intermediate. The relevant portion of an EMSA gel showing that the pacC+/−20205 product from either acidic or neutral growth conditions is recognized by both the α-PacC(529–678) and the α-PacC(301–529) antisera, indicating that, in contrast to the wild-type used as control, this mutant is unable to form the processing intermediate. FLC indicates the unprocessed wild-type and mutant PacC complexes. Download figure Download PowerPoint