title: The CreB deubiquitinating enzyme does not directly target the CreA repressor protein in Aspergillus nidulans.
abstract: Ubiquitination/deubiquitination pathways are now recognized as key components of gene regulatory mechanisms in eukaryotes. The major transcriptional repressor for carbon catabolite repression in Aspergillus nidulans is CreA, and mutational analysis led to the suggestion that a regulatory ubiquitination/deubiquitination pathway is involved. A key unanswered question is if and how this pathway, comprising CreB (deubiquitinating enzyme) and HulA (ubiquitin ligase) and other proteins, is involved in the regulatory mechanism. Previously, missense alleles of creA and creB were analysed for genetic interactions, and here we extended this to complete loss-of-function alleles of creA and creB, and compared morphological and biochemical phenotypes, which confirmed genetic interaction between the genes. We investigated whether CreA, or a protein in a complex with it, is a direct target of the CreB deubiquitination enzyme, using co-purifications of CreA and CreB, first using strains that overexpress the proteins and then using strains that express the proteins from their native promoters. The Phos-tag system was used to show that CreA is a phosphorylated protein, but no ubiquitination was detected using anti-ubiquitin antibodies and Western analysis. These findings were confirmed using mass spectrometry, which confirmed that CreA was differentially phosphorylated but not ubiquitinated. Thus, CreA is not a direct target of CreB, and nor are proteins that form part of a stable complex with CreA a target of CreB. These results open up new questions regarding the molecular mechanism of CreA repressing activity, and how the ubiquitination pathway involving CreB interacts with this regulatory network.
authors.affiliation: Department of Genetics and Evolution, The University of Adelaide, Adelaide, 5005, Australia.
authors.forename: Md Ashiqul
authors.initials: MA
authors.lastname: Alam
authors.affiliation: Department of Genetics and Evolution, The University of Adelaide, Adelaide, 5005, Australia.|Walailak University, 222 Thaiburi Thasala, Nakhonsithamrat, Nakhon Si Thammarat, 80160, Thailand.
authors.forename: Niyom
authors.initials: N
authors.lastname: Kamlangdee
authors.affiliation: Department of Genetics and Evolution, The University of Adelaide, Adelaide, 5005, Australia. joan.kelly@adelaide.edu.au.
authors.forename: Joan M
authors.initials: JM
authors.lastname: Kelly
mesh_terms: D001233:Aspergillus nidulans / Q000235:genetics*; D057465:Catabolite Repression / Q000235:genetics*; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics* / Q000378:metabolism; D000072017:Deubiquitinating Enzymes / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D009154:Mutation; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D054875:Ubiquitination / Q000235:genetics
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D005656:Fungal Proteins; D012097:Repressor Proteins; C071237:CreA protein, Aspergillus nidulans; D000072017:Deubiquitinating Enzymes
keywords: Aspergillus nidulans; Carbon catabolite repression; CreA DNA-binding protein; CreB deubiquitinating enzyme; Regulatory deubiquitination; Transcriptional repression