Structure-function analysis of yeast piD261/Bud32, an atypical protein kinase essential for normal cell life

Dipartimento di Chimica Biologica, Centro Studi delle Biomembrane del Consiglio Nazionale delle Ricerche and Centro Ricerca Interdipartimentale Biotecnologie Innovative, University of Padova, Viale G. Colombo, 3, 35125 Padova, Italy.
Biochemical Journal (Impact Factor: 4.4). 07/2002; 364(Pt 2):457-63. DOI: 10.1042/BJ20011376
Source: PubMed


The Saccharomyces cerevisiae YGR262c/BUD32 gene, whose disruption causes a severe pleiotropic phenotype, encodes a 261-residue putative protein kinase, piD261, whose structural homologues have been identified in a variety of organisms, including humans, and whose function is unknown. We have demonstrated previously that piD261, expressed in Escherichia coli as a recombinant protein, is a Ser/Thr kinase, as judged by its ability to autophosphorylate and to phosphorylate casein. Here we describe a mutational analysis showing that, despite low sequence similarity, the invariant residues representing the signature of protein kinases are conserved in piD261 and in its structural homologues, but are embedded in an altered context, suggestive of unique mechanistic properties. Especially noteworthy are: (i) three unique inserts of unknown function within the N-terminal lobe, (ii) the lack of a lysyl residue which in all other Ser/Thr kinases participates in the catalytic event by interacting with the transferred ATP gamma-phosphate, and which in piD261 is replaced by a threonine, and (iii) an exceedingly short activation loop including two serines, Ser-187 and Ser-189, whose autophosphorylation accounts for the appearance of an upshifted band upon SDS/PAGE. A mutant in which these serines are replaced by alanines was devoid of the upshifted band and displayed reduced catalytic activity. This would include piD261 in the category of protein kinases activated by phosphorylation, although it lacks the RD (Arg-Asp) motif which is typical of these enzymes.

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Available from: Sonia Facchin
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    • "Archaeal PaKae1 has been shown to have DNA-binding and apurinic site endonuclease activities (Hecker et al., 2007). Bud32p is an atypical serine/threonine protein kinase (Facchin et al., 2002) that is homologous to human TP53RK/PRPK, which has been shown to phosphorylate p53 (Abe et al., 2001). In the KEOPS/EKC complex , Kae1p and Bud32p are closely associated (Mao et al., 2008). "
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    ABSTRACT: The kaeA(KAE1) (suDpro) gene, which was identified in Aspergillus nidulans as a suppressor of proline auxotrophic mutations, encodes the orthologue of Saccharomyces cerevisiae Kae1p, a member of the evolutionarily conserved KEOPS/EKC (Kinase, Endopeptidase and Other Proteins of Small size/Endopeptidase-like and Kinase associated to transcribed Chromatin) complex. In yeast, this complex has been shown to be involved in tRNA modification, transcription, and genome maintenance. In A. nidulans, mutations in kaeA result in several phenotypic effects, the derepression of arginine catabolism genes, and changes in the expression levels of several others, including genes involved in amino acid and siderophore metabolism, sulfate transport, carbon/energy metabolism, and translation and transcription regulation, such as rcoA(TUP1), which encodes the global transcriptional corepressor. Copyright © 2015. Published by Elsevier B.V.
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    • "Bud32 was initially described as an RIO-type serine/threonine protein kinase and its phosphotransferase activity was demonstrated in vitro and in vivo (30). We therefore tested if the Bud32 ATPase activity corresponded to the protein kinase activity under the conditions used for the synthesis of t6A in vitro. "
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    Full-text · Article · Aug 2013 · Nucleic Acids Research
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    • "Bud32p is an atypical kinase that lacks some of the canonical domains required for substrate recognition (5,6). In vitro phosphorylation assays have demonstrated that this protein is a functional kinase that is also responsible for its autophosphorylation (8). It has been suggested that the association of Kae1p with Bud32p inhibits the phosphorylation activity of the latter (6). "
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    ABSTRACT: The EKC/KEOPS complex is universally conserved in Archaea and Eukarya and has been implicated in several cellular processes, including transcription, telomere homeostasis and genomic instability. However, the molecular function of the complex has remained elusive so far. We analyzed the transcriptome of EKC/KEOPS mutants and observed a specific profile that is highly enriched in targets of the Gcn4p transcriptional activator. GCN4 expression was found to be activated at the translational level in mutants via the defective recognition of the inhibitory upstream ORFs (uORFs) present in its leader. We show that EKC/KEOPS mutants are defective for the N6-threonylcarbamoyl adenosine modification at position 37 (t(6)A(37)) of tRNAs decoding ANN codons, which affects initiation at the inhibitory uORFs and provokes Gcn4 de-repression. Structural modeling reveals similarities between Kae1 and bacterial enzymes involved in carbamoylation reactions analogous to t(6)A(37) formation, supporting a direct role for the EKC in tRNA modification. These findings are further supported by strong genetic interactions of EKC mutants with a translation initiation factor and with threonine biosynthesis genes. Overall, our data provide a novel twist to understanding the primary function of the EKC/KEOPS and its impact on several essential cellular functions like transcription and telomere homeostasis.
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