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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    • "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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    ABSTRACT: N(6)-threonylcarbamoyladenosine (t(6)A) is a universal tRNA modification essential for normal cell growth and accurate translation. In Archaea and Eukarya, the universal protein Sua5 and the conserved KEOPS/EKC complex together catalyze t(6)A biosynthesis. The KEOPS/EKC complex is composed of Kae1, a universal metalloprotein belonging to the ASHKA superfamily of ATPases; Bud32, an atypical protein kinase and two small proteins, Cgi121 and Pcc1. In this study, we investigated the requirement and functional role of KEOPS/EKC subunits for biosynthesis of t(6)A. We demonstrated that Pcc1, Kae1 and Bud32 form a minimal functional unit, whereas Cgi121 acts as an allosteric regulator. We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex. Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA. Surprisingly, we found that Bud32 is regulated by Kae1 and does not function as a protein kinase but as a P-loop ATPase possibly involved in tRNA dissociation. Overall, our data support a mechanistic model in which the final step in the biosynthesis of t(6)A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.
    Nucleic Acids Research 08/2013; 41(20). DOI:10.1093/nar/gkt720 · 9.11 Impact Factor
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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.
    Nucleic Acids Research 04/2011; 39(14):6148-60. DOI:10.1093/nar/gkr178 · 9.11 Impact Factor
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    • "Strikingly, the C-lobe of Bud32 lacks the infrastructure normally responsible for phospho-acceptor site recognition, including the activation segment (Aloop ), which is substituted by a direct link between strand β8 and helix αF (Figure 2A). Despite its atypical structure, Bud32 retains conserved elements required for ATP binding and catalysis (Figure 2A, Table S2) and indeed can auto-phosphorylate in vitro (Figure 2B) (Facchin et al., 2002) in a manner dependent on the integrity of conserved catalytic elements. "
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    ABSTRACT: Kae1 is a universally conserved ATPase and part of the essential gene set in bacteria. In archaea and eukaryotes, Kae1 is embedded within the protein kinase-containing KEOPS complex. Mutation of KEOPS subunits in yeast leads to striking telomere and transcription defects, but the exact biochemical function of KEOPS is not known. As a first step to elucidating its function, we solved the atomic structure of archaea-derived KEOPS complexes involving Kae1, Bud32, Pcc1, and Cgi121 subunits. Our studies suggest that Kae1 is regulated at two levels by the primordial protein kinase Bud32, which is itself regulated by Cgi121. Moreover, Pcc1 appears to function as a dimerization module, perhaps suggesting that KEOPS may be a processive molecular machine. Lastly, as Bud32 lacks the conventional substrate-recognition infrastructure of eukaryotic protein kinases including an activation segment, Bud32 may provide a glimpse of the evolutionary history of the protein kinase family.
    Molecular cell 11/2008; 32(2):259-75. DOI:10.1016/j.molcel.2008.10.002 · 14.02 Impact Factor
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