Structure-function analysis of yeast piD261/Bud32, an atypical protein kinase essential for normal cell life.
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.
Facchin
Sonia
S
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.
Lopreiato
Raffaele
R
Stocchetto
Silvia
S
Arrigoni
Giorgio
G
Cesaro
Luca
L
Marin
Oriano
O
Carignani
Giovanna
G
Pinna
Lorenzo A
LA
D000595:Amino Acid Sequence; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C108301:BUD32 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases