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

Abstract

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.

Structure and functional characterization of the atypical human kinase haspin

“…In contrast, protein kinases of the divergent RIO/piD261/Bud32 superfamily typically contain serine or threonine at this position. The mutation Thr163Ala does not substantially alter the activity of piD261 (22), and Ser-220 in Rio2 does not directly contact ATP (23). Despite haspin's unique structural properties, kinetic analysis has shown that it utilizes a sequential reaction mechanism like those of ePKs that have been analyzed (24).…”

Protein kinase CK2 phosphorylates and upregulates Akt/PKB

“…The activity of purified Akt (specific activity of 1.2 pmol Pi/min/mg, under basal conditions) was measured towards either the substrate peptide, or the recombinant yeast protein kinase piD261/Bud32, 33 which contains in its C-terminal tail a site conforming to the consensus sequence of Akt and which is not affected by CK2. Neither the peptide nor piD261 are phosphorylated by CK2 to any appreciable extent.…”

“…32 Akt-specific peptide substrate was from Calbiochem, Akt protein substrate piD261 was expressed, purified 33 and kindly provided by Dr. S Facchin (Padova).…”

“…Purified recombinant Akt (0.2-0.5 mg) was incubated with recombinant CK2 (a or a 2 b 2 , 10-20 units, where 1 unit is the amount of enzyme transferring 1 pmol of Pi to the peptide substrate per min) in the presence of 50 mM Tris-HCl, pH 7.5, 12 mM MgCl 2 , 10mM [g- 33 P]ATP (B1500 cpm/pmol), 0.1 M NaCl, in a total volume of 20 ml, for 10 min at 301C.…”

Alk1 and Alk2 are Two New Cell Cycle-Regulated Haspin-Like Proteins in Budding Yeast

“…When assayed in vitro, Alk1 and Alk2 have a weak protein kinase activity, similarly to other atypical kinases. 17 The level of both proteins is highly regulated, exhibiting a striking periodicity during the cell cycle. Alk1 and Alk2 polypeptides, in fact, peak in mitosis and late-S/G 2 , respectively.…”