Important Role for Phylogenetically Invariant PP2Ac Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Corresponding author: David R. H. Evans, Program in Molecular Pharmacology, Mailstop D2-100, Fred Hutchinson Cancer Research Ctr., 1100 Fairview Ave. N., Seattle, WA 98109., drhevans{at}usa.net (E-mail)

Communicating editor: P. RUSSELL

PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Ac functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Ac Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Ac catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Ac C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Ac catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

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