Characterization of the Protein Phosphatase 2A Regulatory Subunit PR70
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Protein phosphatase 2A (PP2A) is a phosphoserine/threonine phosphatase that controls the phosphorylation of numerous proteins in eukaryotic cells. PP2A consists of a core dimer composed of a scaffolding subunit (A-subunit) and a catalytic subunit (C-subunit) that interacts with a variety of regulatory subunits. There are four families of regulatory subunits: R2, R3, R4, and R5. The diversity of regulatory subunits gives rise to multiple PP2A holoenzymes and accounts for the ability of PP2A to regulate diverse cellular processes. Relatively little is known about the molecular basis for the interaction of the regulatory subunits with the core dimer and substrates. A more thorough understanding of these interactions would provide insights into how the regulatory subunits target PP2A to different cellular processes. The R3 regulatory subunit termed PR70 was identified in a yeast two hybrid screen with the DNA replication protein Cdc6 as bait. PR70 interacts with the PP2A core dimer and Cdc6 in vivo and in vitro. Biochemical approaches were used to identify regions and residues within PR70 that are important for mediating protein-protein interactions with the PP2A core dimer and Cdc6. PR70 contains two conserved calcium binding EF hand motifs and binds calcium in vitro. Calcium enhances the binding of PR70 to the A-subunit but not to Cdc6. Although calcium did not enhance the binding of PR70 to Cdc6, it did result in an increase in the amount of PP2A associated with Cdc6. Both calcium binding and enhanced interactions with the A-subunit require functional EF hand motifs. A conserved motif within the conserved R3 family domain was identified that is sufficient for the interaction of PR70 with PP2A. The C-terminal region was shown to be important for the interaction of PR70 with Cdc6, but not with the A-subunit. This result suggested that different portions of PR70 are important for mediating interactions with PP2A and Cdc6. Finally, PR70 is phosphorylated in intact cells at threonine 76 and serine 543. A functional analysis indicated that mutation of these sites does not affect the ability of PR70 to interact with PP2A, suggesting phosphorylation plays some other role in regulating PR70.