Identification and functional analysis of two Ca2+-binding EF-hand motifs in the B/PR72 subunit of protein phosphatase 2A

Protein phosphatase 2A (PP2A) is a multifunctional serine/threonine phosphatase that is critical to many cellular processes including cell cycle regulation and signal transduction. PP2A is a heterotrimer containing a structural (A) and catalytic (C) subunit, associated with one variable regulatory or targeting B-type subunit, of which three families have been described to date (B/PR55, B'/PR61, and B/PR72). We identified two functional and highly conserved Ca2+-binding EF-hand motifs in human B/PR72 (denoted EF1 and EF2), demonstrating for the first time the ability of Ca2+ to interact directly with and regulate PP2A. EF1 and EF2 apparently bind Ca2+ with different affinities. Ca2+ induces a significant conformational change, which is dependent on the integrity of the motifs. We have further evaluated the effects of Ca2+ on subunit composition, subcellular targeting, catalytic activity, and function during the cell cycle of a PR72-containing PP2A trimer (PP2A(T72)) by site-directed mutagenesis of either or both motifs. The results suggest that integrity of EF2 is required for A/PR65 subunit interaction and proper nuclear targeting of PR72, whereas EF1 might mediate the effects of Ca2+ on PP2A(T72) activity in vitro and is at least partially required for the ability of PR72 to alter cell cycle progression upon forced expression.