Identification and Functional Analysis of Two Ca2+-binding EF-hand Motifs in the B“/PR72 Subunit of Protein Phosphatase 2A

Abstract

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 Ca 2؉ -binding EF-hand motifs in human B؆/PR72 (denoted EF1 and EF2), demonstrating for the first time the ability of Ca 2؉ to interact directly with and regulate PP2A. EF1 and EF2 apparently bind Ca 2؉ with different affinities. Ca 2؉ induces a significant conformational change, which is dependent on the integrity of the motifs. We have further evaluated the effects of Ca 2؉ 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 Ca 2؉ 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.

PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail)

“…This behavior is consistent with observed differences in the intrinsic binding affinity of the different B-type subunits for Aa in quantitative mammalian two-hybrid experiments (i.e. PR72/B 0 > PR61/B 0 > PR55/B) [16]. In comparison with Aa, the binding of Ab to B-type and catalytic subunits is weaker [12], whereas no difference is observed between the binding of Ca and Cb to A-or B-type subunits [17].…”

“…Electrostatic interactions between two conserved arginine residues in WD repeat 3 and two glutamic acid residues in the intra-repeat loop of Aa HEAT-repeat 3 are vital for trimeric assembly [27]. In the PR72/B 00 subunits, two highly conserved Ca 2+ -binding, helix-loop-helix EF-hand motifs are probably involved in A-subunit interaction because the mutation of EF-hand 2 abolishes heterotrimer formation [16,40].…”

Critical Role for Protein Phosphatase 2A Heterotrimers in Mammalian Cell Survival

“…Ectopically expressed, monomeric B␥ mutants are rapidly degraded (7), whereas monomeric BЉ/PR72 mutants are reportedly stable (20). The latter finding is consistent with the observed persistence of endogenous BЉ/PR59 levels after A␣ subunit RNAi (Fig.…”

Glutamate regulation of DARPP-32 phosphorylation in neostriatal neurons involves activation of multiple signaling cascades

“…When PP-2B was inhibited by cyclosporin A, the stimulatory effect of nNOS͞NO͞cGMP͞PKG signaling was sustained for a longer time. (25) and by the interaction with calmodulin-binding proteins such as striatin and S͞G 2 nuclear autoantigen (26). Because the phosphorylated form of DARPP-32 at Thr-75 inhibits PKA, the decrease in Thr-75 phosphorylation results in an increase in PKA activity by disinhibition (3).…”