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 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.
Janssens
Veerle
V
Division of Biochemistry, Faculty of Medicine, Katholieke Universiteit Leuven, Herestraat 49, Belgium.
Jordens
Jan
J
Stevens
Ilse
I
Van Hoof
Christine
C
Martens
Ellen
E
De Smedt
Humbert
H
Engelborghs
Yves
Y
Waelkens
Etienne
E
Goris
Jozef
J
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002453:Cell Cycle; D006801:Humans; D010749:Phosphoprotein Phosphatases / Q000032:analysis / Q000737:chemistry* / Q000502:physiology; D054648:Protein Phosphatase 2; D051381:Rats; D013329:Structure-Activity Relationship
C432785:PPP2R1B protein, human; D010749:Phosphoprotein Phosphatases; C522189:Ppp2r1b protein, rat; D054648:Protein Phosphatase 2; D002118:Calcium