Protein phosphatase 2A holoenzyme assembly: identification of contacts between B-family regulatory and scaffolding A subunits

• Published in: The Journal of biological chemistry 2002-06, Vol.277 (23), p.20750-20755
• Main Authors: Strack, Stefan, Ruediger, Ralf, Walter, Gernot, Dagda, Ruben K, Barwacz, Chris A, Cribbs, J Thomas
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Cell Line; Dimerization; DNA, Complementary; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; phosphoprotein phosphatase 2A; Phosphoprotein Phosphatases - chemistry; Phosphoprotein Phosphatases - genetics; Phosphoprotein Phosphatases - metabolism; Precipitin Tests; Protein Conformation; Protein Phosphatase 2; Rats; Sequence Homology, Amino Acid
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M202992200

Protein serine/threonine phosphatase (PP) 2A is a ubiquitous enzyme with pleiotropic functions. Trimeric PP2A consists of a structural A subunit, a catalytic C subunit, and a variable regulatory subunit. Variable subunits (B, B', and B" families) dictate PP2A substrate specificity and subcellular localization. B-family subunits contain seven WD repeats predicted to fold into a beta-propeller structure. We carried out mutagenesis of Bgamma to identify domains important for association with A and C subunits in vivo. Several internal deletions in Bgamma abolished coimmunoprecipitation of A and C subunits expressed in COS-M6 cells. In contrast, small N- and C-terminal Bgamma deletions had no effect on incorporation into the PP2A heterotrimer. Thus, holoenzyme association of B-family subunits requires multiple, precisely aligned contacts within a core beta-propeller domain. Charge-reversal mutagenesis of Bgamma identified a cluster of conserved critical residues in Bgamma WD repeats 3 and 4. Acidic substitution of paired basic residues in Bgamma (RR165EE) abolished association with wild-type A and C subunits, while fostering incorporation of Bgamma into a PP2A heterotrimer containing an A subunit with an opposite charge-reversal mutation (EE100RR). Thus, binding of A and B subunits requires electrostatic interactions between conserved pairs of glutamates and arginines. By expressing complementary charge-reversal mutants in neuronal PC6-3 cells, we further show that holoenzyme incorporation protects Bgamma from rapid degradation by the ubiquitin/proteasome pathway.