Protein phosphatase 2A holoenzyme assembly: identification of contacts between B-family regulatory and scaffolding A subunits.
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.
Strack
Stefan
S
Department of Pharmacology, University of Iowa, Iowa City, Iowa 52242, USA. stefan-strack@uiowa.edu
Ruediger
Ralf
R
Walter
Gernot
G
Dagda
Ruben K
RK
Barwacz
Chris A
CA
Cribbs
J Thomas
JT
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary; D019281:Dimerization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D011487:Protein Conformation; D054648:Protein Phosphatase 2; D051381:Rats; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2