title: The alpha4 regulatory subunit exerts opposing allosteric effects on protein phosphatases PP6 and PP2A.
abstract: The protein Ser/Thr phosphatase family contains three enzymes called PP2A, PP4, and PP6 with separate biological functions inferred from genetics of the yeast homologues Pph21/22, Pph3, and Sit4. These catalytic subunits associate with a common subunit called alpha4 (related to yeast Tap42). Here, we characterized recombinant PP6 and PP2A catalytic monomers and alpha4.phosphatase heterodimers. Monomeric PP6 and PP2A showed identical kinetics using either p-nitrophenyl phosphate (pNPP) or 32P-myelin basic protein (MBP) as substrates, with matching Km and Vmax values. Using pNPP as substrate, PP6 and PP2A gave the same IC50 with active site inhibitors okadaic acid, microcystin-LR, calyculin A, and cantharidin. However, with MBP as substrate, PP6 was inhibited at 5-fold lower concentrations of toxins relative to PP2A, suggesting PP6 might be a preferred in vivo target of toxins. Heterodimeric alpha4.PP6 and alpha4.PP2A were starkly different. With MBP as substrate the alpha4.PP2A heterodimer had a 100-fold higher Vmax than alpha4.PP6, and neither heterodimer was active with pNPP. Thus, these phosphatases are distinguished by their different responses to allosteric binding of the common regulatory subunit alpha4. Transient expression of alpha4 differentially increased or decreased phosphorylation of endogenous phosphoproteins, consistent with opposing effects on PP2A and PP6.
authors.affiliation: Center for Cell Signaling and Department of Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
authors.forename: Todd D
authors.initials: TD
authors.lastname: Prickett
authors.forename: David L
authors.initials: DL
authors.lastname: Brautigan
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002193:Cantharidin / Q000494:pharmacology; D002522:Chlorocebus aethiops; D004791:Enzyme Inhibitors / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins; D008387:Marine Toxins; D051379:Mice; D052998:Microcystins / Q000494:pharmacology; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D019319:Okadaic Acid / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004791:Enzyme Inhibitors; C092377:Igbp1 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; D008387:Marine Toxins; D052998:Microcystins; D018832:Molecular Chaperones; D010080:Oxazoles; D010750:Phosphoproteins; D019319:Okadaic Acid; C059041:calyculin A; D010749:Phosphoprotein Phosphatases; C106079:protein phosphatase 6; C057862:cyanoginosin LR; D002193:Cantharidin