The α4 regulatory subunit exerts opposing allosteric effects on protein phosphatases PP6 and PP2A

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 alpha 4 ( related to yeast Tap42). Here, we characterized recombinant PP6 and PP2A catalytic monomers and alpha 4(.)phosphatase heterodimers. Monomeric PP6 and PP2A showed identical kinetics using either p-nitrophenyl phosphate (pNPP) or P-32-myelin basic protein (MBP) as substrates, with matching K-m and V-max 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 alpha 4(.)PP6 and alpha 4(.)PP2A were starkly different. With MBP as substrate the alpha 4(.)PP2A heterodimer had a 100-fold higher Vmax than alpha 4(.)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 alpha 4. Transient expression of alpha 4 differentially increased or decreased phosphorylation of endogenous phosphoproteins, consistent with opposing effects on PP2A and PP6.