The α4 Regulatory Subunit Exerts Opposing Allosteric Effects on Protein Phosphatases PP6 and PP2A

Todd D. Prickett, David L. Brautigan
2006 Journal of Biological Chemistry  
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 ␣4 (related to yeast Tap42). Here, we characterized recombinant PP6 and PP2A catalytic monomers and ␣4⅐phosphatase heterodimers. Monomeric PP6 and PP2A showed identical kinetics using either p-nitrophenyl phosphate (pNPP) or 32 P-myelin
more » ... ic protein (MBP) as substrates, with matching K m and V max values. Using pNPP as substrate, PP6 and PP2A gave the same IC 50 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 ␣4⅐PP6 and ␣4⅐PP2A were starkly different. With MBP as substrate the ␣4⅐PP2A heterodimer had a 100fold higher V max than ␣4⅐PP6, and neither heterodimer was active with pNPP. Thus, these phosphatases are distinguished by their different responses to allosteric binding of the common regulatory subunit ␣4. Transient expression of ␣4 differentially increased or decreased phosphorylation of endogenous phosphoproteins, consistent with opposing effects on PP2A and PP6. Downloaded from FIGURE 4. Kinetics of MBP dephosphorylation by His 6 -PP6 and His 6 -PP2A. Proteins were assayed using 32 P-labeled MBP. A, a Lineweaver-Burke plot of His 6 -PP6 with a Michaelis-Menten plot as an inset to show near saturation. B, a Lineweaver-Burke plot of His 6 -PP2A with a Michaelis-Menten plot as an inset to show saturation. PP6 and PP2A showed similar activities with MBP. The K m is at least 1000-fold lower for MBP compared with pNPP for both PP2A and PP6. PP6 and PP2A
doi:10.1074/jbc.m601054200 pmid:16895907 fatcat:6wxv2hqvqvcgfd37q7ipc27trq