Inhibition of specific binding of okadaic acid to protein phosphatase 2A by microcystin-LR, calyculin-A and tautomycin: method of analysis of interactions of tight-binding ligands with target protein

Abstract

Several groups have reported that okadaic acid (OA) and some other tight-binding protein phosphatase inhibitors including microcystin-LR (MCLR), calyculin-A and tautomycin prevent each other from binding to protein phosphatase 2A (PP2A). In this paper, we have introduced an improved procedure for examining to what extent the affinity of an enzyme for a labelled tight-binding ligand is reduced by binding of an unlabelled tight-binding, ligand to the enzyme. Using this procedure, we have analysed the dose-dependent reduction of PP2A binding of [24-3H]OA by addition of OA, MCLR, calyculin-A and tautomycin. The results indicate that the binding of the unlabelled inhibitors to the PP2A molecule causes a dramatic (10(6)-10(8)-fold) increase in the dissociation constant associated with the interaction of [24-3H]OA and PP2A. This suggests that OA and the other inhibitors bind to PP2A in a mutually exclusive manner. The protein phosphatase inhibitors may share the same binding site on the PP2A molecule. We have also measured values of the dissociation constant (Ki) for the interaction of these toxins with protein phosphatase 1 (PP1). For MCLR and calyculin-A, the ratio of the Ki value obtained for PP1 to that for PP2A was in the range 4-9, whereas it was 0.01-0.02 for tautomycin. The value of tautomycin is considerably smaller than that (0.4) calculated from previously reported Ki values.

The dNTP triphosphohydrolase activity of SAMHD1 persists during S-phase when the enzyme is phosphorylated at T592

“…Securin and cyclin B disappeared rapidly while the decline of pH3S10 and pThr-CDK substrates was OKA dose dependent (Figure 7(c)). Considering that in human cells OKA doses below 1 µM are reported to be ineffective against PP1 phosphatases [32,33], our data point to an involvement of phosphatase(s) of the PP1 superfamily in the dephosphorylation of SAMHD1 during mitosis.

10.1080/15384101.2018.1480216-F0007Figure 7.Okadaic acid treatment maintains SAMHD1 phosphorylation in normal and transformed cells. Okadaic acid was added to hRPE-1 cells during synchronous mitotic exit (A-C), and to asynchronous populations of hRPE-1 (D) or THP-1 cells (E).

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“…We took advantage of the 10–100 fold differential selectivity of the cell-permeable inhibitor okadaic acid (OKA) for PP2A over PP1 with active concentrations below 1 µM reported as specific for PP2A in human cell lines [32,33]. The requirement for ≥1 µM OKA to fully inhibit SAMHD1 dephosphorylation during mitotic exit suggests the involvement of the PP1 family of phosphatases.…”

Functional analysis of the PP2A subfamily of protein phosphatases in regulating Drosophila S6 kinase

“…Calyculin A is 7-to 9-fold more potent toward PP2A than PP1 in vitro [39] and, when used at concentrations of 50 nM or less, is selective for inhibition of the PP2A subfamily of protein phosphatases in intact cells [40,41]. S2 cells were treated with 50 nM calyculin A for 15 minutes and then starved for amino acids or treated with 20 nM rapamycin to inhibit TOR activity.…”

Phosphorylation and activation of phosphodiesterase type 3B (PDE3B) in adipocytes in response to serine/threonine phosphatase inhibitors: deactivation of PDE3B in vitro by protein phosphatase type 2A

“…Incubation with 1 µM okadaic acid did not affect PP1 activity at all but inhibited PP2A activity by 46 % compared with the activity in untreated cells ; incubation with 1 µM tautomycin inhibited PP1 activity by 60 % but PP2A only by 11 %, and incubation with 300 nM calyculin A was sufficient to inhibit both PP1 and PP2A almost completely (Figure 2). Calyculin A binds tightly to the catalytic subunits of both PP1 (K i , 1.1 nM) and PP2A (K i , 0.13 nM), okadaic acid binds tightly to PP2Ac (K i , 0.032 nM) and tautomycin binds tightly to PP1c (K i , 0.48 nM) [46,47]. It has been shown previously in MCF7 cells that due to the tight binding of the inhibitors to the different phosphatases, cell penetration can be monitored by measuring PP1 and PP2A activities in cell-free extracts [46,47].…”

“…Calyculin A binds tightly to the catalytic subunits of both PP1 (K i , 1.1 nM) and PP2A (K i , 0.13 nM), okadaic acid binds tightly to PP2Ac (K i , 0.032 nM) and tautomycin binds tightly to PP1c (K i , 0.48 nM) [46,47]. It has been shown previously in MCF7 cells that due to the tight binding of the inhibitors to the different phosphatases, cell penetration can be monitored by measuring PP1 and PP2A activities in cell-free extracts [46,47]. One may therefore assume that the complete inhibition of PP1 and PP2A in the homogenates after calyculin-A treatment, the 46 % inhibition of PP2A in the homogenates after okadaic acid treatment and the 60 % inhibition of PP1 in the homogenates after tautomycin treatment correspond to the conditions in the adipocytes before homogenization.…”