Use of Protein Phosphatase Inhibitors

Abstract

Reversible protein phosphorylation is recognized as a major mechanism regulating the physiology of plant and animal cells. Virtually every biochemical process within eukaryotic cells is controlled by the covalent modification of key regulatory proteins. This in turn dictates the cellular response to a variety of physiological and environmental stimuli; errors in signals transduced by phosphoproteins contribute to many human diseases. Thus, defining protein phosphorylation events, and specifically, the phosphoproteins involved, is crucial for obtaining a better understanding of the physiological events that distinguish normal and diseased states. Protein phosphatase inhibitors are useful when deciphering physiological events regulated by reversible protein phosphorylation but the hormonal stimuli or signaling pathways involved are not known. They are also useful in analyzing the impact of hormones and other physiological stimuli on the function of a specific phosphoprotein. This unit describes protocols for inhibiting the cellular PP1/PP2A activity with okadaic acid, microcystin-LR, and PP2B/calcineurin and a widely utilized strategy for inhibiting protein tyrosine phosphatases.

Cross-talk between Serine/Threonine Protein Phosphatase 2A and Protein Tyrosine Phosphatase 1B Regulates Src Activation and Adhesion of Integrin αIIbβ3 to Fibrinogen

“…Finally, to examine the role of PP2Ac in a more physiological context, we examined Src activation in human platelets treated with PP2Ac selective inhibitor-OA. OA at concentrations of 10 -100 nM is required to inhibit PP2Ac in intact cells, while 10 M can inhibit cellular PP1c activity (15). Compared with the DMSO-treated platelets, phosphorylation of Src Tyr-529 was distinctly reduced (p ϭ 0.009), whereas the level of Src Tyr-418 phosphorylation was enhanced (p ϭ 0.05) in response to 50 nM OA treatment (Fig.…”

Greatwall-phosphorylated Endosulfine is both an inhibitor and a substrate of PP2A-B55 heterotrimers

“…The anti-Endos activity of purified PP2A-B55α heterotrimers has the same characteristics as the predominant anti-Endos activity in whole cell extracts. In D , the fostriecin used has lost potency during the ∼6 months of storage after the experiments shown in Figure 2 were performed; fostriecin is well known to be somewhat labile in this time frame ( Weiser et al, 2003 ). It is nonetheless clear that both the anti-Endos and anti-CDKS functions of PP2A-B555α display similar dose responses to this drug.…”

Dopamine D2 Receptor Relies upon PPM/PP2C Protein Phosphatases to Dephosphorylate Huntingtin Protein

“…Pharmacological experiments conducted in HEK293 cells demonstrated that PP1, PP2A, and PP2B were not involved in HTT Ser-421 dephosphorylation downstream of D2R activation. These results, along with a sequence similarity screen of HTT Ser-421 surrounding residues, prompted us to turn our attention toward the members of the PPM/PP2C family, which are insensitive to common phosphatase inhibitors (25). Expression of various members of the PPM/PP2C family and the use of a pharmacological inhibitor (127153) along with shRNA experiments point to PPM1A and/or PPM1B as strong candidates for the D2R-mediated dephosphorylation of HTT Ser-421.…”

“…Even at 10 M, okadaic acid (OA) was still ineffective at preventing HTT dephosphorylation following apomorphine stimulation (Fig. 1D); at this concentration, OA inhibits PP1 and PP2A but also PP4, PP5, PP6, and PP2B (calcineurin-B, PP3) (22)(23)(24)(25). To ascertain that PP2B was not implicated (OA has an IC 50 of 4 M for PP2B (22)), we used two PP2B-specific inhibitors: cypermethrin and cyclosporin A.…”