Inhibition of Protein Phosphatase 2A Activity by Selective Electrophile Alkylation Damage

Abstract

Protein serine/threonine phosphatase 2A (PP2A) is a critical regulator of numerous cellular signaling processes and a potential target for reactive electrophiles that dysregulate phosphorylation-dependent signal transduction cascades. The predominant cellular form of PP2A is a heterotrimeric holoenzyme consisting of a structural A, a variable B, and a catalytic C subunit. We studied the modification of two purified PP2A holoenzyme complexes (ABalpha(FLAG)C and ABdelta(FLAG)C) with two different thiol-reactive electrophiles, biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (PEO-IAB) and the biotinamido-4-[4'-(maleimidomethyl)cyclohexanecarboxamido]butane (BMCC). In vivo treatment of HEK 293 cells with these electrophiles resulted in alkylation of all three PP2A subunits. Electrophile treatment of the immunopurified FLAG-tagged holoenzymes produced a concentration-dependent adduction of PP2A subunits, as observed by Western blot analysis. Although both electrophiles labeled all three PP2A subunits, only BMCC inhibited the catalytic activity of both holoenzymes. Alkylation patterns in the A and B subunits were identical for the two electrophiles, but BMCC alkylated four Cys residues in the C subunit that were not labeled by PEO-IAB. Homology between the catalytic subunits of PP1 and PP2A enabled generation of a comparative model structure for the C subunit of PP2A. The model structure provided additional insight into contributions of specific BMCC-Cys adducts to PP2A enzyme inhibition. The results indicate that site selectivity of protein adduction should be a critical determinant of the ability of electrophiles to affect cellular signaling processes.

Phenylarsine Oxide Binding Reveals Redox-Active and Potential Regulatory Vicinal Thiols on the Catalytic Subunit of Protein Phosphatase 2A

“…The importance of these cysteine residues for the structure, function, and regulation of PP2A are largely unknown although, significantly, the C266-C269 pair appears to form part of the binding site for PP2A inhibitors [32]. A detailed mass spectrometry-based study of the labeling patterns and effects on catalysis of different classes of thiol alkylating agents [13] revealed that the thiols of C266 and C269 were highly and uniquely reactive but selective modification of these thiols did not affect PP2A activity. Peptide fragments containing the C50-C55 thiol pair were not detected in this study so any effects on catalysis due to modification of these thiols remains unknown.…”

“…Nevertheless, findings that thiol-reactive agents [11–13] and oxidants [14–16] can inhibit PP2A activity in vitro suggest that modification of one or more of the thiols of PP2A is sufficient to impair enzyme activity via a presumed conformational change. The roles of PP2A thiol groups in the regulation and dysregulation of PP2A activity are, however, unknown.…”

A FRET-based method to study protein thiol oxidation in histological preparations

“…The protocol uses iodoacetamide to block free thiols, DTT to reduce disulfides and iodoacetamide-conjugated fluorescein as a probe. In our method, we used maleimide to block free thiols as it is known to be more efficient in alkylating cysteines [29]. Maleimide is also the functional group in CPM, the probe we found to be more effective in thiol labeling.…”

Protein Damage by Reactive Electrophiles: Targets and Consequences

“…We previously characterized electrophile modifications of serine-threonine phosphatase PP2A, a three-subunit protein that serves as a key regulator of signal transduction (82). Reaction with the biotinylated electrophile probes PEO-IAB and BMCC (see above and Figure 3) labeled the structural (A), regulatory (B), and catalytic (C) subunits, as analyzed by affinity capture and Western blotting.…”