title: 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.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Simona G
authors.initials: SG
authors.lastname: Codreanu
authors.forename: Deanna G
authors.initials: DG
authors.lastname: Adams
authors.forename: Eric S
authors.initials: ES
authors.lastname: Dawson
authors.forename: Brian E
authors.initials: BE
authors.lastname: Wadzinski
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Liebler
mesh_terms: D000478:Alkylation; D001710:Biotin / Q000031:analogs & derivatives / Q000494:pharmacology; D015153:Blotting, Western; D002384:Catalysis; D002460:Cell Line / Q000187:drug effects; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D020035:Holoenzymes / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D054648:Protein Phosphatase 2; D015398:Signal Transduction / Q000235:genetics; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D020035:Holoenzymes; D013438:Sulfhydryl Compounds; D001710:Biotin; C043329:N-iodoacetyl-N'-biotinylhexylenediamine; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D003545:Cysteine