A Novel, Evolutionarily Conserved Protein Phosphatase Complex Involved in Cisplatin Sensitivity

Abstract

Using a combination of tandem affinity purification tagging and mass spectrometry, we characterized a novel, evolutionarily conserved protein phosphatase 4 (PP4)-containing complex (PP4cs, protein phosphatase 4, cisplatin-sensitive complex) that plays a critical role in the eukaryotic DNA damage response. PP4cs is comprised of the catalytic subunit PP4C; a known regulatory subunit, PP4R2; and a novel protein that we termed PP4R3. The Saccharomyces cerevisiae PP4R3 ortholog Psy2 was identified previously in a screen for sensitivity to the DNAdamaging agent and anticancer drug cisplatin. We demonstrated that deletion of any of the PP4cs complex orthologs in S. cerevisiae elicited cisplatin hypersensitivity. Reversible protein phosphorylation is a highly conserved, essential regulatory mechanism involved in a host of cellular processes. Yet, while the phosphorylation of regulatory molecules by kinases has been studied intensively, their subsequent dephosphorylation is much less well understood. In eukaryotes, dephosphorylation on serine/threonine residues is effected by two distinct groups of functionally diverse phosphatases, the phosphoprotein M (represented by a sole member in higher eukaryotes, PP2C) and PPP 1 families (1 PP2A often functions as a standard trimeric complex with a catalytic (C) subunit (encoded by two genes in mammals) associated with one of many regulatory (or B) subunits via one of two adaptor (A) molecules (4, 5). The regulatory and adaptor subunits are thought to confer substrate specificity to the complex (5).In contrast to PP2A, the supramolecular architecture and subunit composition of PP4 multiprotein complexes remains largely unknown. Two mammalian PP4 regulatory subunits were previously identified (here termed PP4R1 and PP4R2, gene names PPP4R1 and PPP4R2; Refs. 6 and 7). Although PP4R1 shares some sequence homology with the PP2A adaptor proteins (PPP2R1A and PPP2R1B), it does not bridge PP4C and PP4R2; PP4R1 and PP4R2 display mutually exclusive PP4C interactions (Refs. 6 and 7; and see below). Other PP4C-interacting partners have also been reported (e.g. Refs. 8 and 9), but whether these proteins represent bona fide regulatory subunits or phosphatase substrates and how these binding proteins may affect PP4 activity are unclear.To gain a better understanding of the composition, function, and regulation of PP4, we systematically analyzed mammalian and yeast PP4C-interacting proteins. In doing so, we From the ‡Institute for

Protein phosphatase PP4 is overexpressed in human breast and lung tumors

“…The ablation of PP4 leads to embryonic lethality in mice [4]. The human PP4 phosphatase complex, PP4C-PP4R2-PP4R3, was recently identified through mammalian TAP-tagging technique and mass spectrometry [6]. In Saccharomyces cerevisiae, the pph3 (PP4 orthologue), psy2 (PP4R2 orthologue), and yb1046w (PP4R3 orthologue) mutants, but not the pph22 (PP2A orthologue) mutant, are hypersensitive to cisplatin treatment [6,7].…”

Conditional Knockout Mice Reveal an Essential Role of Protein Phosphatase 4 in Thymocyte Development and Pre-T-Cell Receptor Signaling

“…Furthermore, PP4 physically associates with, dephosphorylates, and inhibits histone deacetylase 3 activity (29). Saccharomyces cerevisiae strains mutated for Pph3 (PP4 ortholog), but not Pph22 (PP2A ortholog), become hypersensitive to the cancer drug cisplatin, suggesting a role for PP4 in DNA damage signaling (5). In yeast, Pph3, but not Pph22, dephosphorylates ␥H2AX, whose posttranslational modification is an early hallmark following a double-strand break (15).…”

Restricted Protein Phosphatase 2A Targeting by Merkel Cell Polyomavirus Small T Antigen

“…pMIG-FLAG-B55␣ was digested with NheI/XhoI and inserted into pCEP-4HA vector for hemagglutinin (HA) tagging. FLAG-tagged C subunits of all protein phosphatase (PPase) constructs were kindly provided by A.-C. Gingras (50). MCV sT and SV40 sT antigen plasmids were described previously (42) (Addgene, catalog no.…”