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

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 DNA-damaging agent and anticancer drug cisplatin. We demonstrated that deletion of any of the PP4cs complex orthologs in S. cerevisiae elicited cisplatin hypersensitivity. Furthermore human PP4R3 complemented the yeast psy2 deletion, and Drosophila melanogaster lacking functional PP4R3 (flfl) exhibited cisplatin hypersensitivity, suggesting a highly conserved role for PP4cs in DNA damage repair. Finally we found that PP4R3 may target PP4cs to the DNA damage repair machinery at least in part via an interaction with Rad53 (CHK2). 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 DNA-damaging agent and anticancer drug cisplatin. We demonstrated that deletion of any of the PP4cs complex orthologs in S. cerevisiae elicited cisplatin hypersensitivity. Furthermore human PP4R3 complemented the yeast psy2 deletion, and Drosophila melanogaster lacking functional PP4R3 (flfl) exhibited cisplatin hypersensitivity, suggesting a highly conserved role for PP4cs in DNA damage repair. Finally we found that PP4R3 may target PP4cs to the DNA damage repair machinery at least in part via an interaction with Rad53 (CHK2). 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 1The abbreviations used are: PPP, phosphoprotein phosphatase; TAP, tandem affinity purification; NTAP, N-terminal TAP; CTAP, C-terminal TAP; PP, protein phosphatase; PP4cs, protein phosphatase 4, cisplatin-sensitive; aa, amino acid(s); TEV, tobacco etch virus; HA, hemagglutinin; HEK, human embryonic kidney; HRP, horseradish peroxidase; Pol, polymerase; YPD, yeast-peptone-dextrose growth medium; MGC, mammalian gene collection. families (1Barford D. Das A.K. Egloff M.P. The structure and mechanism of protein phosphatases: insights into catalysis and regulation.Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 133-164Google Scholar). Within the much larger PPP class, a common catalytic domain (of 280 aa) is highly conserved, whereas the N and C termini are more divergent and further separate the PPP proteins into subfamilies. A number of distinct PPP subfamilies have thus been characterized (PP1, PP2A, PP2B, PP5, and PP7; 2Andreeva A.V. Kutuzov M.A. PPP family of protein Ser/Thr phosphatases: two distinct branches?.Mol. Biol. Evol. 2001; 18: 448-452Google Scholar) based on sequence homology, associated regulatory subunits, sensitivity to different types of chemical inhibitors, and metal ion requirements. A major PPP subfamily that plays a variety of critical roles in a multitude of physiological processes is the PP2A-type phosphatases, PP2AC (the catalytic subunit of PP2A; human gene names PPP2CA and PPP2CB), PP4C (gene name PPP4C, formerly known as PPX), and PP6C (PPP6C; 3Cohen P.T. Novel protein serine/threonine phosphatases: variety is the spice of life.Trends Biochem. Sci. 1997; 22: 245-251Google Scholar). 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 (4Janssens V. Goris J. Van Hoof C. PP2A: the expected tumor suppressor.Curr. Opin. Genet. Dev. 2005; 15: 34-41Google Scholar, 5Sontag E. Protein phosphatase 2A: the Trojan Horse of cellular signaling.Cell. Signal. 2001; 13: 7-16Google Scholar). The regulatory and adaptor subunits are thought to confer substrate specificity to the complex (5Sontag E. Protein phosphatase 2A: the Trojan Horse of cellular signaling.Cell. Signal. 2001; 13: 7-16Google Scholar). 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; 6Kloeker S. Wadzinski B.E. Purification and identification of a novel subunit of protein serine/threonine phosphatase 4.J. Biol. Chem. 1999; 274: 5339-5347Google Scholar and 7Hastie C.J. Carnegie G.K. Morrice N. Cohen P.T. A novel 50 kDa protein forms complexes with protein phosphatase 4 and is located at centrosomal microtubule organizing centres.Biochem. J. 2000; 347: 845-855Google Scholar). 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 (6Kloeker S. Wadzinski B.E. Purification and identification of a novel subunit of protein serine/threonine phosphatase 4.J. Biol. Chem. 1999; 274: 5339-5347Google Scholar and 7Hastie C.J. Carnegie G.K. Morrice N. Cohen P.T. A novel 50 kDa protein forms complexes with protein phosphatase 4 and is located at centrosomal microtubule organizing centres.Biochem. J. 2000; 347: 845-855Google Scholar; and see below). Other PP4C-interacting partners have also been reported (e.g. 8Carnegie G.K. Sleeman J.E. Morrice N. Hastie C.J. Peggie M.W. Philp A. Lamond A.I. Cohen P.T. Protein phosphatase 4 interacts with the Survival of Motor Neurons complex and enhances the temporal localisation of snRNPs.J. Cell Sci. 2003; 116: 1905-1913Google Scholar and 9Zhang X. Ozawa Y. Lee H. Wen Y.D. Tan T.H. Wadzinski B.E. Seto E. Histone deacetylase 3 (HDAC3) activity is regulated by interaction with protein serine/threonine phosphatase 4.Genes Dev. 2005; 19: 827-839Google Scholar), but whether these proteins represent bona fide regulatory subunits phosphatase and these proteins may PP4 activity are a of the and of we mammalian and yeast PP4C-interacting In we were to different mutually exclusive complexes and identified a novel, evolutionarily conserved PP4C we termed that into a complex with PP4C and deletion of the yeast PP4R3 ortholog was demonstrated previously to to the DNA drug cisplatin identification of genes to the anticancer and Scholar). We found that deletion of the of the yeast complex also cisplatin we termed complex PP4cs phosphatase 4, cisplatin-sensitive; see below). anticancer as cisplatin and display a of of and of 2003; 22: Scholar, D. of anticancer Rev. 2005; Scholar). the to DNA damage is not well are involved in DNA repair D. of anticancer Rev. 2005; Scholar). A common with is to cisplatin. A of the cellular processes and involved in the to cisplatin may thus to was a S. and and the were H. was and the yeast were previously D. V. A. Y. D. S. A of interactions in Saccharomyces 2000; Scholar, of interactions by a a Scholar). The yeast tandem affinity purification as well as deletion in the and in the were The yeast and as well as the and were previously D. V. A. Y. D. S. 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A protein purification for protein complex and 1999; Scholar) with and for may found at yeast were in and were to as were with of of protein purification was with of yeast to were with of for at 4 with were and with and with and were in of of and to with at 4 for the were and the was to a were with and and were with the of a was to the was more and to a of was for at 4 with The was into and the was Two in were by in and The of were with and were to Two with of and were and the was into for two was to the was at the was and in A in to the the was at for and the was to a were to a of and were by were with number a to the were in A. of the was to the a and in A for The was in of a mass for of the the the mass 3 was on an for 3 were the a and The of the was in the was on a by were to the E. H. S. E. A common of mass and to 22: Scholar), and the were the human Protein the Saccharomyces yeast were for the number of with a mass on the ion of and as a was with J. H. of proteins affinity and mass 2001; 19: Scholar), A. A.I. E. to the of by and Chem. Scholar), and A.I. A. E. A for proteins by tandem mass Chem. 2003; Scholar) the of the of and are and the for of DNA a were used to of a to the proteins were to and to a The was for in was used to the proteins the with 3 with was were by the with and with of with the was in protein lacking agent by for at agent was and the was and and at are a were on at and to for were and for at were and to standard into the cisplatin was to was in to and for of the were also for cisplatin The as well as the in cisplatin sensitivity as with tagging and mass spectrometry, in a protein complex of is in a affinity and are identified by the ion of have to in the of protein complexes A. A protein purification for protein complex and 1999; Scholar, in protein complex mass 2005; Scholar, E. E. The tandem affinity purification a of protein complex 2001; Scholar, J. The substrate in Scholar). with purification by tagging are the of are also not for of of is that are to the of interaction in partners for proteins in into the supramolecular architecture of we the catalytic subunit at N with a protein in and the protein with partners for purification and proteins were to and identified via by of the a of and A. A.I. E. to the of by and Chem. Scholar and A.I. A. E. A for proteins by tandem mass Chem. 2003; Scholar; see a of we the In proteins in were as and to the a of PP4C-interacting proteins see The are also in protein interactions by mass in a In to the of the protein we a number of the known PP4C regulatory subunits PP4R1 and PP4R2 We also many previously demonstrated to with of the phosphatase catalytic subunits J. with protein 4, and Biophys. 1998; Scholar, The role of in in Scholar). is the ortholog of the yeast the target of to PP2A-type C.J. via the the of with Dev. Scholar, J. protein to the catalytic subunit of protein phosphatase Sci. S. A. 1997; Scholar, Y. S. N. of protein phosphatase catalytic activity by protein and yeast Biophys. 1998; Scholar). the role of the PP2A-type in in is not well understood. also identified subunits of an the The complex was thought to highly for the and of and but that it in the and of a of J. The substrate in Scholar). The interaction with to evolutionarily conserved as (the ortholog of and also with complex in a Y. A. A. A. C. S. J. J. C. D. H. H. A. E. J. V. J. D. D. identification of protein complexes in Saccharomyces cerevisiae by mass Scholar). The functional of the complex interaction with is at Other the known PP4C-interacting partners and the two proteins were in the and The human is in and is The two proteins sequence and homology at the amino with the of the PP2A-type phosphatases, we to as and to as for further proteins are conserved the Drosophila (flfl) protein shares with at the amino and the S. cerevisiae Psy2 protein with amino The and structure of the PP4R3 protein family that a at the N shares homology with a domain found in The structure of is also of homology The is the conserved of the protein at the amino with yeast and with in the Biol. 2000; Scholar) that the and proteins also a number of PP4R3 with is at we were to interaction by in in not A.K. D. S. H. H. screen in human genes essential for Scholar). (flfl) was identified in a screen to novel of in in a also associated with deletion E. a novel domain protein involved in Drosophila The of Scholar; see below). Finally the yeast PP4R3 was based on of to the anticancer drug cisplatin identification of genes to the anticancer and Scholar; see below). the novel interactions and to the supramolecular architecture of complexes in we the human PP4R1 PP4R2 and into these and the purification and mass identification for of these proteins and their partners A and and The PP4R1 the and the were in these complex proteins in to a PP4C and a the known partners one and In a PP4R2 the protein and PP4C were identified as well as many and as with PP4R2 to in a complex with In to these the PP4R2 also the human of S. cerevisiae mass of a protein involved in phosphatase E. interacts with and the 2001; Scholar, S. S. Wadzinski B.E. of protein serine/threonine phosphatase J. 1997; Scholar). We the to as into the and the with PP4C and PP4R2 to phosphatase subunits, reported were is the yeast protein was reported to with the yeast and to complexes with E. interacts with and the 2001; Scholar; see Finally a of the interaction with PP4C and proteins were identified in the PP4R3 these that PP4C is in distinct a complex with a complex and the and a complex PP4R2 and may also The complex to as PP4cs below). also an may used to the of multiprotein We that PP4R2, and a protein was at N with a different to for and DNA for of the proteins was used to a by and to a were to proteins were PP4R2 and PP4R3 were with PP4C often two with different is of the does not with the a protein and of PP4R3 the on the interaction and complex and is not on PP4R3. of the of with suggesting that PP4R2 is to the interaction PP4C and PP4R3. with a the yeast orthologs of PP4R2 and PP4R3 was reported previously in a Ozawa Y. A to the yeast protein Sci. S. A. 2001; Scholar; also see below). a interaction PP4R2 and PP4R3 in the is not for the of a complex as of the of with 4 and thus that a complex PP4C and PP4R2 PP4R3 as in the of trimeric complexes in that the subunit, the catalytic the regulatory subunits (e.g. X. Two conserved in regulatory subunits to the A subunit of protein phosphatase J. Biochem. Scholar). tagging that the of PP4R3 and PP4R2 is to were purification of PP4R2 PP4R3. PP4R2 PP4R3 were purification of not these we whether the two regulatory proteins with the mammalian PP2A-type The for and PP6C were into the and in a with and catalytic subunits were PP4R2 and PP4R3 were on an with 3 and The interaction with PP4C is thus and PP4R3 to a bona fide PP4 regulatory The specificity of the PP4cs interactions with that of with of the phosphatase catalytic subunits in with with the in the was with and the serine/threonine phosphatase (the PPP phosphatase to PP2A with to was to In the human interactions with PP2A-type J. with protein 4, and Biophys. 1998; Scholar). In contrast to a an interaction the S. cerevisiae and proteins E. interacts with and the 2001; Scholar), was to these that with of the PP2A-type phosphatase catalytic subunits and is not a for the PP4 The S. cerevisiae orthologs of the mammalian PP4R2, and PP4R3 The protein the homology to PP4R3 in S. cerevisiae is Psy2 The yeast protein is to the of the S. cerevisiae PP2A-type and are also Although yeast protein sequence homology to PP4R2 the a of homology in N have for interactions these yeast a interaction was reported Psy2 and Ozawa Y. A to the yeast protein Sci. S. A. 2001; Scholar), and complexes and Psy2 with have been Y. A. A. A. C. S. J. J. C. D. H. H. A. E. J. V. J. D. D. identification of protein complexes in Saccharomyces cerevisiae by mass Scholar, A. J. C. H. A. D. V. A. S. C. M.A. A. E. V. of the yeast by of protein Scholar). We thus to whether yeast complexes were in a to mammalian complexes and whether a complex has been conserved we yeast and proteins and used the purification and mass to partners for these in and interactions the yeast proteins were to we for the human PP4cs and Psy2 in the mammalian was also associated with the that it was in the We not the complex in the may to the of the at the C of the protein (the mammalian PP4C was at N as and were previously to with the complex Y. A. A. A. C. S. J. J. C. D. H. H. A. E. J. V. J. D. D. identification of protein complexes in Saccharomyces cerevisiae by mass protein interactions by mass in a of yeast and human PP4C interaction and cisplatin sensitivity of yeast deletion A and of the multiprotein complexes and identified by tagging and mass are also in and yeast are of the interaction and are as in represent proteins used as in the tagging whereas represent proteins that were not The the complex conserved yeast to C and cisplatin sensitivity of yeast for PP4cs complex (C) S. cerevisiae deletion were to and with cisplatin (or for 4 were YPD, and growth was is a yeast of the PP2A catalytic subunit, whereas is a of the human catalytic was used as a we also the complex and in of Psy2 and was also previously as a Psy2 and in a A. J. C. H. A. D. V. A. S. C. M.A. A. E. V. of the yeast by of protein Scholar). purification of and subunits, as in to for and A role for PP4cs may thus to target the phosphatase to the A for to the DNA-damaging cisplatin and identification of genes to the anticancer and Scholar) novel sensitivity identification of genes to the anticancer and Scholar). deletion was also found to to cisplatin identification of genes to the anticancer and Scholar). of was also reported to the sensitivity of S. cerevisiae to D. genes in the and DNA damage in Sci. S. A. Scholar). in mammalian PP4R2 is an for the of PP4C and and with PP4cs in yeast and we characterized the sensitivity of yeast lacking the genes to cisplatin yeast were with cisplatin and the growth of yeast was expected the deletion of the protein to cisplatin and with a role for the yeast PP4cs complex in cisplatin deletion of also elicited cisplatin hypersensitivity. deletion of an more sensitivity a less sensitivity the for the cisplatin sensitivity to the in for to we the with a deletion and the yeast PP4 complex and Psy2 plays a role in DNA may a role as a by the PP4 complex more to DNA damage and with and in the and was in the of DNA damage via dephosphorylation of A.I. J. of in to DNA Scholar), we that the of the PP4cs proteins in cisplatin may effected and and and deletion were for cisplatin In the of the of the exhibited to cisplatin C and and are not to of the cisplatin sensitivity by PP4cs In an to PP4cs and are to the DNA damage repair we to a yeast Psy2 was previously reported to with Rad53 in the yeast was used as D. V. A. Y. D. S. A of interactions in Saccharomyces 2000; Scholar). We to interaction the with Rad53 into a and Psy2 a In we interactions of the of yeast PP4 complex and interactions with of the DNA damage A of yeast were in to and Rad53 the Although a Rad53 as Rad53 and interactions not for The were in a and the growth of on lacking was the yeast on of of that growth of (e.g. as a to previously interactions were Psy2 and 4, A and In with and We also a previously interaction and with Psy2 with and in the Finally a was also Psy2 and Rad53 was for Rad53 with any of the was any of the and proteins involved in DNA damage that the yeast PP4cs complex is to the DNA damage machinery via an interaction Psy2 and whether the yeast and human PP4cs are functionally in the cisplatin sensitivity we a Psy2 mammalian PP4R3 (or into the We cisplatin sensitivity as the with was much more to cisplatin the with the of was less on the used for on as used in with exhibited a of cisplatin sensitivity to the of mammalian PP4R3 also the cisplatin of a that Psy2 and PP4R3 are functionally in to cisplatin. whether human PP4R3 the with the yeast proteins as we a yeast human PP4R3 to Although we not interactions with PP4R3 with yeast Rad53 in the interaction we thus that an interaction and the DNA damage machinery has been evolutionarily whether a in PP4R3 activity a to we in the Drosophila (flfl) the of the protein Two and were identified in a for an and and E. in Although for were of the combination and a in as with their of in and in were standard with of cisplatin and were 3 of is in the of these were to the of in the of the Survival for and on of cisplatin are in at cisplatin with to whereas some were with not that in higher is for to cisplatin and further for a critical and evolutionarily conserved role of PP4C-interacting in the to DNA an we that the mammalian PP4 catalytic subunit is a of different mutually exclusive one and the and a PP4R2 and a novel protein that we termed PP4R3. The PP4cs complex is evolutionarily conserved and in yeast is of the and Psy2 The in S. cerevisiae a role for PP4cs in to of the yeast deletion with the DNA-damaging and have the of and in to these identification of genes to the anticancer and Scholar, D. genes in the and DNA damage in Sci. S. A. Scholar). We demonstrated that yeast for are to cisplatin to the as for The mammalian ortholog of for the yeast protein in cisplatin sensitivity suggesting that the mammalian complex plays a Furthermore in of the the ortholog of to cisplatin the complex to the DNA damage A yeast screen for partners Psy2 D. V. A. Y. D. S. A of interactions in Saccharomyces 2000; Scholar). We and demonstrated that the interaction is of Rad53 activity not Rad53 (or in is a critical in a DNA damage conserved yeast to ortholog N. DNA damage tumor genes and Opin. Genet. Dev. Scholar and a the of 2003; Scholar). We were not to human interactions by suggesting that these interactions are purification that the interactions may Other PP4cs and DNA damage also PP4cs with and proteins to DNA dephosphorylation at and subsequent A.I. J. of in to DNA Scholar). deletion of not in to that the interaction is not to the cisplatin sensitivity Psy2 was also reported previously to and with and two proteins to a role in the of D. functions of and in the DNA damage Scholar). In human PP4C the PP4cs complex and DNA damage may to at least in by dephosphorylation of at of enhances by protein phosphatase Biol. Chem. Scholar). The of of these to the cisplatin of the PP4cs remains to and as are many types of and a see and of Rev. 1999; Scholar). have also for the a see D. of anticancer Rev. 2005; Scholar). to cisplatin is the of of D. of anticancer Rev. 2005; Scholar). may to drug cellular DNA to DNA and in of and of 2003; 22: Scholar). is thus a major in and a in anticancer has been to anticancer and Rev. 2003; Scholar). The to cisplatin of S. cerevisiae for any of the PP4cs that of the human PP4cs cisplatin may tumor to the In of two different for cisplatin cisplatin demonstrated a to a with of two PP2A interaction and 2005; Scholar, X. Protein phosphatase and of cisplatin by novel anticancer Chem. Scholar). PP4C is also to these C.J. Cohen P.T. Purification of protein phosphatase 4 catalytic by the drug and and 1998; Scholar) and was in these Novel comprised of two the and elicited sensitivity of these the exhibited activity PP2A interaction and 2005; Scholar, X. Protein phosphatase and of cisplatin by novel anticancer Chem. Scholar). on the sensitivity of PP2AC and PP4C to PP4C is also by these A drug that the of PP4cs, by PP4R2 to PP4C thus with cisplatin while PP2A-type Finally a different of anticancer was also demonstrated to cisplatin sensitivity in and (e.g. Y. A. enhances and sensitivity to cisplatin in Scholar). are conserved yeast to and to of PP2A-type phosphatase activity and least in are used in the to and as anticancer see N. N. and of Dev. 18: Scholar and S. S. D. E. of with 2005; Scholar). the cisplatin and was demonstrated to to in the of the protein of a in by A. S. S. J. The tumor to of 2005; Scholar). PP4cs plays a role in the of and the of remains to of the partners and supramolecular architecture of the PP2A-type in the roles that of these proteins plays in cellular processes. that these critical roles in DNA repair and tumor (4Janssens V. Goris J. Van Hoof C. PP2A: the expected tumor suppressor.Curr. Opin. Genet. Dev. 2005; 15: 34-41Google Scholar), a more of their and with for types of We H. and S. for the of and N. H. and of the and for We S. J. A. and for critical of the with

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