Two conserved domains in regulatory B subunits mediate binding to the A subunit of protein phosphatase 2A Xinghai Li 1 and David M. Virshup 1,2 1 Department of Oncological Sciences, Center for Children, Huntsman Cancer Institute, and 2 Department of Pediatrics, University of Utah, Salt Lake City, UT, USA Protein phosphatase 2A (PP2A) is an abundant heterotri- meric serine/threonine phosphatase containing highly con- served structural (A) and catalytic (C) subunits. Its diverse functions in the cell are determined by its association with a highly variable regulatory and targeting B subunit. At least three distinct g ene families encoding B subunits are known: B/B55/CDC55, B¢/B56/RTS1 and B¢¢/PR72/130. No homology has been identi®ed among the B families, and little is known a bout how these B subunits interact with the P P2A A and C subunits. In vitro expression of a series of B56a fragments identi®ed two distinct domains that bound inde- pendently to the A subunit. Sequence alignment of these A subunit b inding domains (ASBD) identi®ed conserved resi- dues in B/B55 and PR72 family members. The alignment successfully predicted domains in B55 and PR72 subunits that similarly bound to the PP2A A subunit. These results suggest t hat these B s ubunits share a common core structure and mode of interaction with the PP2A ho loenzyme. Keywords: phosphoprotein phosphatase; PP2A; subunit interactions; phosphorylation. Protein phosphatase 2A (PP2A) is an abundant cellular serine/threonine-speci®c phosphatase that regulates a sig- ni®cant array of cellular events. The PP2A holoenzyme is a heterotrimer, containing a 65-kDa regulatory A subunit (A/PR65), a 36-kDa catalytic C subunit, and one of a variety of r egulatory B subunits. These diverse B subunits in the P P2A heterotrimer allow the phosphatase to localize t o distinct regions of the cell and to dephosphorylate speci®c substrates, thereby allowing PP2A to regulate diverse processes in the cell such as DNA replication, Wnt signaling, apoptosis, and cytoskeletal function (reviewed in [1,2]). The importance o f B subunits in cellular r egulation is illustrated by the effect of mutations that alter B subunit function. Over-expression of B56 blocks Wnt signaling in Xenopus embryos [3±5], mutations in a Drosophilia B/B55 subunit l eads to imaginal disc duplication and defects in mitosis [6,7], t ransposon insertions in B56c enhance the metastatic ability of mouse melanoma cell lines [8], muta- tions in the A s ubunit that a lter B subunit binding are found in lung, breast, colorectal and s kin cancers [9,10], and decreases in A subunit e xpression are seen in neuronal tumors [11]. Despite the signi®cant role t he B subunits play in cellular homeostasis, little is known about how they physically interact with the PP2A holoenzyme to target the phosphatase to its substrates. The P P2A A subunit serves as a scaffold for assembly of the B and C subunits. I t i s c omposed of 15 imperfect HEAT repeats, each of 39 amino acids, which form a hook-shaped molecule [12]. T he repeats consist of t wo a helices connected by an intrarepeat loop, and m utations in distinct lo ops alter the binding of the B and C subunits [13]. The B subunits bind to repeats 1±10 of the A subunit, whereas the C s ubunit binds to repeats 11±15. Interactions between the B and C subunits are a lso important for heterotrimer formation, as loss of C subunit binding sites prevents B subunit binding [14,15], and modi®cation of the C-term inus of the C s ubunit regulates B subunit binding [16±18]. To date, at least three families of PP2A B subunits have been identi®ed in e ukaryotes. They are designated B (PR55, B55, CDC55), B¢ (PR61, B56, RTS1), and B¢¢ (PR72/130). Each B subunit family is encoded by m ultiple genes, with multiple splice variants, generating an extraordinary diver- sity of these regulatory subunits [1,2]. Although the three families o f B subunits do not share a pparent sequence similarities between the families, they do have signi®cant sequence homology within each family. For example, within the B56 family, each isoform shares a common core r egion of 241 amino acids with 71±88% identity by protein sequence, while both the N- and C -termini are signi®cantly more divergent [19±21]. The conserved core region h as been proposed to interact with the AC h eterodimer, while the nonconserved N- and C-ends may perform different functions, such as r egulation of s ubstrate speci®city and subcellular targeting [20,22]. Two additional classes of polypeptides also interact with the AC core of PP2A. Both the small and middle T antigens encoded by polyomavirus and SV40, and the calmodulin-binding proteins striatin and SG2NA [ 23], bind to the AC c ore of P P2A. However, unlike the B subunits, T antigens and s triatin do not require interaction with, nor methylation, of the PP2A C subunit [17]. Little is known about the molecular basis for the interaction of the B subunits with the AC heterodimer. None of the B subunits have been mapped to de®ne the Correspondence to D. M. Virshup, Huntsman Cancer Institu te, University of Utah, Salt Lake City, UT 84112. Fax: + 801 587 9415, Tel.: + 801 585 3408, david.virshup@hci.utah.edu Abbreviations: PP2A, protein phosphatase 2A; ASBD, A subunit binding dom a in; GST-A, glu t athione S-transferase A s ubunit; NP-40, nonidet p40; CMV, cyto megalovirus. (Received 19 S eptember 2001, revised 8 November 2001, accepted 16 November 2001) Eur. J. Biochem. 269, 546±552 (2002) Ó FEBS 2002 A subunit binding domains. I n t his s tudy, w e u sed t he B56 a isoform as a model regulatory protein to identify structural elements involved in the interaction with PP2A. We identi®ed two distinct domains within the B56a core region that are each suf®cient for interaction with the A subunit. Sequence alignment analyses demonstrated that these two distinct regions are signi®cantly conserved among the three eukaryotic B subunit f amilies. The predicted A subunit binding domains in B/B55 and B¢¢/PR7 2 were also able to interact with the PP2A A subunit. The p resence of a conserved motif in the highly divergent B subunits suggests a common ancestry, structure, and mode of A subunit interaction for these i mportant regulatory proteins. EXPERIMENTAL PROCEDURES Synthesis of [ 35 S]protein [ 35 S]Methionine-labeled B subunits and t heir fragments, and SV40 small t antigen a nd its mutant were g enerated by coupled in vitro transcription and translation in r abbit reticulocyte lysates ( TNT, Promega) using PCR-generated templates. All N-terminal PCR primers incorporated a T3 or T7 promoter sequence. Ampli®ed PCR products were puri®ed using a PCR puri®cation kit (Qiagen) and 200±400 ng of puri®ed DNA was added to 50 lLof reticulocyte lysate in the presence of [ 35 S]methionine. The reaction was incubated at 30 °C for 2 h. In several cases, additional lower molecular mass bands were seen which a re likely to b e due to either premature termination o r partial proteolysis of the [ 35 S]methionine-labeled proteins. Preparation of glutathione S -transferase (GST) and GST-A fusion proteins The GST-A subunit o f PP2A ( GST-A) construct was a generous gift from M. Mumby (UT Southwestern, Dallas, TX, USA) [24]. Puri®cation of GST-A and GST proteins from Escherichia coli was p erformed as described previously [24]. The puri®ed proteins were thoroughly dialyzed against buffer A (50 m M Tris/HCl pH 7.5, 20 m M NaCl, 2 m M EDTA, 1 m M dithiothreitol, containing 3 lgámL )1 pepsta- tin and leupeptin, 2 m M benzamidine, and 1 m M phen- ylmethanesulfonyl ¯uoride). The resultant protein preparation was stored at )70 °C in buffer A containing 50% glycerol until use. GST precipitation assay The binding reactions contained 10 lLof[ 35 S]methionine- labeled polypeptides from programmed reticulocyte lysates, 2 lg of GST or GST-A and buffer A in a ®nal volume of 50 lL. After i ncubation for 2 h at ambient temperature (or 4hat30°C, where indicated), the reaction was diluted to 500 lL with buffer B [buffer A containing 0.1% nonidet p40 (NP-40) and 0.25% BSA] and 20 lLofapre- washed 1 : 1 slurry of glutathione±Sepharose (Amersham Pharmacia) was added. Incubation continued f or 2 h at 4 °C . The beads we re then washed four times w ith 1 mL of buffer B , o r R IPA buffer (50 m M Tris, pH 7 .5, 150 m M NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS) where indicated, for 10 min each wash. Bound proteins were then eluted by incubating the beads with 20 lLof10m M reduced glutathione in buffer A on ice for 30 min The eluted polypeptides w ere analyzed by either conventional SDS/PAGE or on tricine/glycine gels for small molecular mass peptides [25] and i maged u sing a Molecular D ynamics PhosphorImager. RESULTS AND DISCUSSION Identi®cation of two A subunit-binding domains in B56a To determine the minimal r egion of B56 that interacted with the PP2A subunit, we utilized an in vitro binding assay using GST-A subunit and reticulocyte lysate-synthesized B f rag- ments [10,21]. To optimize c onditions for the assay, full- length B56a was ®rst tested for binding to GST-A. B56a full-length protein bound well to GST-A, but not to GST alone (Fig. 1A). To f urther con® rm the speci®c binding, SV40 small t antigen and a truncation mutant w e re used a s a binding control. Consistent with p revious reports, GST-A speci®cally bound to wild-type small t, but not to a mutant small t antigen l acking the A subunit binding site (m#3, Fig. 1. Binding of B56a to the A subunit of PP2A. [ 35 S]Methionine- labeled proteins generated in vitro w ere incu ba ted with GST or GST-A for 2 h at ambient temperature, and precipitated w ith glutathione± Sepharose beads. The bound proteins were eluted with the reduced glutathione and analyzed b y SDS /PAGE followed b y Pho sphorIm ager analysis. (A) Added C subunit does not enhance the GST-A:B56a interaction. Binding of B56a wild type pr otein to PP2A A was assessed in the presence or a bsence of 1 lg of puri®e d PP2A C and/or 10 lLof 35 S-labeled PP2A C synthesized in vitro. ( B) GS T-A bound spe ci®cally to the full-length SV40 small t, but not to the m#3 mutant small t (1±110 fragment). Ó FEBS 2002 Conserved PP2A A subunit binding domains (Eur. J. Biochem. 269) 547 small t 1±11 0 f ragment, Fig. 1B) [ 24,26]. A lso c onsistent with previous reports, we saw no enhancement of B56a binding when the reactions were supplemented w ith puri®ed C subunit or [ 35 S]methionine-labeled C subunit synthesized in the reticu locyte lysate (Fig. 1A), suggesting the C subun it present in the reticulocyte lysate may contribute t o the formation of heterotrimers [27]. To map the region(s) of B56a responsible for binding to the A subunit, multiple B56 fragments were generated by PCR followed by in vitro transcription and translation. The ability of t he fragments to bind to GST-A was assessed as described above and the results shown in Fig. 2. Two distinct domains that interacted with GST-A but not the GST control w ere identi®ed. Generally less than 10% of input B56a was recovered from the glutathione±Sepharose beads w hen GST-A s ub unit was included. This low r ecovery may be due to a high level of nonspeci®c adsorption of the B56a polypeptides to the beads, and suboptimal binding in the absence of cotranslation of the A and C subunits. The smallest N-terminal fragmen t of B56a tha t interacted with GST-A encompasses r esidues 200±303 (Fig. 2). A second domain e xtending from amino acids 325 ±383 wa s capable of independently binding to GST-A ( Fig. 2). These regions were named A subunit b inding domains (ASBD) 1 and 2. Given that the two distinct regions can bind to the structural A subunit, an effort was undertaken to express these domains in vivo. We reasoned that over-expression o f an A subunit binding domain at high levels might displace endogenous B subunits, thereby blocking speci®c interac- tions with substrates and leading to alterations in speci®c signaling pathways. A series of epitope-tagged B56a frag- ments (amino acids 1±142, 142±303, 200±383, 303±383, and 383±486) were expressed in human embryonic kidney (HEK293) cells using a cytomegalovirus (CMV)-promoter driven construct. Unfortunately, only the 1±142 fragment was highly expressed by immunoblot analysis, w hile the 142±303 fragment was barely e xpressed in comparison with expression of the full-length protein (1±486). Expression of other B56a fragments was not detectable (data not shown). Similar r esults were obtained with t wo additional expression vectors. In addition, fusion of green ¯uorescent protein to either end of a polypeptide containing B56a amino acids 180±383 did not result in detectable protein. Considering that these fragments can be well expressed in reticulocyte lysates, it seems likely that t he failure to detect the e xpressed fragments in c ultured cells is due to enhanced degradation by intracellular proteases. One possibility is that these B56a fragments h ave substantially lower af®nity for the PP2A AC heterodimer than does full-length B56a. As B56 subunits over-expressed in v ivo are detected only in PP2A he terotri- mers [20], B subunits and their fragments unable to be stabilized by PP2A b inding in vivo may be inherently unstable and rapidly l ost. Identi®cation of two conserved regions present in all three families of B subunits Although no apparent sequence homology has been discovered among B s ubunits of the t hree families identi®ed thus far, all B subunits do bind to overlapping N-terminal regions of PP2A A (intraloop repeats 1±10) [13,27]. These data sugge st t he possibility that B subunits contain common structural elements that are responsible for t he PP2A A binding. To test whether these two PP2A A binding domains identi®ed in B56a are conserved among different B subunits, the CLUSTALW multiple sequence alignment program (available at http://workbench.sdsc.edu) was used to align a diverse collection o f B subunits (either functionally identi®ed or characterized by sequence homology from various species) against these two domains. While full- length B56 failed to produce a signi®cant alignment with other B subunits, homology with B/B55 and P R72 family members was found when only the B56 binding domains were used in the a lignment ( Fig. 3). For ASBD 1, the region of homology ( amino a cids 188±292 of hsB56a) substantially overlaps the experimentally deduced A subunit b inding domain (amino acids 200±303), while for A SBD 2, t he overlap is even tighter (homology, 329±386; binding 325± 383). Conserved hydrophobic, charged, and po lar residues are distributed along the length of the two domains. The two domains are s eparated by a l ess-conserved region o f between 20 and 41 amino acids. A conserved amino-acid pro®le ( Fig. 3) was generated by visual inspection of the two aligned s equences, a nd used to search the nonredundant protein database at the Swiss I nstitute for Experimental Cancer Research web site (http://www.isrec.isb-sib.ch). Each pro®le identi®ed over 9 5% of the approximately 1 05 B/B55/CDC55, B¢/B56/RTS1, and B¢¢/PR72 related seq- uences contained in the database. Neither pro®le identi®ed any novel types o f B subunits, strongly suggesting no additional conventional B subunit families exist, at least in the n onredundant protein database. Neither pro®le identi- ®ed irrelevant proteins. The pro®les did not match SV40 and polyomavirus t antigens nor members of the striatin/ SG2NA families, implying the se PP2A-interacting proteins have a distinct ancestry and mechanism of interaction. Notably, the pro®les identi®ed B, B56, and PR72-type B subunits in organisms as diverse as Neurospora crassa, Candida tropicalis, Dictyostelium discoideum, Medicago varia (alfalfa), Arabidopsis t haliana, Oryza sativa (rice), Caenorhabditis elegans, Drosophila m elanogaster, Xenopus laevis, a nd mammals. Combining the ASBD 1 a nd 2 pro®les with a variable linker b etween them also identi®ed o ver 90% of the B subunits in the database. Similar results were obtained when a PROSITE pro®le, generated from the multiple sequence alignment data using the MOTIF program at http://www.motif.genome.ad.jp was used to search t he Swiss-Prot protein database. We conclude that these pro®les accurately re¯ect con served amino acid s in the P P2A B subunit families. Fig. 2. Binding of full-length and truncated B56a to the A subunit of PP2A. [ 35 S]Methionine-labeled reticulocyte lysate-synthesized B56a and f ragments were mixed with GST-A or GST for 2 h at the ambient temperature as described, and the resultant complexes were precipi- tated with glutathione±Sepharose b eads. After washing, bound com- plexes were elute d with re duced glu tathion e and analyzed by SDS/PAGE and PhosphorImager. (A) Schematic summary of the binding pro perties of the B56a fragmen ts. Th e emp ty bar represents full-length B56a or its fragments, and the gray boxes represent the deduced A s ubunit-bin ding d omains. (B) Representative autoradio- graphs from the binding assays. The left panel shows 5 lL of input reticulocyte lysate, and the r ight panel d emonstrates which B 56a fragments precipitated with GST-A and GST beads. Each experiment was repeated at least thre e times with sim ilar results. 548 X. Li and D. M. Virshup (Eur. J. Biochem. 269) Ó FEBS 2002 Ó FEBS 2002 Conserved PP2A A subunit binding domains (Eur. J. Biochem. 269) 549 The binding of the two conserved regions from B and PR72 to GST-A To test whether these two conserved regions of B subunits found in B/B55/CDC55 and PR72/B¢¢ family members indeed form domains capable of interaction with the PP2A A subunit, the corresponding regions from rat Ba and human PR72 were expressed a nd [ 35 S]methionine- labeled in reticulocyte lysates, and tested in the G ST precipitation assay. As shown in Fig. 4, polypeptides encompassing the two conserved regions from Ba and PR72 bound well to GST-A, but not to GST alone. Unrelated fragments of Ba and PR72 lying outside the deduced A subunit binding domains did n ot bind to GST-A (data not shown). S V40 small t antigen was used as positive control for GST-A binding, while the C-terminal truncated small t ant igen (m#3) was used f or a negative control. The fact that the sequence alignment presented in Fig. 3 correctly predicted domains in B/CDC55 and PR72 family members that interact with PP2A A subunit strongly suggest that the sequence conservation is biolog- ically relevant. How do B subunits bind to the A subunit? The A subunit is comprised of 1 5 imperfect repeats, and the B subunits interact with repeats 1 through 10. Detailed mutagenesis and structural s tudies have shown that intrarepeat loops are binding s ites f or different types of B subunits [13,27]. Substitution of certain amino acids in the intrarepeat loops abrogates the binding of some B subunits but not others [13,28]. The results here de®ne two distinct PP2A binding domains in the B subunits that are signi®cantly conserved among all B subunits of the three known families. These conserved residues in the B s ubunits are likely to r e¯ect a common conserved s tructure, w hile the variable residues and spacing may allow the B subunits to contact different residues on th e A subunit i ntrarepeat loops. One further implication of the sequence conservation is that th e B subunits may h ave evolved from a single ancestral B subunit. In summary, in this study we have de®ned t wo separate PP2A b inding domains in the regulatory a nd targeting B 56a subunit, which a re conserved in sequence a nd function in all three families of regulatory B subunits. This ®nding may facilitate i denti®cation of new B subunits and p rovide Fig. 3. PP2A B s ubunits have two conserved ASBDs. Representative B sub units of the three families (B, B56, and PR72) f rom evolutionarily distant organisms were a ligned against the t wo ASBD domains i n human B56a (residues 2 00±303 and 325±383). The ®rst two characters on the left are the name of an organism (hs, homo sapie ns;oc,Oryctolagus c unic ulus;dm,Drosophila melanogaster;xl,Xenopus laevis;ce,Caenorhabditis elegans;sc, Saccharomyces cerevisiae;sp.,Schizosaccharomyces pombe;at,Arabidopsis thaliana;rn,Rattus norvegicus ;dd,Dictyostelium discoideum;os,Oryza sativa;mm,Mus musculus). Nu mb ers in parentheses indicate the ®rst and last o f the aligned amino-acid residues in the individual protein sequence, followed by the GenBank accession number. Amino acids that are invariant are highlighted in black. Identical residues conserved in more than 50% of the aligned B subunits are highlighted in dark gray, while conserved similar residues are highlighted in light gray. The two deduced ASBD pro®les are listed underneath the alignments. R esidues marked with asterisks were included in the pro®les. 550 X. Li and D. M. Virshup (Eur. J. 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Virshup 1,2 1 Department. bunit -binding dom ains (ASB D 1 and ASBD 2 ) in human B 56 a, ratBa, and human PR72, highlighted in gray. Ó FEBS 2002 Conserved PP 2A A subunit binding domains