Báo cáo khoa học: Two conserved domains in regulatory B subunits mediate binding to the A subunit of protein phosphatase 2A pdf

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. Biochem. 269) Ó FEBS 2002 information for further elucidating the structural basis of interactions in the PP2A holoenzyme. ACKNOWLEDGEMENTS We thank Dr Marc Mumby, Estelle Sontag, and Matthew Movsesian for plasmids and Joni Seeling a nd other members of the V irshup lab for their assistance. Oligonuc leotide synthesis was supported by NIH grant 3P30 CA42014. This research was supported by NIH R01 C A80809 and the Huntsman Cancer Foundation REFERENCES 1. Janssens, V. & Goris, J. (2001) Protein phosphatase 2A: a highly regulated family of serine /threonin e phosphatase s implicated in cell growth and signalling. Biochem. J. 353, 417±439. 2. Virshup, D.M. (2000) Protein phosphatase 2A: a panoply of enzymes. Curr. Opin. C ell Biol. 12, 180±185. 3. Seeling, J.M., Miller, J.R., Gil, R., Moon, R.T., White, R. & Virshup, D.M. ( 1999) R egulation o f b eta-catenin signaling b y t he B56 subunit of protein phosph atase 2A. Science 283, 2089±2091. 4. 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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