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

Eur. J. Biochem. 269, 546–552 (2002) (cid:211) FEBS 2002

Two conserved domains in regulatory B subunits mediate binding to the A subunit of protein phosphatase 2A

Xinghai Li1 and David M. Virshup1,2

1Department of Oncological Sciences, Center for Children, Huntsman Cancer Institute, and 2Department of Pediatrics, University of Utah, Salt Lake City, UT, USA

pendently to the A subunit. Sequence alignment of these A subunit binding domains (ASBD) identified 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 that these B subunits share a common core structure and mode of interaction with the PP2A holoenzyme.

Keywords: phosphoprotein phosphatase; PP2A; subunit interactions; phosphorylation.

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 gene families encoding B subunits are known: B/B55/CDC55, B¢/B56/RTS1 and B¢¢/PR72/130. No homology has been identified among the B families, and little is known about how these B subunits interact with the PP2A A and C subunits. In vitro expression of a series of B56a fragments identified two distinct domains that bound inde-

repeats, each of 39 amino acids, which form a hook-shaped molecule [12]. The repeats consist of two a helices connected by an intrarepeat loop, and mutations in distinct loops 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 subunit binds to repeats 11–15. Interactions between the B and C subunits are also important for heterotrimer formation, as loss of C subunit binding sites prevents B subunit binding [14,15], and modification of the C-terminus of the C subunit regulates B subunit binding [16–18].

Protein phosphatase 2A (PP2A) is an abundant cellular serine/threonine-specific phosphatase that regulates a sig- nificant 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 regulatory B subunits. These diverse B subunits in the PP2A heterotrimer allow the phosphatase to localize to distinct regions of the cell and to dephosphorylate specific 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 of B subunits in cellular regulation 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 leads to imaginal disc duplication and defects in mitosis [6,7], transposon insertions in B56c enhance the metastatic ability of mouse melanoma cell lines [8], muta- tions in the A subunit that alter B subunit binding are found in lung, breast, colorectal and skin cancers [9,10], and decreases in A subunit expression are seen in neuronal tumors [11]. Despite the significant role the 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 PP2A A subunit serves as a scaffold for assembly of the B and C subunits. It is composed of 15 imperfect HEAT

To date, at least three families of PP2A B subunits have been identified in eukaryotes. They are designated B (PR55, B55, CDC55), B¢ (PR61, B56, RTS1), and B¢¢ (PR72/130). Each B subunit family is encoded by multiple genes, with multiple splice variants, generating an extraordinary diver- sity of these regulatory subunits [1,2]. Although the three families of B subunits do not share apparent sequence similarities between the families, they do have significant sequence homology within each family. For example, within the B56 family, each isoform shares a common core region of 241 amino acids with 71–88% identity by protein sequence, while both the N- and C-termini are significantly more divergent [19–21]. The conserved core region has been proposed to interact with the AC heterodimer, while the nonconserved N- and C-ends may perform different functions, such as regulation of substrate specificity 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 core of PP2A. However, unlike the B subunits, T antigens and striatin 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 define the

Correspondence to D. M. Virshup, Huntsman Cancer Institute, 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 domain; GST-A, glutathione S-transferase A subunit; NP-40, nonidet p40; CMV, cytomegalovirus. (Received 19 September 2001, revised 8 November 2001, accepted 16 November 2001)

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reduced glutathione in buffer A on ice for 30 min The eluted polypeptides were analyzed by either conventional SDS/PAGE or on tricine/glycine gels for small molecular mass peptides [25] and imaged using a Molecular Dynamics PhosphorImager.

R E S U L T S A N D D I S C U S S I O N

Identification of two A subunit-binding domains in B56a

A subunit binding domains. In this study, we used the B56a isoform as a model regulatory protein to identify structural elements involved in the interaction with PP2A. We identified two distinct domains within the B56a core region that are each sufficient for interaction with the A subunit. Sequence alignment analyses demonstrated that these two distinct regions are significantly conserved among the three eukaryotic B subunit families. The predicted A subunit binding domains in B/B55 and B¢¢/PR72 were also able to interact with the PP2A A subunit. The presence of a conserved motif in the highly divergent B subunits suggests a common ancestry, structure, and mode of A subunit interaction for these important regulatory proteins.

E X P E R I M E N T A L P R O C E D U R E S

Synthesis of [35S]protein

To determine the minimal region of B56 that interacted with the PP2A subunit, we utilized an in vitro binding assay using GST-A subunit and reticulocyte lysate-synthesized B frag- ments [10,21]. To optimize conditions for the assay, full- length B56a was first tested for binding to GST-A. B56a full-length protein bound well to GST-A, but not to GST alone (Fig. 1A). To further confirm the specific binding, SV40 small t antigen and a truncation mutant were used as a binding control. Consistent with previous reports, GST-A specifically bound to wild-type small t, but not to a mutant small t antigen lacking the A subunit binding site (m#3,

[35S]Methionine-labeled B subunits and their fragments, and SV40 small t antigen and its mutant were generated by coupled in vitro transcription and translation in rabbit reticulocyte lysates (TNT, Promega) using PCR-generated templates. All N-terminal PCR primers incorporated a T3 or T7 promoter sequence. Amplified PCR products were purified using a PCR purification kit (Qiagen) and 200–400 ng of purified DNA was added to 50 lL of reticulocyte lysate in the presence of [35S]methionine. The reaction was incubated at 30 (cid:176)C for 2 h. In several cases, additional lower molecular mass bands were seen which are likely to be due to either premature termination or partial proteolysis of the [35S]methionine-labeled proteins.

Preparation of glutathione S-transferase (GST) and GST-A fusion proteins

resultant

The GST-A subunit of PP2A (GST-A) construct was a generous gift from M. Mumby (UT Southwestern, Dallas, TX, USA) [24]. Purification of GST-A and GST proteins from Escherichia coli was performed as described previously [24]. The purified proteins were thoroughly dialyzed against buffer A (50 mM Tris/HCl pH 7.5, 20 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol, containing 3 lgÆmL)1 pepsta- tin and leupeptin, 2 mM benzamidine, and 1 mM phen- protein fluoride). The ylmethanesulfonyl preparation was stored at )70 (cid:176)C in buffer A containing 50% glycerol until use.

GST precipitation assay

The binding reactions contained 10 lL of [35S]methionine- labeled polypeptides from programmed reticulocyte lysates, 2 lg of GST or GST-A and buffer A in a final volume of 50 lL. After incubation for 2 h at ambient temperature (or 4 h at 30 (cid:176)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 lL of a pre- washed 1 : 1 slurry of glutathione–Sepharose (Amersham Pharmacia) was added. Incubation continued for 2 h at 4 (cid:176)C. The beads were then washed four times with 1 mL of buffer B, or RIPA buffer (50 mM Tris, pH 7.5, 150 mM 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 lL of 10 mM

Fig. 1. Binding of B56a to the A subunit of PP2A. [35S]Methionine- labeled proteins generated in vitro were incubated with GST or GST-A for 2 h at ambient temperature, and precipitated with glutathione– Sepharose beads. The bound proteins were eluted with the reduced glutathione and analyzed by SDS/PAGE followed by PhosphorImager analysis. (A) Added C subunit does not enhance the GST-A:B56a interaction. Binding of B56a wild type protein to PP2A A was assessed in the presence or absence of 1 lg of purified PP2A C and/or 10 lL of 35S-labeled PP2A C synthesized in vitro. (B) GST-A bound specifically to the full-length SV40 small t, but not to the m#3 mutant small t (1–110 fragment).

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small t 1–110 fragment, Fig. 1B) [24,26]. Also consistent with previous reports, we saw no enhancement of B56a binding when the reactions were supplemented with purified C subunit or [35S]methionine-labeled C subunit synthesized in the reticulocyte lysate (Fig. 1A), suggesting the C subunit present in the reticulocyte lysate may contribute to the formation of heterotrimers [27].

binding. To test whether these two PP2A A binding domains identified 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 of B subunits (either functionally identified or characterized by sequence homology from various species) against these two domains. While full- length B56 failed to produce a significant alignment with other B subunits, homology with B/B55 and PR72 family members was found when only the B56 binding domains were used in the alignment (Fig. 3). For ASBD 1, the region of homology (amino acids 188–292 of hsB56a) substantially overlaps the experimentally deduced A subunit binding domain (amino acids 200–303), while for ASBD 2, the overlap is even tighter (homology, 329–386; binding 325– 383). Conserved hydrophobic, charged, and polar residues are distributed along the length of the two domains. The two domains are separated by a less-conserved region of between 20 and 41 amino acids. A conserved amino-acid profile (Fig. 3) was generated by visual inspection of the two aligned sequences, and used to search the nonredundant protein database at the Swiss Institute for Experimental Cancer Research web site (http://www.isrec.isb-sib.ch). Each profile identified over 95% of the approximately 105 B/B55/CDC55, B¢/B56/RTS1, and B¢¢/PR72 related seq- uences contained in the database. Neither profile identified any novel types of B subunits, strongly suggesting no additional conventional B subunit families exist, at least in the nonredundant protein database. Neither profile identi- fied irrelevant proteins. The profiles did not match SV40 and polyomavirus t antigens nor members of the striatin/ SG2NA families, implying these PP2A-interacting proteins have a distinct ancestry and mechanism of interaction. Notably, the profiles identified B, B56, and PR72-type B subunits in organisms as diverse as Neurospora crassa, Candida tropicalis, Dictyostelium discoideum, Medicago varia (alfalfa), Arabidopsis thaliana, Oryza sativa (rice), Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, and mammals. Combining the ASBD 1 and 2 profiles with a variable linker between them also identified over 90% of the B subunits in the database. Similar results were obtained when a PROSITE profile, generated from the multiple sequence alignment data using the MOTIF program at http://www.motif.genome.ad.jp was used to search the Swiss-Prot protein database. We conclude that these profiles accurately reflect conserved amino acids in the PP2A B subunit families.

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 the 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 were identified. Generally less than 10% of input B56a was recovered from the glutathione–Sepharose beads when GST-A subunit was included. This low recovery may be due to a high level of nonspecific 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 fragment of B56a that interacted with GST-A encompasses residues 200–303 (Fig. 2). A second domain extending from amino acids 325–383 was capable of independently binding to GST-A (Fig. 2). These regions were named A subunit binding 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 of an A subunit binding domain at high levels might displace endogenous B subunits, thereby blocking specific interac- tions with substrates and leading to alterations in specific 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, while the 142–303 fragment was barely expressed in comparison with expression of the full-length protein (1–486). Expression of other B56a fragments was not detectable (data not shown). Similar results were obtained with two additional expression vectors. In addition, fusion of green fluorescent 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 the failure to detect the expressed fragments in cultured cells is due to enhanced degradation by intracellular proteases. One possibility is that these B56a fragments have substantially lower affinity for the PP2A AC heterodimer than does full-length B56a. As B56 subunits over-expressed in vivo are detected only in PP2A heterotri- mers [20], B subunits and their fragments unable to be stabilized by PP2A binding in vivo may be inherently unstable and rapidly lost.

Identification of two conserved regions present in all three families of B subunits

Although no apparent sequence homology has been discovered among B subunits of the three families identified thus far, all B subunits do bind to overlapping N-terminal regions of PP2A A (intraloop repeats 1–10) [13,27]. These data suggest the possibility that B subunits contain common structural elements that are responsible for the PP2A A

Fig. 2. Binding of full-length and truncated B56a to the A subunit of PP2A. [35S]Methionine-labeled reticulocyte lysate-synthesized B56a and fragments 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 beads. After washing, bound com- plexes were eluted with reduced glutathione and analyzed by SDS/PAGE and PhosphorImager. (A) Schematic summary of the binding properties of the B56a fragments. The empty bar represents full-length B56a or its fragments, and the gray boxes represent the deduced A subunit-binding domains. (B) Representative autoradio- graphs from the binding assays. The left panel shows 5 lL of input reticulocyte lysate, and the right panel demonstrates which B56a fragments precipitated with GST-A and GST beads. Each experiment was repeated at least three times with similar results.

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The binding of the two conserved regions from B and PR72 to GST-A

How do B subunits bind to the A subunit? The A subunit is comprised of 15 imperfect repeats, and the B subunits interact with repeats 1 through 10. Detailed mutagenesis and structural studies have shown that intrarepeat loops are binding sites for 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 define two distinct PP2A binding domains in the B subunits that are significantly conserved among all B subunits of the three known families. These conserved residues in the B subunits are likely to reflect a common conserved structure, while the variable residues and spacing may allow the B subunits to contact different residues on the A subunit intrarepeat loops. One further implication of the sequence conservation is that the B subunits may have evolved from a single ancestral B subunit.

In summary, in this study we have defined two separate PP2A binding domains in the regulatory and targeting B56a subunit, which are conserved in sequence and function in all three families of regulatory B subunits. This finding may facilitate identification of new B subunits and provide

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 and [35S]methionine- labeled in reticulocyte lysates, and tested in the GST 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 not bind to GST-A (data not shown). SV40 small t antigen was used as positive control for GST-A binding, while the C-terminal truncated small t antigen (m#3) was used for 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.

Fig. 3. PP2A B subunits have two conserved ASBDs. Representative B subunits of the three families (B, B56, and PR72) from evolutionarily distant organisms were aligned against the two ASBD domains in human B56a (residues 200–303 and 325–383). The first two characters on the left are the name of an organism (hs, homo sapiens; oc, Oryctolagus cuniculus; 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). Numbers in parentheses indicate the first and last of 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 profiles are listed underneath the alignments. Residues marked with asterisks were included in the profiles.

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information for further elucidating the structural basis of interactions in the PP2A holoenzyme.

Fig. 4. Binding of the two conserved domains in Ba and PR72 to the A subunit of PP2A. Fragments of rat Ba and human PR72 encompassing ASBD 1 and 2 were tested for binding to GST and GST-A by incubation for 4 h at 30 (cid:176)C. The precipitated proteins were washed with RIPA bu(cid:128)er four times prior to elution from the glutathione–Sepharose beads. (A) Binding of rat Ba ASBD 1 and 2; (B) binding of human PR72 ASBD 1 and 2. SV40 small t antigen and truncation m#3 were used for a specificity control. The data shown are representative of five independent experiments. (C) Diagrammatic representation of the two conserved A subunit-binding domains (ASBD 1 and ASBD 2) in human B56a, rat Ba, and human PR72, highlighted in gray.

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