Molecular and Cellular Biochemistry 132: 101-108,1994. 9 1994 Kluwer Academic Publishers. Printed in the Netherlands.
Immunological approach to identify Calmodulin-stimulated phosphatase isozymes from bovine brain Noriko Yokoyama, Takayoshi Kuno, 2 Shunsuke Furuyama and Jerry H. Wang 3 1Department of Physiology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, 271, Japan; 2 Department of Pharmacology, Kobe University School of Medicine, Kobe, 650, Japan; 3 Department of Medical Biochemistry, MRC Group in Signal Transduction, University of Calgary, Calgary, Alberta, T2N 4N1, Canada Received 31 May 1993; accepted 20 December 1993
Abstract Molecular cloning of human, mouse and rat brain CaM-stimulated phosphatase has suggested the existence of two genes for the ct subunit of the enzymes. Act and A~ fragments of Act and A[~ from rat brain library have been expressed in bacteria to produce specific anti-calcineurin Act and anti-calcineurin AI3 antibodies (Kuno et aL, J Neurochem 58: 1643-1651, 1992). Alternative mRNA splicing gives rise to additional calcineurin isozymes with some containing an insertion sequence of ATVEAIEADE. Antibody against synthetic peptide of this insertion sequence has been raised in this study. Three CaM-stimulated phosphatase isozymes previously purified from bovine brain (BPI, BPII, BPIII) (Yokoyama & Wang, J Biol Chem 266: 14822-14829, 1991), along with the bacterially expressed rat Act and A~ fragments, were analyzed by two calcineurin ct subunit monoclonal antibodies V J6 and VD3, the rat anti-calcineurin Act and anti-calcineurin A[~ specific polyclonal antibodies, and the insertion peptide antibody. The bovine brain CaMstimulated phosphatase isozymes BPI and BPIII reacted with both anti-calcineurin Act and anti-calcineurin A[~ antibodies. While BPII reacted with anti-calcineurin Act but not anti-calcineurin A~ antibody, it differed from the expressed Act fragment in immunoreactivity towards the monoclonal antibodies. The results show that the bovine brain CaM-stimulated phosphatase isozymes cannot be simply categorized as derived from Act or A[~ genes products. On the other hand, on the basis of immunoreactivity toward the insertion antibody, the isozymes can be readily classified into those containing the insertion sequence (BPI, LPI) and those without the inserting (BPII, BPIII, LPII, LPIII). (Mol Cell Biochem 132: 101-108, 1994)
Key words: CaM-stimulated phosphatase isozymes, PP2B, calcineurin Abbreviations." C a M - calmodulin, SDS - sodium dodecyl sulfate, B P - brain peak, LP - lung peak, PAGE - polyacrylamide gel electrophoresis, EGTA - [ethylenebis(oxyethylenenitrilo)]-tetra acetic acid, cDNA - complementary DNA, mAb - monoclonal antibody, NBT - nitro blue tetrazolium chloride, BCIP - 5-bromo 4-chloro-3-indolyl phosphate
Address for offprints: N. Yokoyama, Department of Physiology,Nihon University School of Dentistry at Matsudo, 2-870-1,Sakaecho-nishi, Matsudo, Chiba, 271, Japan
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Introduction CaM-stimulated phosphatase, also known as calcineurin [1] or phosphatase 2B [2], has been purified from bovine brain and shown to be composed of two subunits, ~ (or A), molecular weight 60,000 and ~ (or B), molecular weight 20,000 [3-6]. The c~ subunit contains the CaMbinding domain, catalytic domain and autoinhibitory domain [1, 4, 7], whereas the [~subunit has the Ca 2+binding domain [1, 6]. CaM-stimulated phosphatase is ubiquitously distributed in eukaryotes [8] and exists as tissue-specific isozymes [9, 10]. Molecular cloning studies using human, mouse and rat brain libraries have suggested that there are at least two genes for CaM-stimulated phosphatase ~ subunit, designated as PP2Bc~ [1113] and PP2BI3 [14,151, or Am [12] and A[3 [15]. Through alternative mRNA splicing, these two genes may give rise to as many as 5 isoforms of the o~subunit: PP2B0q, PP2Ba 2, PP2B~I, PP2B[32 and PP2BIS3 [13, 14]. The third gene (PP2B%) for CaM-stimulated phosphatase (x subunit is a testis specific form and also has been cloned from murine and human libraries [16, 17]. In addition, a distinct o~ subunit of CaM-stimulated phosphatase (PP2Bw) has been cloned from a rabbit brain library and has been identified as a yeast phosphatase [18,19]. None of the protein products of these genes have been identified and isolated in pure state. Recently, carboxyterminal fragments of calcineurin Ao~ and A~ have been expressed in E. coli [20]. Antibodies displaying strict specificities toward their respective protein antigens have been raised against these protein fragments. We have recently purified three isozymes of CaMstimulated phosphatase from bovine brain or bovine lung which are designated as BPI, BPII, and BPIII, or LPI, LPII and LPIII, respectively [21]. These isozymes exhibit differentia][ reactivities towards two specific monoclonal antibodies VJ6 and VD3. While all isoforms react about quantitatively equally with VD3, only BPI shows strong reactivity and BPIII, LPI, weak reactivity toward VJ6. The relationship between the multiple bovine brain isozymes and the multiple cDNA clones of calcineurin from human, mouse and rat brain libraries is not clear. In the present study, attempts are made to elucidate the relationship between the purified bovine brain CaM-stimulated phosphatase isozymes and the various calcineurin c~subunit clones by using an immunological approach. Five antibody preparations were used, these are the monoclonal antibodies produced against the bo-
vine brain enzyme. V J6 and VD3 [22[, the polyclonal antibodies produced against the bacterially expressed rat Am and AI3 fragments, respectively, and an antibody against a synthetic peptide corresponding to the insertion sequence found in PP2Ba 1. From the comparative analysis of the immunoreactivities of the various antibodies with the purified bovine brain CaM-stimulated phosphatase isozymes and the bacterially expressed rat brain Am and A[3 fragments, it is suggested that isozyme patterns in mammalian brain are species specific.
Materials and methods Materials
Nitro blue tetrazolium chloride (NBT) was purchased from Sigma. Immobilon was from Milipore. Affi-gel 10 and affinity purified goat anti-mouse IgG (H + L) horseradish peroxidase or alkaline phosphatase conjugate were from Bio Rad. 5-bromo 4-chloro-3-indolyl-phosphate (BCIP) was from Boehringer Mannheim GmbH. Peroxidase conjugated IgG Fraction goat anti rabbit IgG was from Cappel. Alkaline-phosphatase APA to rabbit IgG (H + L) was from Biomaker. Insertion peptide (ATVEAIEADE) was from Takara Biomedicals (Japan).
Proteins
Three isoforms of CaM-stimulated phosphatase from bovine brain and bovine lung (BPI, BPII, BPIII, LPI, LPII and LPIII) were purified as described previously [21]. Briefly, bovine brain or lung CaM-binding protein fractions [23] were applied to a heparin-agarose column pre-equilibrated with buffer A (20raM Tris-HC1, pH 7.0, i mM Mg(Ac)2, i mM imidazole, 10 mM 2-mercaptoethanol and 0.1 mM EGTA) containing protease inhibitors: 200mg/1 phenylmethylsulfonyl fluoride, 400 mg/1 benzamidine, 10 mg/1 trypsin inhibitor and 2 mg/1 antipain. PI and PII were eluted in the flowthrough fraction or subsequent washing fraction, respectively PIII was eluted by buffer A containing NaCh Ech peak was repurified by heparin-agarose column chromatography and further purified by FPLC gel filtration [21].
103
Expression of Ac~ and A~ fragments in bacteria Expression was performed as described previously [20]. The cDNA fragments encoding amino acids 179-521 of Acz [12] or amino acids 179-525 or 406-525 of A~3 [15] were amplified with 23-28 base oligonucleotide primers containing NcoI or BamHI linkers using polymerase chain reaction [24]. Amplified cDNA fragments were digested with NcoI and BamHI and ligated into the pET8c backbone digested with NcoI and BamHI. Bacterial cells containing these plasmidos expressed 343, 347 or 120 amino acid residues as nonfusion proteins, respectively. Expressed protein in E. coli were solubilized in the SDS-PAGE sample buffer and subjected to SDSPAGE.
calcineurin Acz and calcineurin A[3 were prepared as described before [20] using the bacterially synthesized fragments of Ao~ (amino acids 179-521) or A[3 (amino acids 406-525) as immunogens and affinity purified using antigen-coupled Sepharose 4B conjugates [20]. Anti-bovine brain CaM-stimulated phosphatase polyclonal antibody was raised and affinity purified as described before [10]. For anti-insertion peptide antibody production, purified insertion peptide was coupled to keyhole limpet hemocyanin (KLH) and the peptide-KLH conjugate was used to immunize a rabbit. Antibody was partially purified by 50% ammonium sulfate precipitation and the precipitate dissolved and dialyzed against 10 mM Tris-HC1 (pH 7.0).
Antibodies
SDS-polyacrylamide gel electrophoresis and immunoblotting analysis
c~subunit specific mAbs, VJ6 and VD3, were produced [22] and purified from mouse ascites as previously described [25]. Polyclonal antibodies which distinguished
SDS-polyacrylamide gel electrophoresis was carried out according to the method of Laemmli [26]. Proteins were stained with Coomassie Brilliant Blue. For immunoblot-
Fig. 1. Comparison of immunoreactivity of CaM-stimulated phosphatase isozymes toward mAbs, VD3 and VJ6, and polyclonal antibodies, anticalcineurin Ac~ and anti-calcineurin A[3. Purified bovine brain CaM-stimulated phosphatase isozymes, BPI, BPII and BPIII, and purified bovine lung CaM-stimulated phosphatase isozymes, LPI, LPII and LPIII, were subjected to Western immunoblot analysis using mAb VD3 (A), mAb VJ6 (B), anti-calcineurin Ac~ antibody (C), or anti-calcineurin A[~ antibody (D). Approximately 0.6 gg of each purified sample was analyzed except 1.7 gg of BPIII and 2.0 p.g of LPIII were used. Lane 1-6 are BPI, BPII, BPIII, LPI, LPII and LPIII, respectively. Position of the c~subunit is marked as c~.
104
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Fig.2. Immunologicalanalysis of the bacterially synthesizedfragments of calcineurinA(x or calcineurinA[3.0.5 gg of each bacterially synthesized fragments of calcineurin Ac~ (amino acids 179-521,lane 1) or calcineurin A[3 (amino acids 179-525, lane 2) and BPI phosphatase (lane 3) were subjected to SDS-PAGE.The proteins were analyzedby immunotransblotwith anti-calcineurinAc(antibody (A), anti-calcineurinAt3antibody (B), polyclonalantibody against bovine brain native calcineurin(C), mAb VD3 (D), mAb VJ6 (E) or by protein stain with CoomassieBrilliantBlue (F). Positions of subunits were marked as ct and [3. ting analysis, proteins were transfered to Immobilon m e m b r a n e in the presence of 0.1% SDS by the method of Towbin et al. [27]. The membranes were incubated with antibodies and immunostained using either peroxidase conjugated or alkaline phosphatase conjugated second antibodies with 4-methoxy-l-naphthol or NBT/ BCIP as a substrate, respectively.
Immunoprecipitation of BPI by VJ6 or VD3 immunoaffinity gel BPI phosphatase (20 gg) was immunoprecipitated by using 100 gl of VJ6 or VD3 immunoaffinity gel as described before [21]. The immunoprecipitates were boiled for 5 min with SDS-sample buffer, centrifuged, and the supernatants used for immunological analysis.
Immunoprecipitation of BPI by anti-calcineurin Ao~ or anti-calcineurin A~ antibody BPI (3.7 gg) was incubated with anti-calcineurin Ac~ antibody or anti-calcineurin A[~ antibody for 1 hr at 30 ~ C. Anti-rabbit IgG coupled protein A-Sepharose gel was added to the reaction mixture and further incubated for 2 hr at 4 ~ C. The immunoprecipitates were boiled for 5 min with SDS-sample buffer, centrifuged, and the supernatants used for immunoblot analysis.
Results and discussion Immunoreactivity of o~subunit isoforms of CaM-stimulated phosphatase Since antibodies produced against the bacterial expressed Ao~ and A[3 fragments display strict specificity towards the respective protein antigens, the possibility of using these antibodies to classify bovine brain CaMstimulated phosphatase isozymes into 0~ and [~ gene
105 products has been examined. Figure 1 shows immunoreactivities of the purified CaM-stimulated phosphatase isozymes from bovine brain and bovine lung towards these antibodies. For comparison, mAbs VJ6 and VD3, which have been shown previously to exhibit differential immunoreactivities towards these isozymes, have also been used in this experiment. In agreement with our previous observation, all isoforms from bovine brain and lung immunostained equally by mAb VD3 (Fig. 1A). On the other hand, BPI showed strong reactivity toward mAb VJ6, BPIII and LPI exhibited weak reactivity toward mAb VJ6 (Fig. 1B), whereas BPII, LPII and LPIII showed no reactivity towards mAb VJ6. Analysis of the immunoreactivity of the various bovine isozymes towards anti-calcineurin Act and anti-calcineurin A[3 antibodies showed that anti-calcineurin Act and anti-calcineurin A[3 antibodies were similar to mAb VD3 or mAb V J6, respectively. Thus BPII, LPII and LPIII did not react with either mAb VJ6 or anti-calcineurin AI3 antibody on the blot, whereas BPIII showed a weak stain with mAb V J6 or anti-calcineurin AI3 antibody (Fig. 1B and 1D). On the other hand, like mAb VD3, anti-calcineurin Act displayed similar reactivity towards all isozymes (Fig. 1C).
Immunoreactivity of the bacterially synthesized fragments While comparison of the immunoreactivities of bovine CaM-stimulated phosphatase isozymes suggested that the immunoreactivity of mAbs VD3 and V J6 are similar to anti-calcineurin Act and anti-calcineurin A[3 antibodies, respectively, further investigation of the relationship between these antibodies (Act and A[3), with respect to their reactions with the bacterially synthesized fragments of the isoforms (amino acids 179-521 of Act or amino acids 179-525 of A~) has led to different conclusions. Anti-calcineurin Act and anti-calcineurin AI3 antibodies were found to display strict specificity, i.e. showing immunoreactivity only towards their respective antigens (Fig. 2). Both mAb VJ6 and mA[3 VD3, on the other hand, exhibited strong immunoreactivity toward the bacterially synthesized fragment of Act, with no immunoreactivity towards the fragment of A[3 (Fig. 2D and 2E). From the results of Fig. i and Fig. 2, it may be concluded that the four antibodies used in these experiments possess distinct sites of reaction on CaM-stimulated
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Fig.3. ImmunoprecipitationofBPI phosphataseby a VJ6-immunoaffinitygel. PurifiedBPI phosphatase(20 ktg)was immunoprecipitated by using100 gl of VJ6-immunoaffinitygel for 4 hr at 4~C. A control experimentwas done underthe conditionswithoutBPI phosphatase. The immunoprecipitatewas analyzedby immunotransblotusinganticalcineurinAcr antibody(A), anti-calcineurinA[~antibody(B), mAb VD3 (C),or mAbVJ6 (D). Lane1-3 are originalBPIphosphatase,the immunoprecipitateand VJ6-immunoaffinitygel control,respectively. Positionsof the c~subunit,heavychain of IgG and lightchainof IgG were markedas c~,H and L, respectively. phosphatase isozymes. Analysis of the immunoreactivity of the purified bovine brain isozymes with these antibodies shows that none of isolated bovine isozymes can be classified into the Act or AI] gene product. Isozymes BPI and BPIII reacted with both anti-calcineurin Act and anti-calcineurin A[3 antibodies, so that they possess characteristic of both Act and A~ gene products. Although BPII shows reactivity toward anti-calcineurin Act but not anti-calcineurin A~ antibodies, it differ from the expressed Act fragment in showing immunoreactivity toward the two monoclonal antibodies, mAbs VJ6 and VD3 (Fig. 1 and Fig. 2).
106
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Fig. 4. Immunoprecipitationof BPI phosphataseby anti-calcineurinAc~and anti-calcineurinA[3 antibodies.Immunoprecipitationprotocolwas describedunder 'Methods'.BPI immunoprecipitatedby either anti-calcineurinAc~antibody(lane 2) or anti-calcineurinA[3antibody(lane3) was stainedwithanti-calcineurinAa antibody(A), anti-calcineurinA[~antibody(B),mAbVD3 (C) or mAbVJ6 (D). Lane1is originalBPIphosphatase sample. Positionof the c~subunit is marked as a.
Immunological analysis of the immunoprecipitation of BPI Although the observation that BPI, BPIII and LPI react with both anti-calcineurin A a and anti-calcineurin A~ antibodies suggests that these isozymes are neither Ao~ nor A[~ gene product, an alternative explanation that these isozyme preparations contained mixtures of A a and A[3 gene-derived isozymes has to be considered. To test this possibility; a sample of BPI was subjected to immunoprecipitation by mAb VJ6 and the precipitate was probed by Western immunoblot analysis. The result showed that the a subunit of the precipitate reacted with both anti-calcineurin A a antibody and anti-calcineurin A[~ antibody, as well as with mAbs V J6 and VD3 (Fig. 3). If BPI is a mixture of an anti-calcineurin Ac~ antibody reactive and an anti-calcineurin A~ antibody reactive isozymes, only the former should be immunoprecipitated by a mAb VJ6 immunoabsorbant. Thus, the immunoprecipitate would be expected to react with the anticalcineurin A a antibody only. Similar results were obtained in the immunoprecipitation using a mAb VD3 immunoabsorbant (result not shown). In addition, phosphatase activity of BPI was quantitatively precipitated by either the mAb V J6 or mAb VD3 immunoabsorbant (results not shown), further supporting the notion that the BPI sample contained a single isozyme species. Similarly, BPI immunoprecipitated by either anti-calcineurin Ac~ antibody or anti-calcineurin A~ antibody immunoabsorbant was found to react with all four antibodies (Fig. 4), and the phosphatase activity was essentially completely removed from the supernatant upon immunoprecipitation (results not shown). These results shows
clearly that BPI is distinct from both rat A a and A[3 gene products. Thus it may be suggested that bovine brain CaM-stimulated phosphatase isozymes cannot be classified simply into the categories of Ac~ and A[~ gene products.
Immunostaining of CaM-stimulated phosphatase isozymes using anti-insertion peptide antibody In addition to the multiplicity in structure genes, alternative mRNA splicing also cotributes to multiple protein isoforms. Inspection of the sequence alignment of various mouse and human brain CaM-stimulated phosphatase isozymes shows that in some isoforms, a sequence of ten amino acids, ATVEAIEADE, is inserted near the C-terminus [13, 28]. Thus, this sequence is found in PP2Bc h and PP2B[~2but not in PP2Bc~2, PP2B[31 and PP2BI33. An antibody against a synthetic peptide corresponding to this sequence has been raised and used to discriminate the bovine brain and bovine lung CaMstimulated phosphatase isoforms. As shown in Fig. 5, BPI and LPI showed good reactivity towards the antiinsertion peptide antibody. Whereas BPII, BPIII, LPII and LPIII did not react with this antibody.
General conclusion
Results of the present immunological characterization of bovine brain CaM-stimulated phosphatase isozymes and the bacterial expressed rat Ac~ and A[~ fragments are summarized in Table 1. The major conclusion from
107 zyme patterns may also exist. The identification of the various bovine CaM-stimulated phosphatase isozymes in terms of their protein structures, therefore requires the molecular cloning of CaM-stimulated phosphatase from bovine brain libraries.
References
9
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Fig. 5. Immunoreactivity of CaM-stimulated phosphatase isozymes towards anti-insertion peptide antibody. Immunotransblot analysis of the various samples used for the experiment in Fig. 1 were carried out with anti-insertion peptide antibody. Same amounts of protein as described in the legend of Fig. i were analyzed. Lane 1-6 are BPI, BPII, BPIII, LPI, LPII, and LPIII, respectively. Position of the a subunit is marked as a.
Table i is that none of the purified bovine brain and lung CaM-stimulated phosphatase isozymes can be classified simply as A a or A[3 gene product on the basis of their immunoreactivity towards mAbs VD3 and VJ6, and anti-calcineurin A a and anti-calcineurin A]3 polyclonal antibodies. On the other hand, the antibody raised against the insertion peptide can easily distinguish the isozymes containing the insertion (BPI and LPI) from those without such an insertion (BPII, BPIII, LPII and LPIII). The study has demonstrated the general usefulness of the immunological approach in the study of isozymes. It seems that the calcineurin isozymes pattern is more complex than that has been revealed by the molecular cloning studies. In addition to tissue specific CaMstimulated phosphatase isozymes, species specific isoTable 1. Immunoreactivities of brain, lung CaM-stimulated phosphatases and synthesized fragment of calcineurin Ac~ or calcineurin AlL Symbols used to detect the strength of reactivities are: +, positive stain; -, nondetectable; N/T, not tested. BPI BPII BPIII LPI LPII LPIII Ac~ A~ fragment fragment (179-521) (406-525) Antibody VD3 V J6 Ac~ A[3 Insert antibody
+ + +
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