©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification in the Calcineurin A Subunit of the Domain That Binds the Regulatory B Subunit (*)

(Received for publication, September 6, 1994)

Yasuo Watanabe Brian A. Perrino Bill H. Chang Thomas R. Soderling (§)

From the Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Calcineurin (CaN) is the serine/threonine protein phosphatase (phosphatase 2B) that is activated by binding of Ca to its B subunit and to calmodulin (CaM). This paper identifies residues between the catalytic region and the CaM-binding domain of the A subunit as the domain that binds the regulatory B subunit.

A purified fusion protein containing residues 328-390 of the A subunit 1) binds CaN B subunit, and 2) inhibits (IC = 0.1 µM) the in vitro stimulation of CaN A phosphatase activity by purified CaN B subunit. A synthetic peptide corresponding to residues 341-360 blocked the binding of CaN B to residues 328-390 in the fusion protein, so 4 hydrophobic residues within this region (Val-Phe and Phe-Val) were mutated to either Glu (E mutant) or Gln (Q mutant). The wild-type and mutant A subunits were expressed individually or co-expressed with B subunit in Sf9 cells, purified and characterized. The mutant A subunits were similar to wild-type A subunit in terms of basal phosphatase activity (1-3 nmol/min/mg) and activation by Mn/CaM. Addition of purified B subunit to purified wild-type A subunit at a 1:1 molar ratio gave a 40-fold increase in phosphatase activity whereas addition of B subunit to either of the mutant A subunits had no effect on phosphatase activity, even at a 3:1 molar excess of B subunit. Furthermore, when wild-type or mutant A subunits were co-expressed with B subunit and purified on CaM-Sepharose, the B subunit co-eluted with the wild-type A subunit but not with either mutant A subunit. These results demonstrate that residues 328-390 in the A subunit bind B subunit and that the mutated hydrophobic residues are essential.


INTRODUCTION

Calcineurin (CaN), (^1)a Ca/calmodulin (CaM)-dependent protein Ser/Thr phosphatase (protein phosphatase 2B), is a major brain protein (1% of total brain protein as opposed to 0.1-0.05% in other tissues)(1, 2) . Although the substrate specificity of CaN is limited compared to protein phosphatases 1 and 2A, CaN is involved in the regulation of diverse cellular functions such as ion channels(3) , protein phosphatase 1 activity(4) , gene transcription(5, 6) , and neuronal long term depression(7) .

CaN is a heterodimer of a catalytic A subunit (58-61 kDa) and a regulatory B subunit (19 kDa). In addition to the NH(2)-terminal catalytic domain (residues 71-325, numbering based on the rat brain alpha isoform(8) ), the A subunit contains a central CaM-binding domain (residues 390-414) (9) and a COOH-terminal autoinhibitory region (residues 420-482) (10) . (^2)The myristylated B subunit has four ``EF'' hand high affinity Ca-binding sites(2) . The isolated CaN A subunit has very low phosphatase activity, which is synergistically activated 200-fold by CaM plus B subunit(11) . Kinetic studies have shown that Ca/CaM activates CaN by increasing the V(max), whereas Ca-binding to the B subunit decreases the K and increases the V(max).^2 Thus, binding of Ca to the B subunit is essential for enzymatic activity and for the cellular regulation of CaN.

Studies on limited proteolysis of CaN in the absence of Ca/CaM demonstrated cleavage of the CaN A subunit NH(2)-terminal of the CaM-binding domain with retention of phosphatase activity and B subunit binding(12) . This result demonstrated that the binding domain for the B subunit was located between the catalytic domain and the CaM-binding domain. Dissociation of the CaN A and B subunits requires buffer containing 6 M urea(13) , indicating that hydrophobic interactions are important in the subunit association. Two regions (residues 347-357 and 363-372) whose hydrophobicity are highly conserved in CaN A isoforms from human to Drosophila are likely candidates for binding the B subunit. Using peptide chemistry, site-specific mutagenesis and enzymology, this paper identifies this region as important for B subunit interaction.


EXPERIMENTAL PROCEDURES

cDNA Construction and Mutagenesis

The cDNA encoding the -isoform rat brain CaN A (-CaN A) was introduced into SmaI- and EcoRI-digested pVL1393 transfer vector resulting in pVLCaN A(11) . The KpnI/SphI fragment of the cDNA for the -CaN A was inserted into M13mp18 to produce single-strand DNA for mutagenesis. Two different oligonucleotides, 5`-TTTCTCCCCTTCTTCTGGCAGCGACCAGGTTTCTTCATCCATGAA-3` and 5`-TTTCTCCCCTTGTTGTGGCAGCGACCAGGTTTGTTGATCCATGAA-3` designed for mutation of (Val/Phe and Phe/Val) to (Glu/Glu and Glu/Glu) and (Gln/Gln and Gln/Gln) (underlined codons), respectively, were synthesized. The Amersham oligonucleotide-directed mutagenesis system (version 2.1) was used to generate mutant cDNAs. Mutant clones were isolated, and cDNA sequences were reconfirmed by dideoxynucleotide sequencing (Sequenase version 2.0, U. S. Biochemical Corp.)(14) . The mutant cDNAs were ligated into pVLCaN A and transfected into Sf9 cells. The BamHI/PstI fragment of the cDNA encoding rat brain CaN B was ligated into pVL1393 to generate pVLCaN B.

Construction and Expression of the CaN A 328-390 Fusion Protein

The CaN 328-390 fragment was constructed by PCR from alpha-CaN A plus engineered BamHI and EcoRI restriction sites. This fragment was subcloned into pGEX-2T vector which contains GST (Pharmacia Biotech Inc.), and the fusion protein was expressed in Escherichia coli and purified by affinity chromatography on glutathione-Sepharose (Pharmacia Biotech Inc.).

Cell Culture and Selection of Recombinant Baculovirus

Sf9 cells were cotransfected with linear baculovirus DNA (In Vitrogen) and pVLCaN A or pVLCaN B. Recombinant baculovirus were identified by screening for occlusion-negative plaques, and expressed CaN A or CaN B was detected by Western blots of cell homogenates with rabbit anti-CaN A or anti-CaN A/B. Second-passage recombinant baculovirus was obtained and titered by end point dilution. Large scale protein expression of CaN A was done in 100-150-ml Corning spinner flasks at an initial cell density of 3 times 10 ^6 cells/ml and m.o.i. of 5. For co-expression of CaN A/B, the m.o.i. of CaN A and CaN B were 4 and 6, respectively.

Purification of Expressed CaN A and Co-expressed CaN A/B

Purification procedures were performed as described previously except that the 100,000 times g centrifugation was omitted and protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 5 mg of leupeptin/liter, 5 mg of aprotinin/liter, and 5 mg of soybean trypsin inhibitor/liter) were added to the (NH(4))(2)SO(4) pellet(11) . Protein concentration was determined by the method of Bradford (15) using BSA as the standard.

Synthetic Peptides

R peptide (DLDVPIPGRFDRRVSVAAE), a synthetic peptide substrate for CaN, was synthesized, and the purity, amino acid composition, and concentration were determined as described(16) . CaN A 341-360 (YWLPNFMDVFTWSLPFVGEK) was synthesized by Macromolecular Resources.

In Vitro Reconstitution of CaN A and CaN B and Phosphatase Assays

Reconstitution of CaN A and Can B was carried out as described previously(11) . P-R peptide was phosphorylated by cAMP kinase (17) and used as substrate for CaN with previously described assay conditions(11) .

Gel Electrophoresis and Western Blot Analysis

SDS-PAGE was carried out according to the method of Laemmli(18) . The electrophoretic transfer of proteins from the SDS-PAGE to the PVDF membrane was performed as described by Towbin et al.(19) . For immunodetection of the transferred proteins, the procedure of Burnette (20) was used except that the second antibody was linked to horseradish peroxidase. Antigen-antibody complexes were visualized by reacting the bound peroxidase with the chemiluminescence reagent (DuPont).

Materials

[-P]ATP (6,000 Ci/mmol) was purchased from DuPont NEN. Restriction enzymes and DNA-modifying enzymes were from Promega or Life Technologies, Inc. Grace's insect medium, lactalbumin hydrolysate, yeastolate, Pluronic F-68, and antibiotics were from Life Technologies, Inc. Fetal bovine serum was purchased from HyClone. Dowex resins were from Bio-Rad. CaM-Sepharose were obtained from Pharmacia Biotech Inc. The CaN antibodies that recognize the CaN A and CaN A/B were gifts from Dr. K. Fukunaga (Kumamoto University, Japan) and Dr. R. Kincaid (NIAA, National Institutes of Health), respectively. Anti-CaN B was from Upstate Biotechnologies, Inc. The bacterial expressed myristylated CaN B was kindly provided by Dr. F. Rusnak (Mayo Foundation, Rochester, MN). All other materials and reagents were of the highest quality available from commercial suppliers.


RESULTS

Interaction of CaN B with CaN A328-390

CaN A subunit sequences from Drosophila to human brain show a highly conserved region of unknown function between the catalytic domain and the CaM-binding domain (Fig. 1A). This CaN A sequence has two highly hydrophobic motifs (Fig. 1B), which may be involved in binding of the B subunit since dissociation of native CaN into its A and B subunits requires 6 M urea (13) . Therefore, we expressed and purified a fusion protein of glutathione S-transferase (GST) and sequences 328-390 of CaN A (GST/A328-390). We tested the ability of residues 328-390 in this fusion protein to interact with CaN B by two independent techniques. Direct binding of CaN B to residues 328-390 was demonstrated in a mixture containing B subunit, fusion protein GST/A328-390, and glutathione-Sepharose. When this mixture was centrifuged and washed, the B subunit was bound to the fusion protein in the pellet (Fig. 2, lane 1). This interaction did not occur with just the GST protein alone (Fig. 2, lane 2), demonstrating that the B subunit was binding to residues 328-390. Furthermore, binding of CaN B to the GST/A328-390 was blocked by the presence of 10 µM synthetic peptide corresponding to residues 341-360 of CaN A (data not shown).


Figure 1: Schematic of rat brain alpha CaN A subunit. A, the localization of the domains for catalytic phosphatase activity (residues 71-325), CaM binding (390-414), and one autoinhibitory element (457-482) are depicted. The sequence 328-390 is also shown with the residues mutated in this study underlined. B, the hydropathy index of residues 328-390 for wild-type (upper panel) and Q mutant (lower panel) were analyzed by the method of Kyte and Doolittle (22) with a window average of 6 residues. The asterisks indicate the residues mutated in this study.




Figure 2: Binding of CaN B to residues 328-390 of CaN A. The CaN B subunit (1 µg) was incubated for 1 h at 4 °C in 0.5 ml containing 40 mM Tris-HCl (pH 7.5), 0.5 mM CaCl(2), 150 mM 2-mercaptoethanol, 0.2 mg/ml BSA, glutathione-Sepharose 4B (40 µl of 50% (v/v) slurry), and either 8 µg of fusion protein GST/A328-390 plus 8 µg of GST (lane 1) or 16 µg of GST (lane 2). The Sepharose beads were collected by centrifugation, washed three times with 40 mM Tris-HCl (pH 7.5), 0.5 mM CaCl(2), 300 mM NaCl, resuspended in 50 µl of 2 times SDS-PAGE sample buffer, and boiled for 3 min. Eluted proteins were analyzed by SDS-PAGE (15%), stained by Coomassie Blue (panel A), or transferred to PVDF membrane and immunoblotted with anti-CaN B antibody (panel B).



Since CaN A residues 328-390 bind CaN B subunit, GST/A328-390 should block the in vitro interaction between the CaN A and B subunits. In agreement with our previous study(11) , the low phosphatase activity of baculovirus/Sf9-expressed CaN A subunit (8 nmol/min/mg, Fig. 3A) was greatly enhanced (300-350 nmol/min/mg, Fig. 3A) after in vitro reconstitution with the B subunit (Fig. 3A). When the CaN A and B subunits were co-expressed in Sf9 cells, the purified CaN had an activity of about 700 nmol/min/mg CaN A, and purified brain CaN had an activity of 410 nmol/min/mg CaN A under the same assay conditions(11) . Thus, reconstitution with the B subunit resulted in a 40-fold increase in the phosphatase activity of the A subunit to a value equivalent to purified brain CaN and about half that of co-expressed CaN. However, increasing concentrations of GST/A328-390 inhibited this in vitro reconstitution with an IC of 50-100 nM (Fig. 3B). This represents potent inhibition since the concentrations of CaN A and B subunits were each 30 nM. Inhibition by GST/A328-390 was specific since 1) GST alone was without effect (Fig. 3B), and 2) addition of 600 nM fusion protein to co-expressed CaN A/B gave a small stimulation of phosphatase activity (not shown).


Figure 3: Reconstitution of CaN A and B subunits in the absence and presence of fusion protein GST/A328-390. Panel A, time course of subunit reconstitution. CaN A (50 nM) was incubated with CaN B (50 nM) at 30 °C in 40 mM Tris-HCl (pH 7.5), 1.5 mM Mn, 150 mM beta-mercaptoethanol, and 2 mg/ml BSA. At the indicated times aliquots were removed and assayed 10 min for phosphatase activity using 60 µMP-R peptide and 500 nM CaM as described under ``Experimental Procedures.'' The phosphatase activity of co-expressed CaN A/B using the same conditions was 668 ± 33 nmol/min/mg CaN A. Panel B, inhibition of CaN A and B subunit reconstitution by GST/A328-390. CaN A (30 nM) was incubated with the indicated concentrations of GST/A328-390 or GST for 1 h prior to incubation for another hour with CaN B (30 nM). The reconstitution conditions and phosphatase assay were the same as in panel A. Assays in panels A and B were performed in duplicate, and each point represents the mean ± S.D. from two experiments.



Construction and Expression of CaN A Mutants

The above results demonstrated that residues 328-390 in the CaN A subunit bind CaN B subunit. Since binding of the A and B subunits probably involves hydrophobic interactions and peptide 341-360 blocked binding of B subunit, we mutated two clusters of hydrophobic residues within CaN A 341-360: Val-Phe and Phe-Val. All 4 residues were mutated to either Gln (Q mutant) or Glu (E mutant). Computer analysis of predicted hydrophobicity indicated that these mutations would dramatically reduce the hydrophobicity of this region (Fig. 1B, lower panel).

These mutant CaN A subunits were expressed individually in Sf9 cells and purified on CaM-Sepharose. Analysis by SDS-PAGE and Western blot indicated highly purified subunits (Fig. 4). The low basal phosphatase activities of these expressed mutant CaN A subunits, assayed in the presence of Mn, were equivalent to the basal activity of the wild-type A subunit (Fig. 5, left bars). Wild-type and mutant A subunit phosphatase activities were each stimulated 5-6-fold by Mn/CaM (Fig. 5, middle bars). Thus, the mutant CaN A subunits had indistinguishable properties relative to wild-type A subunit in terms of basal phosphatase activity and activation by CaM. However, the mutant A subunits did not exhibit activation by B subunit upon in vitro reconstitution whereas the wild-type A subunit was stimulated 40-fold (Fig. 5, right bars). Fig. 5(inset) shows that wild-type A subunit was maximally activated by a 1:1 molar ratio of B subunit to A subunit, but the mutants were not affected by 3.4-fold molar excess of B subunit. Our reconstitution conditions included 1.5 mM Mn, and we previously determined that the presence of Ca had no effect. Since the B subunit is a Ca-binding protein, we also tested for reconstitution of the mutant A subunits with the B subunit in the presence of 0.5 mM Ca, but there was still no detectable activation of the mutant A subunits by B subunit (data not shown). Because the reconstitution is relatively slow (Fig. 3A), there may be refolding of either or both subunits during the incubation. If the mutant A subunits were improperly folded, they may not be able to properly unfold to interact with B subunit. We added 6 M urea, which is known to cause dissociation of the A and B subunits(13) , to a mixture of B subunit plus either wild-type or mutant A subunits, incubated them overnight and then dialyzed out the urea prior to assaying phosphatase activity. The resulting phosphatase activities of wild-type and mutant A subunit plus B subunit incubations were essentially identical to the far right bars in Fig. 5(data not shown).


Figure 4: SDS-PAGE and Western blot analyses of purified wild-type and mutant CaN A subunits. Lane 1, wild-type CaN A; lane 2, mutant E CaN A; lane 3, mutant Q CaN A. Proteins (2 µg) were separated by 10% SDS-PAGE and stained by Coomassie Blue (panel A) or transferred to PVDF membrane and immunoblotted with anti-CaN A antibody (panel B).




Figure 5: Effects of mutagenesis of CaN A subunit on phosphatase activities and reconstitution with B subunit. Purified wild-type CaN A and mutants Gln (Q) and Glu (E) at 50 nM were assayed at 30 °C for 10 min without (left bars) or with (middle bars) CaM (500 nM) in the presence of 1.5 mM Mn and 60 µMP-RII peptide. Each subunit (50 nM) was reconstituted for 2 h with 170 nM CaN B subunit (see ``Experimental Procedures'') prior to determining phosphatase activities (right bars). The inset shows the effect of the molar ratio of B subunit to A subunit on the reconstitution. All assays were performed in duplicate, and each point represents the mean ± S.D. from two experiments.



Co-expression of CaN B Subunit with Wild-type and Mutant CaN A Subunits

The above results suggest that the hydrophobic residues mutated in the CaN A subunit are essential to in vitro association of the A and B subunits. To further test the role of these hydrophobic residues, the wild-type and mutant CaN A subunits were co-expressed with the B subunit in Sf9 cells. The Western blot in Fig. 6A shows that both the A and B subunits were expressed in the cells. The doublet of immunoreactive bands at about 19 kDa was due to the presence of both the myristylated (lower band) and non-myristylated (upper band) B subunits.^2 The Sf9 extracts were subjected to CaM-Sepharose, which purifies the CaN A subunits due to their CaM-binding domains (Fig. 6B). With co-expressed wild-type subunits, the B subunit co-purifies with the CaN A subunit due to their interaction, and the Western blot of the CaN eluted from the CaM-Sepharose detects both A and B subunits. Note that both the myristylated and non-myristylated B subunits are present in the holoenzyme (Fig. 6B). However, the B subunit did not co-purify with either of the mutant CaN A subunits, confirming their lack of interaction (Fig. 6B). Thus, the B subunit does not interact with the mutant A subunits either in the in vitro reconstitution assay or when co-expressed in Sf9 cells.


Figure 6: Co-expression of CaN B with wild-type and mutant CaN A subunits. Panel A, Sf-9 cells were co-infected with wild-type CaN A (lane 2), mutant E CaN A (lane 3), or mutant Q CaN A (lane 4) and CaN B at a m.o.i. of 4 and 6, respectively. Cells were harvested 72 h post-infection, homogenized in buffer A and centrifuged at 10,000 times g for 60 min, and the supernatants were subjected to SDS-PAGE. Lane 1 is a crude extract from non-infected Sf9 cells. Proteins were transferred to PVDF membranes and treated with anti-CaN A and anti-CaN B. Panel B, the supernatants were purified on CaM-Sepharose chromatography, and the EGTA eluants were analyzed by SDS-PAGE. Western blot analysis was performed as above.




DISCUSSION

This study used multiple approaches to identify the region of the CaN A subunit that is involved in binding of the regulatory B subunit. The B subunit binding domain was likely to be located between the catalytic domain and the CaM-binding domain, since limited proteolysis in the absence of Ca/CaM cleaves the A subunit NH(2)-terminal of the CaM-binding domain but the B subunit is still associated with the catalytic A subunit(21) . We focused on the highly conserved (Fig. 1) sequence 328-390 between the catalytic domain and the CaM-binding domain.

The fusion protein GST/A328-390 containing the putative B subunit-binding domain specifically bound CaN B (Fig. 2) and potently inhibited in vitro interaction of the A and B subunits (Fig. 3B). Interaction of the CaN A and B subunits requires 6 M urea for dissociation(13) , suggesting the potential importance of hydrophobic interactions. Since the synthetic peptide corresponding to CaN A sequence 341-360 blocked binding of CaN B to GST/A328-390 (data not shown), we focused on hydrophobic residues (Phe and Val) within 341-360 and mutated two hydrophobic clusters to either Gln or Glu. Glu and Gln were chosen because they are both very hydrophilic and they have side-chain bulkiness close to Phe and Val. The mutant A subunits were well expressed in Sf9 cells and purified on CaM-Sepharose (Fig. 4). However, the two mutant CaN A subunits did not interact with the B subunit as determined by both enzymatic assay after in vitro reconstitution (Fig. 5) and physical detection after co-expression and purification on CaM-Sepharose (Fig. 6). The inability of the Gln mutant to bind the B subunit was presumably due to the decreased hydrophobicity of residues 347-357, whereas the lack of B subunit binding of the Glu mutant could additionally involve the acidic charges. The CaN B subunit is an acidic protein(2) . The inability of the mutant A subunits to bind B subunit was probably not due to major conformational changes induced by the mutations since 1) both mutant A subunits alone had Mn-stimulated phosphatase activities similar to wild-type A subunit (Fig. 5, far left bars), and 2) both mutant A subunits bound (i.e. were purified on CaM-Sepharose) and were stimulated by (Fig. 5, middle bars) Ca/CaM. Since the catalytic (residues 71-325) and CaM-binding (residues 391-414) domains are normal in the mutant A subunits and they flank the region of mutagenesis (residues 349-357), any putative structural changes would be confined to the B subunit-binding domain. Furthermore, in vitro reconstitution after exposure of both subunits to 6 M urea was normal for wild-type A subunit but did not occur with either mutant. Finally, the mutant A subunits, unlike wild-type A subunit, did not interact with B subunit when they were co-expressed in Sf9 cells (Fig. 6).

Binding of the B subunit to the catalytic CaN A subunit is essential to its activity and regulation of its phosphatase activity. We have recently determined that Ca binding to the B subunit activates CaN by both decreasing its K(m) and increasing the V(max), whereas Ca binding to CaM increases only V(max).^2 It will be important to determine the mechanism by which binding of the B subunit to the A subunit alters the phosphatase kinetic properties, and this identification of the domain in the A subunit which binds the B subunit will facilitate further study. Our results identifying hydrophobic residues 349/350 and 356/357 in the A subunit as essential for binding of the B subunit do not exclude the potential involvement of other residues (e.g. residues 365-371, LVNVLNI, Fig. 1B) in B subunit binding. We are also initiating a study to determine residues in the B subunit involved in binding to the A subunit.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM41292.

§
To whom correspondence should be addressed: Vollum Institute, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Rd., Portland, OR, 97201. Tel.: 503-494-6931.

(^1)
The abbreviations used are: CaN, calcineurin; BSA, bovine serum albumin; CaM, calmodulin; GST, glutathione S-transferase; m.o.i., multiplicity of infection; PCR, polymerase chain reaction; PVDF, polyvinylidene; P-R peptide, P-labeled peptide derived from the R subunit of cAMP kinase; PAGE, polyacrylamide gel electrophoresis.

(^2)
Perrino, B. A., Ng, L. Y., and Soderling, T. R. (1995) J. Biol. Chem.270, 340-346


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.