©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calcium Regulation of Calcineurin Phosphatase Activity by Its B Subunit and Calmodulin
ROLE OF THE AUTOINHIBITORY DOMAIN (*)

(Received for publication, March 18, 1994; and in revised form, October 20, 1994)

Brian A. Perrino (§) Lilly Y. Ng Thomas R. Soderling (¶)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Calcineurin (CaN) contains an autoinhibitory element (residues 457-482) 43 residues COOH-terminal of the calmodulin-binding domain (Hashimoto, Y., Perrino, B. A., and Soderling, T. R.(1990) J. Biol. Chem.265, 1924-1927) that regulates the Ca-dependent activation of its phosphatase activity. Substitution of Arg and Arg or Asp to Ala in the autoinhibitory peptide 457-482 significantly decreased its inhibitory potency. CaN A subunits with these residues mutated to Ala were co-expressed with the Ca-binding B subunit using the baculovirus/Sf9 cell system. Kinetic analysis showed that although the purified mutants had no activity in the absence of calcium, they were less dependent than the wild-type enzyme on calcium and calmodulin for activity. To determine if additional autoinhibitory motifs were present in the COOH terminus of calcineurin, the A subunit was truncated at residues 457 or 420 and co-expressed with B subunit. The V(max) values of both truncation mutants with or without Ca were increased relative to wild-type calcineurin. The increased Ca-independent activity of CaN relative to CaN indicates the presence of additional autoinhibitory element(s) within residues 420-457. CaN had similar high V(max) values with or without Ca, but the K value for peptide substrate was increased 5-fold to 125 µM in the absence of Ca. The K values of all the expressed calcineurin species were increased in the absence of Ca. The CaN A or CaN A subunits alone have low V(max) and high K (115 µM) values even in the presence of Ca. These results indicate that 1) there are several autoinhibitory motifs between the CaM-binding domain and the COOH terminus that are relieved by Ca binding to CaM and the B subunit, 2) Ca binding to the B subunit also regulates enzyme activity by lowering the K of the catalytic subunit for substrate, 3) binding of the B subunit is required for high V(max) values even after removal of the autoinhibitory domain. These results are consistent with synergistic activation of calcineurin by Ca acting through both CaM and the B subunit.


INTRODUCTION

Calcineurin (CaN) (^1)is the neuronal form of the widely dis-tributed Ca/CaM-dependent Ser/Thr phosphoprotein phosphatase 2B (PP-2B) (reviewed in (1) ). CaN is involved in diverse physiological functions such as induction of long term depression in area CA1 of the hippocampus and mediating the immunosuppresant functions of cyclosporin and FK506 which inhibit dephosphorylation of the transcription factor NF-AT(p) by CaN(2, 3, 4) . Studies with FK506 also implicate regulatory roles for CaN in Ca-dependent transcription of other genes(5, 6, 7) . There is also evidence that the activities of Na channels, L-type Ca channels, N-methyl-D-aspartate; receptors, and the heat-stable inhibitors of PP-1 (inhibitor-1 and DARRP-32) are modulated by CaN(1, 8, 9, 10) .

Type 2B phosphatases are heterodimers composed of the catalytic A subunit (57-61 kDa) and a regulatory B subunit (19 kDa)(11) . The CaN B subunit is an ``EF-hand'' Ca-binding protein which remains tightly associated with the A subunit in the presence or absence of Ca(1) . Ca binding to the B subunit stimulates CaN phosphatase activity, but this activity is low compared with that attained in the presence of Ca/CaM. CaN A has very low phosphatase activity by itself, but addition of Mn/CaM or Mn/B subunit gave 5- or 50-fold activations, respectively(12) . However, addition of CaM to reconstituted A and B subunits gave a synergistic 600-fold activation. CaM increased the V(max) whereas B subunit primarily decreased the K with a smaller effect on V(max)(12) .

The catalytic A subunit is composed of several functional domains(13, 14) . The catalytic domain is presumed to be located between residues 71-325 (all numbering will be based on the rat brain alpha isoform (15) ) because of its sequence homology to PP-1 and PP-2A(16) . The CaM-binding domain encompasses residues 391-414(13) . Limited proteolysis of CaN in the presence of Ca/CaM removes the residues COOH-terminal of the CaM-binding domain and generates a 57-kDa A subunit which still binds B subunit and CaM but which no longer requires Ca or CaM for full activity(14) . These results suggested the presence of a COOH-terminal autoinhibitory domain which was localized to residues 457-482 by use of overlapping synthetic peptides(17) . In this report we present the results of our studies, primarily by use of site-specific and truncation mutagenesis, to identify which residues are critical for the autoinhibitory interaction and to determine whether additional autoinhibitory motifs are present in the COOH terminus. Kinetic analysis of the purified mutants also allowed us to determine mechanisms by which Ca binding to its B subunit and to CaM activates CaN.


EXPERIMENTAL PROCEDURES

Isolation of Rat Brain CaN B cDNA

The open reading frame of CaN B was isolated by PCR from an adult rat brain first strand cDNA synthesis preparation. For the PCR reaction the 5` and 3` oligonucleotides used were 5`-GACGGATCCGCAAAATGGGAAATGAGGC-3` and 5`-CGTCTGCAGTCACACATCTACCACCATC-3`, respectively. The amplified cDNA was purified by agarose gel electrophoresis and Gene Clean (Bio 101), ligated into BamHI and PstI cut pVL1393 vector, and sequenced using the Sequenase Version 2.0 Sequencing Kit. This sequence has been reported to GenBank, accession no. LO3554. The predicted amino acid sequence of the rat brain B subunit is identical to the previously published sequence of the human brain B subunit(18) .

Construction of -CaN A Site-directed Mutants and COOH-terminal Deletion Mutants

The EcoRI fragment of full-length -CaN A cDNA (15) was ligated into M13mp18. Using the Amersham mutagenesis kit (version 2.1)(19) , the oligonucleotides 5`-AGGGCTTAGCCCGAATTAAC-3` and 5`-GCATGGCGTCAGCTGCAGGCGGCAT-3` were used to generate the D467A and R476/477A mutants, respectively. The oligonucleotides 5`-TTCAGAGTTAGTCAGCTCTCACTCT-3`, and 5`-TCGAAGCTGGTTCACTTATGTTGTG-3` were used to generate -CaN A and -CaN A, respectively. The -CaN A mutants were sequenced as described above and ligated into pVL1393 transfer vector(12) .

Generation of Recombinant Baculoviruses

Sf9 cells were co-transfected with linear baculovirus DNA (InVitrogen), and pVL1393 containing mutated -CaN A subunits or the CaN B subunit and recombinant baculoviruses were purified by two rounds of plaque purification as described(12) . Sf9 cell cultures were infected with occlusion negative plaques and screened for expression of mutated -CaN A subunit or CaN B by Western blotting of cell homogenates with rabbit anti-CaN antibodies(20) . Second-passage recombinant baculoviruses were titered by end point dilution as described(12) .

Purification of Recombinant Wild-type or Mutated CaN

Sf9 cells at an initial density of 1-2 times 10^6 cells/ml in 100-150 ml of complete Grace's medium were infected with the appropriate -CaN A second passage recombinant baculovirus and second passage CaN B recombinant baculovirus at multiplicities of infection of 4 and 6, respectively, as described(12) . Recombinant CaN as well as the -CaN A and -CaN A subunits alone were purified as described(12) , except that the 100,000 times g centrifugation step was omitted, and protease inhibitors were added to the resuspended ammonium sulfate pellet. Protein concentration was determined by the method of Bradford(21) , using bovine -globulin as standard.

[^3H]Myristate Labeling of Sf9 Cells

A 100-ml spinner culture of Sf9 cells in serum-free medium (Sf900 II, Life Technologies, Inc) was co-infected with recombinant -CaN A and CaN B baculoviruses as described above. [^3H]myristate (0.4 mCi) was added to the culture 70-h post-infection. The cells were harvested 72-h post-infection and recombinant CaN purified as described above. SDS-PAGE (12%) was done using stacking and resolving gels containing 1 mM EGTA. Following SDS-PAGE the proteins were stained with Coomassie Brilliant Blue, destained, and the gel treated with Amplify (Amersham Corp.) for autoradiography using Kodak X-Omat AR film.

Proteins and Synthetic Peptides

CaM and CaN were purified from bovine brain(22, 23) . The concentration of bovine brain or recombinant CaN was determined using the Bradford assay and CaM concentration determined by amino acid analysis. CaN inhibitor peptide (ITSFEEAKGLDRINERMPPRRDAMP), as well as the peptides containing amino acid substitutions, and R pep (DLDVPIPGRFDRVRRVSVAAE), a peptide substrate for CaN (24) were synthesized and their purity, amino acid compositions, and concentrations determined as described(25) . R pep was P-labeled as described(12) .

Phosphatase Assays

Dephosphorylation of [P]R pep by purified bovine brain CaN was measured as described(12) . The phosphatase activities of recombinant wild-type and mutant CaNs toward [P]R pep were determined as described(12) , except for the following alterations. For determining their activities in the absence of Ca, the reactions contained CaN assay buffer (40 mM Tris-HCl, pH 7.5, 0.1 M KCl, 0.5 mM dithiothreitol), and 1 mM EGTA, 6 mM Mg(CH(2)COOH)(2), plus 0.1 mg/ml bovine serum albumin. The reactions for determining their activities in the presence of Ca contained CaN assay buffer with 0.1 mM CaCl(2) and 6 mM Mg(CH(2)COOH)(2). The concentrations of bovine brain CaN, recombinant wild-type and mutant CaN, -CaN A, -CaN A, and CaM are indicated in the figure legends. The K(m) and V(max) values were determined from Lineweaver-Burk plots of data from phosphatase assays in which the [P]R pep concentrations were varied. The concentration ranges of [P]R pep are indicated in the figure legends.

Immunodetection of CaN Subunits in Western Blots

The purified proteins were separated by 15% SDS-PAGE and transferred to nitrocellulose. Blocking, washing, and antibody dilution was done with TBS buffer (100 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20). The stock solution (3 mg/ml methanol) of 4-chloro-1-naphthol was diluted 5-fold in TBS containing 0.1% H(2)0(2) but without Tween-20. The polyclonal rabbit anti-CaN antibody (Upstate Biotechnology Inc.) was diluted 1000-fold, the monoclonal anti-CaN B antibody (Upstate Biotechnology Inc.) diluted 3000-fold, the horseradish peroxidase-conjugated donkey anti-mouse antibody (Bio-Rad) and the horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Bio-Rad) were each diluted 600-fold.

Materials

[-P]ATP (6000 Ci/mmol) was purchased from DuPont NEN. [^3H]Myristate (1 mCi) was purchased from Amersham. Restriction enzymes and DNA-modifying enzymes were from Life Technologies, Inc. or Promega. Oligonucleotides were obtained from Midland Scientific. Vent polymerase was purchased from New England Biolabs. Sf9 tissue culture supplies were from Life Technologies, Inc. Fetal bovine serum was purchased from Hyclone. The CaN antibody was a generous gift from Dr. Randall Kincaid (NIH, NIDAA). Dr. Rich Maurer (Oregon Health Sciences University) kindly provided cyclic-AMP-dependent protein kinase catalytic subunit. All other materials and reagents were of the highest quality available from commercial suppliers.


RESULTS

Identification of Autoinhibitory Residues Using Synthetic Peptide Substitution

In studies of the CaM-kinase II autoinhibitory domain, we identified essential autoinhibitory residues by substitutions in the synthetic autoinhibitory peptide (26) followed by site-specific mutagenesis of the kinase(27, 28) . Following this same strategy, we substituted to Ala the following residues in the synthetic autoinhibitory peptide 457-482: Arg, Asp, Leu, and Glu. Although the CaN autoinhibitory peptide 457-482 inhibits non-competitively with respect to [P]myosin light chain (17) and [P]R pep(29) , and the specificity determinants of CaN substrates have not been identified in their primary amino acid sequences, the rationale for these substitutions was based partly on their sequence homologies to known substrates of CaN (see Fig. 5 of (17) ) as illustrated below with DARPP-32 and the R subunit of cAMP-kinase (S/T indicates the phosphorylation site): Autoinhibitory peptide 457-482, ITSFEEAKGLDRINERMPPRRDAMP; DARPP-32, residues 11-38, FSVPAPPSQLDPRQVEMIRRRRPTPAML; R, residues 72-102, ADSESEDEEDLDVPIPGRFDRRVSVCAE.

Since many substrates of CaN contain basic residues just NH(2)-terminal of the phosphorylated Ser/Thr, we substituted Arg with Ala. With the R synthetic peptide, addition of residues DLDV to the sequence PIPGRFDRRVSVAAE dramatically decreased the K(m) and increased V(max) for dephosphorylation by CaN(24) , so we substituted the analogous Leu and Asp in the autoinhibitory CaN peptide. The 2 Glu residues were chosen since R peptide has similarly positioned acidic residues. In addition, we synthesized a peptide with a 4-residue NH(2)-terminal extension to determine if this would increase inhibitory potency. Fig. 1shows the abilities of these substituted peptides to inhibit purified bovine brain CaN. Compared to the parent peptide, the NH(2)-terminal extension (peptide F) and the L466A substitution (peptide C) had little or no effect on IC The D467A and R476/477A substitutions (peptides D and E, respectively) strongly decreased inhibitory potency, and the E461/462A substitution (peptide B) was intermediate in effect.


Figure 1: Effects of amino acid substitutions on the inhibitory potency of the CaN autoinhibitory peptide. Purified bovine brain CaN (50 nM) was assayed using 30 µM [P]R pep and 150 nM CaM in the presence of 0.5 mM MnCl(2) (see ``Experimental Procedures''). The indicated concentrations of the following peptides were also included: peptide A (residues 457-482 of the -CaN A subunit) (up triangle), peptide B (bullet), peptide C (), peptide D (down triangle), peptide E (box), and peptide F (circle).



Site-specific Mutagenesis of the Autoinhibitory Domain

To confirm the results of the peptide substitutions which indicated that Asp and Arg were important for the inhibitory potency of the autoinhibitory domain, these residues were mutated to Ala in the CaN A subunit (Fig. 2). The Coomassie stain and Western blot of CaM-Sepharose purified wild-type and mutant CaNs are shown in Fig. 3. The purified heterodimeric CaN was not further stimulated by the addition of purified B subunit (data not shown), indicating that the CaN A and B subunits were present at a 1:1 molar ratio. Two B subunit species were present, and the faster migrating one co-migrated with the B subunit of purified brain CaN. Brain CaN B is myristoylated on its NH(2)-terminal glycine(1) , while bacterially expressed CaN B is non-myristoylated and migrates slower than brain CaN B in SDS-PAGE (30) . To determine whether the CaN B doublet consists of myristoylated and non-myristoylated species, Sf9 cells were co-infected with recombinant CaN A and CaN B baculoviruses and labeled with [^3H]myristic acid. The cells were harvested, and the expressed CaN was purified by CaM-Sepharose chromatography and analyzed by SDS-PAGE and autoradiography. As seen in the Coomassie Blue-stained gel of Fig. 4A, baculovirus-expressed CaN contained the 58-kDa CaN A subunit and the low molecular mass B subunit present as a doublet. The autoradiogram in Fig. 4B contained a single ^3H-labeled band corresponding to the lower M(r) band of the CaN B doublet. This result showed that the two immunoreactive B subunits from Sf9 cells consist of myristoylated and non-myristoylated species. Because the baculovirus-expressed CaN was purified by CaM-Sepharose affinity chromatography, these data demonstrate that myristoylation of the CaN B subunit was not necessary for the formation of active CaN A/B heterodimer.


Figure 2: Domain structure of -CaN A subunit. Catalytic, conserved phosphatase domain; CaM, calmodulin-binding domain; INH, autoinhibitory element. Amino acid residues are numbered according to the -CaN A isoform (15) . The positions of the D467A and R467/477A mutants are shown as well as the truncation mutants A and A.




Figure 3: SDS-PAGE and Western blot of wild-type and mutant CaNs purified from Sf9 cells. A, proteins (4 µg/lane) were separated by SDS-PAGE (15%) and stained with Coomassie Brilliant Blue. Lane 1, molecular mass standards; lane 2, bovine brain CaN; lane 3, wild-type CaN; lane 4, CaN D467A; lane 5, CaN R476/477/A; lane 6, CaN; lane 7, CaN. B, proteins (4 µg/lane) were separated by SDS-PAGE (15%) and transferred to nitrocellulose. Immunostaining was carried out as described under ``Experimental Procedures.'' Lane 1, bovine brain CaN; lane 2, CaN; lane 3, CaN D467A; lane 4, CaN R476/477A; lane 5, CaN; lane 6, CaN.




Figure 4: SDS-PAGE and autoradiography of wild-type CaN purified from [^3H]-myristate-labeled Sf9 cells. A, bovine brain CaN (lane 1, 6 µg) and wild-type CaN (lane 2, 6 µg) were separated by 12% SDS-PAGE and stained with Coomassie Brilliant Blue. B, autoradiogram of gel shown in panel A.



Fig. 5shows the K(m) and V(max) values in the absence and presence of Ca and CaM for the wild-type and mutant CaNs. Similar to purified bovine brain CaN, Ca binding to the B subunit increased the wild-type CaN V(max) (Fig. 5A). An additional 3-5-fold increase in the V(max) with little change in the K(m) resulted from binding of Ca/CaM, consistent with displacement of an autoinhibitory domain. Since the D467A and RR476/477AA peptides were poor inhibitors, we expected the corresponding mutants to have increased phosphatase activity in the absence of Ca. However, the D467A and R476A/R477A mutants were inactive in the absence of Ca, but the V(max) values in the presence of Ca alone were increased 2- and 3.5-fold, respectively, compared to wild-type CaN. The mutants also exhibited significantly higher total phosphatase activities in the presence of Ca/CaM compared to wild-type enzyme. The K(m) values of the mutants were similar to the K(m) values obtained for the wild-type phosphatase (Fig. 5B). The increases in V(max) in the presence of Ca or Ca/CaM indicate that Asp and Arg may be involved in the interaction of the autoinhibitory domain with the catalytic domain, but the autoinhibitory interaction most likely involves additional residues since mutation of Asp and Arg by themselves had little or no effect on Ca-independent activity.


Figure 5: Kinetic analysis of wild-type and mutant CaNs for dephosphorylation of [P]R pep. CaN was assayed (see ``Experimental Procedures'') at the following nM concentrations in the presence of 1 mM EGTA (20 min) or 0.1 mM Ca (10 min): EGTA: 500, wild-type; 300, D467A; 300, R476/477A; 30, CaN; 30, CaN; N.D. A N.D. A; Ca: 30, wild-type; 30, D467A; 30, R476/477A; 30, CaN 30, CaN; 100 A 100 A. CaM was present at a 3-fold molar excess of CaN. Five concentrations of substrate were used in the presence of EGTA (30-300 µM [P]R pep) or Ca (4-80 µM [P]R pep), The solid black bars represent the kinetic analyses of the wild-type A subunit and A subunits alone (20-250 µM [P]R pep). The K and V(max) values were determined by linear regression analysis of Lineweaver-Burk data plots. The assays were performed in triplicate, and the activities shown are the mean ± S.D. (n = 3). A, V(max). B, K.



Truncation Mutants of the CaN A Subunit

The autoinhibitory peptide 457-482 has a relatively low IC of 10-20 µM which could indicate that other autoinhibitory elements are present in CaN A. To explore this possibility, two COOH-terminal truncated CaN A subunits were generated by insertion of stop codons at residues 457 (mutant A) and 420 (mutant A) (Fig. 2). The presence of an autoinhibitory domain in CaN was first revealed when limited proteolysis removed the region COOH-terminal of the CaM-binding domain and converted the enzyme from a form with no activity in the absence of Ca to a form with full activity in the absence of Ca(14) . By similar reasoning, the presence of regulatory elements within residues 420-457 would be apparent by increased Ca-independent activity of CaN relative to CaN. Truncation at residue 457 generated an A subunit lacking the autoinhibitory element previously identified within residues 457-482, while truncation at residue 420 generated an A subunit which additionally lacked the NH(2)-terminal region up to the CaM-binding domain. Fig. 3shows a Coomassie-stained SDS-polyacrylamide gel and the corresponding Western blot of the purified truncation mutants CaN CaN, and wild-type CaN.

To examine the effects of step-wise COOH-terminal truncations of the A subunit on phosphatase activity, the kinetic parameters of mutants CaN and CaN were compared to CaN. As seen in Fig. 5A, CaN has an extremely low specific activity of 3-10 nmol/min/mg in the absence of Ca. The concentration of enzyme was increased 10-17-fold (300 nM or 500 nMversus 30 nM) and the assay time doubled in order to detect significant phosphatase activity in the presence of EGTA. Compared to CaN, truncation of the A subunit at residue 457 increased the V(max) in the absence of Ca 10-fold, while truncation at residue 420 resulted in an additional 6-fold increase in the V(max) value (Fig. 5A). In contrast to CaN, the Ca-independent activity of CaN was greater than the Ca/CaM-stimulated activity of CaN, indicating that the phosphatase activity of CaN was totally Ca/CaM-independent. The elevated Ca- and Ca/CaM-stimulated activity of CaN relative to CaN is similar to previous findings showing that proteolysis of CaN results in levels of activity slightly higher than the Ca/CaM-stimulated activities of the non-proteolyzed enzymes(14) . Thus, removal of the autoinhibitory element located within residues 457-482 by truncation at residue 457 gave partial Ca-independence, while truncation at residue 420 generated completely Ca-independent activity. These results indicate that the A subunit contains an additional autoinhibitory element(s) within residues 420-457. Relative to the wild-type enzyme, the activities of CaN as well as the point mutants were elevated by Ca or Ca/CaM. Although the Ca-stimulated activities of the two point mutants and CaN were elevated to similar levels, an additional 2-fold increase in activity which was also Ca-independent was seen with CaN. These results provide further evidence for the presence of additional autoinhibitory elements within residues 420-457.

The very low phosphatase activity of the A subunit is synergistically stimulated by the B subunit and CaM(12) . However, since the high V(max) of the CaN mutant does not require Ca (Fig. 5A), does this mutant also require the presence of the B subunit? As shown by the solid black bars in Fig. 5A, the wild-type A subunit and A mutant subunit without co-expressed B subunit exhibited very low V(max) values of less than 10 nmol/min/mg. These low V(max) values are not an artifact of improper folding of the A subunits in the absence of B subunit since in vitro reconstitution with B subunit gave 25- and 60-fold increases in phosphatase activities for the wild-type A and A subunits, respectively (data not shown). These results indicate that CaN still requires the B subunit to attain high V(max) values in the absence of Ca.

Similar to purified brain enzyme, CaM increased the V(max) of Sf9-expressed CaN without affecting the K(m) (27.7 ± 4.2 versus 29.7 ± 2.4 nmol/min/mg) (Fig. 5B). The K(m) values of the CaN point mutants and truncation mutants in the presence of Ca were similar to the values of wild-type CaN and were also unaffected by CaM (Fig. 5B). These results indicate that the K(m) was unaffected by COOH-terminal alterations and deletions of the A subunit. However, in the absence of Ca the K(m) values of the wild-type, site-specific, and truncation CaN mutants were 4-5-fold higher (Fig. 5B). This suggested that Ca decreased the K(m) by binding to the B subunit, and this hypothesis was confirmed by kinetic analysis of A subunits expressed without the B subunit. Both the wild-type and A truncated A subunits alone had K(m) values of 100-120 µM which were not decreased by Ca (Fig. 5B, solid black bars). These results demonstrate that the ability of Ca to lower the K(m) required the B subunit.

Since the Ca concentration used (100 µM) in these assays was saturating, we tested whether these mutants would also exhibit an increased sensitivity to activation by lower concentrations of Ca. Fig. 6shows their Ca-dependent activation in the absence of CaM. Wild-type CaN was half-maximally activated at 0.35 µM which is similar to a previous report(31) . All of the mutants were significantly more sensitive to activation by lower concentrations of Ca. The CaN mutant was the most sensitive, and in this experiment it was only about 50% active in the presence of excess EGTA because a K(m) concentration of substrate (i.e. not a V(max) value as in Fig. 5A) was used.


Figure 6: Ca activation of wild-type and mutant CaNs. The phosphatases were assayed without or with the indicated Ca concentrations at 30 °C for 10 min with 70 µM [P]R pep. Free [Ca] was calculated for Ca/EGTA buffers. The phosphatase activities are plotted as the ratio of the activity at 100 µM Ca to the activity at the indicated Ca concentrations. Each point represents the mean of two experiments performed in triplicate. The phosphatase activity (nmol/min/mg) at 100 µM Ca is indicated prior to the appropriate symbol. Wild-type CaN, (19 ± 0.1, up triangle); CaN D467A, (17 ± 0.4, ); CaN R476/477A, (59 ± 1.0, box); CaN, (61 ± 1.6, ); CaN, (90 ± 0.7, bullet).



Both the A and B subunits undergo conformational changes in the presence of Ca(32) . Our finding that the K(m) of the A subunit for substrate was regulated by Ca-binding to the B subunit suggests that a Ca-induced conformational change in the B subunit caused a conformational change in the catalytic domain that increased its affinity for substrate (i.e. decreased the K(m)). Could such a conformational change in the catalytic domain also weaken the interaction between the autoinhibitory and catalytic domains? If so, one would predict that the IC of the autoinhibitory peptide 457-482 would be higher in the presence of Ca. As seen in Fig. 7, similar inhibition curves of CaN were obtained in the absence and presence of Ca. This suggests that the region of the catalytic domain that interacted with CaN inhibitor peptide 457-482 was unaffected by the Ca/B subunit-induced decrease in K(m). This result is consistent with our previous demonstration that peptide 457-482 had similar IC values for proteolyzed CaN in the presence of EGTA and for native CaN in the presence of Ca/CaM(17) . However, that experiment used a single substrate concentration ([P]myosin light chain, 2.8 µM) that would be limiting in EGTA but near K(m) in the presence of Ca/CaM. In the present experiment the substrate concentrations were K(m) under both conditions.


Figure 7: Effect of CaN inhibitor peptide 457-482 on CaN phosphatase activity in the presence or absence of Ca. CaN (30 nM) was assayed with the indicated concentrations of autoinhibitory peptide for 10 min at 30 °C in the absence (circle) or presence (up triangle) of Ca. The concentrations of [P]R pep used in the absence or presence of Ca were 115 µM and 30 µM, respectively. The 100% activities of CaN were 58.5 ± 3.2 and 80.5 ± 3.2 nmol/min/mg without and with Ca, respectively. The assays were performed in triplicate, and the data shown are the mean ± S.D. from three experiments.




DISCUSSION

We previously utilized overlapping synthetic peptides to localize an autoinhibitory element within residues 457-482 of the CaN A subunit (17) . In the present study we made substitutions in the autoinhibitory peptide 457-482 to identify essential autoinhibitory residues. The D467A and R476/477A substitutions strongly decreased the inhibitory potency, indicating their importance for inhibitory function. However, the corresponding mutations in the A subunit generated little or no Ca-independent activity in the expressed CaN mutants, while the Ca-dependent activity was significantly higher than wild-type CaN (Fig. 5A). It is likely that multiple intrasubunit interactions occur between the autoinhibitory and catalytic domains such that disruption of one interaction by site-specific mutagenesis minimally effects inhibitory potency. Since the intermolecular interaction of the synthetic peptide with the A subunit catalytic domain is not subject to intrasubunit structural constraints, single substitutions in the peptide may be more effective at disrupting inhibitory interactions. These results are similar to those observed with the autoinhibitory domain of CaM-kinase II where single substitutions in the autoinhibitory peptide have large effects, but the corresponding site-specific mutations in the enzyme are less dramatic(27, 28) .

The CaN mutants were activated by lower Ca concentrations than wild-type CaN (Fig. 6). Since the B subunit has four EF hand Ca-binding domains(1) , activation of wild-type CaN may require all four sites to be occupied by Ca, whereas fewer occupied sites might be required to activate the mutants. Alternatively, there could be synergistic interactions between the B subunit and the autoinhibitory domain such that deletions or disruptions of the autoinhibitory domain increase the affinity of the B subunit for Ca. These findings are relevant to understanding structure-function aspects of CaN, since several different isoforms of the A subunit have been described(11) . Notably, the A subunit carboxyl-terminal region is less conserved than other functional domains, suggesting that these differences may impart different substrate specificities, distinct tissue or subcellular distribution, or variable Ca sensitivity(33) .

The site-specific mutants were not completely Ca/CaM-independent, so we constructed a truncation mutant (CaN) by inserting a stop codon at residue 457, the NH(2)-terminal boundary of the previously identified autoinhibitory element. Since Ile is located 43 residues COOH-terminal of the CaM-binding domain (residues 391-414), we also made a truncation at residue 420 (CaN) to ascertain if additional autoinhibitory elements were present in this region. Limited proteolysis of CaN removes the region COOH-terminal of the CaM-binding domain and results in levels of Ca-independent activity similar to the Ca/CaM-stimulated activity of the non-proteolyzed enzyme(14) . These findings provided strong evidence for the presence of an autoinhibitory domain COOH-terminal of the CaM-binding domain. Similarly, stepwise deletion of the A subunit COOH-terminal region elevated the V(max) values of both CaN and CaN in the absence of Ca . However, the observation that CaN showed partial Ca-independence while CaN was fully Ca-independent demonstrated that additional inhibitory elements were present within the sequence 420-457. The finding that Ca/CaM was required to further increase the activity of CaN to similar levels of phosphatase activity as CaN with Ca alone is also consistent with displacement of an autoinhibitory domain.

Kinetic analysis of wild-type and mutant CaNs also confirmed that Ca regulates the displacement of the autoinhibitory domain from the catalytic domain by binding both the B subunit and CaM. The Ca-induced increase in V(max) in the absence of CaM can only be mediated by the B subunit. Binding of Ca to CaM further increased the V(max) of recombinant CaN (Fig. 5A). The findings that the activities of the D467A, RR476/477AA mutants, and CaN in the presence of Ca alone are increased relative to wild-type CaN indicate that the autoinhibitory region is sensitive to Ca binding to the B subunit. Similar to Ca/CaM, the Ca/B subunit-induced increase in V(max) may be mediated by conformational changes in the COOH-terminal autoinhibitory domain. Furthermore, the findings that the D467A, RR476/477AA mutants and CaN457 had elevated Ca-stimulated activities but still required CaM for full activation indicate that the effects of Ca on the autoinhibitory elements within residues 420-457 and 457-482 are mediated by both the B subunit and CaM. In addition, the wild-type and truncated A subunits alone had very little phosphatase activity in the presence of Ca (Fig. 5A, inset), but their activities were increased 25- and 60-fold respectively, by in vitro reconstitution with purified B subunit. These results provide strong evidence that the autoinhibitory domain of CaN is regulated by Ca/CaM as well as Ca/B subunit.

Ca also decreased the K(m), but this effect was mediated only by the B subunit and not through CaM. Thus, wild-type CaN and all the mutants had K(m) values of 100-125 µM in the absence of Ca which was reduced to 20-30 µM in the presence of Ca regardless of whether CaM was present (Fig. 5B). Even the CaN mutant which did not require Ca for its maximal V(max) still required Ca to decrease its K(m). The fact that the expressed A subunits alone had K(m) values of 100-125 µM in the presence of Ca documents the need for the B subunit to mediate the decrease in K(m) (Fig. 5B, inset).

Thus, binding of Ca to the B subunit appears to change the conformation of the catalytic pocket to decrease the K(m) for substrate. One could imagine that a conformational change in the catalytic domain which alters K(m) might also partially disrupt the interaction with the autoinhibitory domain and thereby account for the increase in V(max) mediated by Ca binding to B subunit. However, the results shown in Fig. 7are not consistent with this interpretation. The IC of autoinhibitory peptide 457-482 for CaN was the same in the absence or presence of Ca, indicating that the affinity of the catalytic domain for the autoinhibitory peptide was unaffected by Ca. This result suggests that CaN substrates and the autoinhibitory region interact with the catalytic domain at distinct sites, and is consistent with our previous reports that inhibition of CaN by peptide 457-482 was not competitive with substrate(17, 29) . This possibility is being tested with the complete autoinhibitory domain including the CaM-binding domain (i.e. residues 390-482).

Our previous study on in vitro reconstitution of CaN phosphatase activity using expressed wild-type A subunit and purified brain B subunit and/or CaM showed that both Ca-binding proteins were required for synergistic activation(12) . CaM had a strictly V(max) effect whereas the B subunit primarily effected K(m) with a small effect on V(max). The more extensive kinetic analysis presented in this study clearly demonstrates that Ca-binding to the B subunit lowered the K(m) for substrate and increased the V(max). Ca/CaM further increased the V(max) by abolishing the interaction between the catalytic domain and the COOH-terminal autoinhibitory domain. In summary, Ca binding to both the intrinsic B subunit and extrinsic CaM is required to displace the autoinhibitory domain and increase the V(max), while the K(m) is regulated by Ca binding to the B subunit. It is also clear that our previously defined autoinhibitory element (residues 457-482) represents a minimal sequence, and additional elements are present between residues 420-457. Ongoing studies of the interactions between the A and B subunits as well as the interactions between the catalytic and autoinhibitory domains should further our understanding of the mechanisms of Ca regulation of CaN phosphatase activity.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants DK17808 and GM41292. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by Neuroendocrinology Training Grant DK07680.

To whom correspondence should be addressed. Tel.: 503-494-6931. Fax: 503-494-6934.

(^1)
The abbreviations used are: A, A subunit of CaN truncated at residue 457; A, A subunit of CaN truncated at residue 420; CaM, calmodulin; CaN, calcineurin; CaN, wild-type CaN containing the full-length A subunit; CaN, CaN containing the A subunit and B subunit; CaN, CaN containing the A subunit and B subunit; N-methyl-D-aspartate; PCR, polymerase chain reaction; PP-1, type-1 phosphoprotein phosphatase; PP-2A, type-2A phosphoprotein phosphatase; [P]R pep, P-labeled peptide derived from the R subunit of cAMP-kinase; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. Randall Kincaid (NIH, NIDAA) for the gift of the rabbit-anti CaN antibody and Dr. Richard Maurer (Oregon Health Sciences University) and Dr. John Scott (Vollum Institute) for the catalytic subunit of cAMP-dependent kinase. We are grateful to Dr. Roger Cone (Vollum Institute) for his assistance with the PCR. We also thank Dr. Frank Rusnak (Mayo Clinic) for his gift of purified recombinant myristoylated CaN B.

Addendum-Since submission of this manuscript, a report has been published demonstrating that the B subunit of proteolytically activated CaN has a higher affinity for Ca than the native enzyme (Stemmer, P. M., and Klee, C. B.(1994) Biochemistry33, 6859-6866).


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