Eur. J. Biochem. 269, 3540–3548 (2002) Ó FEBS 2002
doi:10.1046/j.1432-1033.2002.03040.x
Substrate selectivity and sensitivity to inhibition by FK506 and cyclosporin A of calcineurin heterodimers composed of the a or b catalytic subunit Brian A. Perrino1, Andrew J. Wilson2, Patricia Ellison3 and Lucie H. Clapp2 1
Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, NV, USA; 2Center for Clinical Pharmacology, University College London, UK; 3Department of Biochemistry, University of Nevada School of Medicine, Reno, NV, USA
The calcineurin (CaN) a and b catalytic subunit isoforms are coexpressed within almost all cell types. The enzymatic properties of CaN heterodimers comprised of the regulatory B subunit (CnB) with either the a or b catalytic subunit were compared using in vitro phosphatase assays. CaN containing the a isoform (CnAa) has lower Km and higher Vmax values than CaN containing the b isoform (CnAb) toward the PO4RII, PO4-DARPP-32(20–38) peptides, and p-nitrophenylphosphate (pNPP). CaN heterodimers containing the a or b catalytic subunit isoform displayed identical calmodulin dissociation rates. Similar inhibition curves for each CaN heterodimer were obtained with the CaN autoinhibitory peptide (CaP) and cyclophilin A/cyclosporin A (CyPA/CsA) using each peptide substrate at Km concentrations, except for a five- to ninefold higher IC50 value measured for CaN containing the b isoform with p-nitrophenylphosphate as substrate. No difference in stimulation of phosphatase
activity toward p-nitrophenylphosphate by FKBP12/FK506 was observed. At low concentrations of FKBP12/FK506, CaN containing the a isoform is more sensitive to inhibition than CaN containing the b isoform using the phosphopeptide substrates. Higher concentrations of FKBP12/FK506 are required for maximal inhibition of b CaN using PO4DARPP-32(20–38) as substrate. The functional differences conferred upon CaN by the a or b catalytic subunit isoforms suggest that the a:b and CaN:substrate ratios may determine the levels of CaN phosphatase activity toward specific substrates within tissues and specific cell types. These findings also indicate that the a and b catalytic subunit isoforms give rise to substrate-dependent differences in sensitivity toward FKBP12/FK506.
Calcineurin (CaN) is a ubiquitously expressed Ca2+/CaMdependent protein phosphatase that is a critical component of several Ca2+-dependent signaling pathways. CaN regulates a number of transcription factors and ion channels and is involved in the regulation of T-cell activation, long-term depression of postsynaptic potential, synaptic vesicle recycling, and cardiac and skeletal muscle hypertrophy [1,2]. CaN is a heterodimer of a catalytic A subunit (CnA) (58–61 kDa) and a Ca2+-binding B subunit (19 kDa). Three CnA isoforms (a, b, c) have been described. The expression of CnAc is restricted to testis, while the CnAa and CnAb isoforms are present in all tissues examined [3]. The physiological significance of the expression of multiple CaN catalytic subunits within the same cell or tissue is unknown. Overall, the aminoacid sequences of CnAa and CnAb are 81% identical [4]. However, the amino-acid sequence identity is 90% within
the core catalytic region, the CnB-binding helix, the CaM-binding domain, and the autoinhibitory domain [4]. Mammalian CnAa or CnAb subunits exhibit extensive sequence homologies, with only one or five amino-acid changes between human and rat CnAa and CnAb, respectively [4]. The most striking differences between the CnAa and CnAb catalytic subunit isoforms are the 12 Pro residues within the first 24 amino-acid residues of CnAb and multiple amino-acid differences C-terminal of the autoinhibitory domain [4]. It has been proposed that the two isoforms may exhibit substrate preferences and may also be selectively targeted to distinct subcellular locations [2]. Variations in the amount and ratio of CnAa:CnAb have been noted within and between tissues [3]. For example, although CnAa is more abundant than CnAb in mammalian brain, the CnAa/CnAb ratio in the striatum is 4 : 1, while in the cerebellum the ratio is 2.5 : 1 [5]. Similarly, CnAa is more abundant in kidney, but its expression is restricted to the tubules, while CnAb expression was observed only in the glomerular region [6]. In contrast, CnAb is more abundant in T and B cells [6]. In addition, in hepatocytes and some neurons, CnAa is found in the cytoplasm and nucleus, while CnAb is found only in the cytoplasm [7,8]. Together these findings raise the possibility of substrate-dependent functional differences between the a and b CaN catalytic subunit isoforms. To determine whether the CnA a and b isoforms impart functional differences to CaN phosphatase activity, we have
Correspondence to B. A. Perrino, Department of Physiology & Cell Biology, Anderson Medical Bldg. MS352, University of Nevada School of Medicine, Reno, Nevada, 89557, Tel.: + 1 775 784 6396, Fax: + 1 775 784 6903, E-mail:
[email protected] Abbreviations: CaN, calcineurin; CnB, calcineurin regulatory B subunit. (Received 12 March 2002, revised 16 May 2002, accepted 10 June 2002)
Keywords: calcineurin; calmodulin; dephosphorylation; Ser/ Thr protein phosphatase.
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Enzymatic characteristics of calcineurin isoforms (Eur. J. Biochem. 269) 3541
initiated in vitro studies of the enzymatic characteristics of CaN heterodimers composed of either CnAa or CnAb. CaNa or CaNb heterodimers were obtained by coexpressing each CnA isoform with CnB in the Sf21/baculovirusexpression system. CaM binding to each CnA isoform within the CaN heterodimer was measured using stoppedflow techniques. We compared Km and Vmax values obtained from assays of the phosphatase activities of both CaN heterodimers towards two peptide substrates, and pNPP. We also compared the inhibition of CaN phosphatase activity toward the two peptide substrates by the CaN autoinhibitory peptide, and the FKBP12/FK506 complex. The activation of CaN phosphatase activity towards pNPP by the FKBP12/FK506 complex was also measured. Our results are the first indication that CaN heterodimers composed of either CnAa or CnAb exhibit differences in substrate selectivity and sensitivity to immunophilin/immunosuppressant inhibition.
EXPERIMENTAL PROCEDURES Materials Rabbit anti-CnAa IgG, rabbit anti-CnAb IgG, and horseradish peroxidase-conjugated goat anti-(rabbit IgG) Ig were purchased from Chemicon. T-4 DNA ligase, restriction enzymes, Grace’s supplemented insect cell medium, antibiotic/antimycotic solution, Pluronic F-68, and bacterial culture media, were obtained from Gibco/ BRL. Fetal bovine serum was purchased from Atlanta Biologicals. CaM–Sepharose was purchased from Amersham Pharmacia. Antibiotics, FKBP12, and pNPP were obtained from Sigma. Cyclophilin A, cyclosporine A, and FK506 were obtained from Calbiochem. Phospho-RII peptide, CaP, and BioMol Green reagent were purchased from BioMol. Phospho-DARPP-32(20–38) [LDPRQVEMIRRRRPT(PO4)PAML] was purchased from American Peptide Company. Human CnAb cDNA was generously provided by M. M. Lai and S. Snyder (The Johns Hopkins University School of Medicine [9]). All other materials and reagents were of the highest quality available commercially. Recombinant CaNa and CaNb expression and purification The 1.6 kb Sal1-Not1 CnAb fragment was ligated into SalINotI cut pSE420 (InVitrogen). The pSE420/CnAb construct was restriction digested with EcoRI–NotI and the EcoRI–NotI CnAb cDNA ligated into EcoRI–NotI cut pVL1393 (InVitrogen). Sf21 cells were transfected with the pVL1393/CnAb construct using the Bac-N-Blue Transfection kit from InVitrogen. Recombinant CnAb baculoviruses were screened by plaque assay and Western blotting using anti-CnAb Ig, and amplified and titered by plaque assay as described [10]. The coinfection, expression and purification of baculovirus-expressed CaN containing the rat brain CnAa subunit and rat brain CnB, or the human CnAb subunit and rat brain CnB was carried out as described, except that monolayer cultures of Sf21 cells were used for CaN expression [11]. The phosphatase activities of the purified CaN heterodimers were not further stimulated by the addition of purified CnB, indicating that the CaN heterodimers are composed of a 1 : 1 molar ratio of CnA/ CnB (data not shown) [10].
Phosphatase assays Dephosphorylation of PO4-RII peptide, PO4-DARPP32(20–38) peptide, and pNPP by CaN was carried out at 30 °C in 50 lL reaction volumes in duplicate. The assays were carried out in CaN assay buffer (40 mM Tris/HCl, pH 7.5, 6 mM Mg(C2H3O2)2, 8 mM ascorbic acid, 100 mM NaCl, 0.1 mM CaCl2, 0.5 mM MnCl2, 0.5 mM dithiothreitol, 0.1 mgÆmL)1 bovine serum albumin). The reactions were initiated by addition of substrate, and the peptide dephosphorylation assays terminated by the addition of 100 lL of BioMol Green reagent, while the pNPP dephosphorylation assays were terminated by the addition of 2 lL of 65% K2HPO4 [12]. The assay times are indicated in the Figure legends. The concentrations of CaN, CaM, CaP, FKBP12, and substrates are indicated in the Figure legends. The Km and Vmax values were determined by linear regression analysis (PRISM software) of inverse plots of the data from phosphatase assays in which the concentrations of substrates were varied. The FK506 or CsA stocks (1 mM in dimethylsulfoxide) were diluted 80-fold in H20 in a glass tube before being added to the samples. The final dimethylsulfoxide concentration of 0.05% in the assays had no effect on CaN phosphatase activity. FKBP12 and FK506 or CyPA and CsA were preincubated together on ice for 10 min, followed by incubation with CaN in assay buffer for 10 min prior to the start of the phosphatase assays. The amount of phosphate released from the peptide substrates was determined by comparing the A620 values obtained from the experimental samples to the values generated from the K2HPO4 standard curve according to the manufacturer’s (BioMol) instructions. Dephosphorylation of pNPP was monitored by measuring the A410 values [13]. The data were best fit to a second-order polynomial equation by nonlinear regression analysis. Rate constant measurements The Lys75 to Cys CaM mutant (CaM C75) was labeled at Cys75 with the fluorescent probe acrylodan (Molecular Probes) essentially as described by Waxham et al. [14,15]. Dissociation rates of CaM from CaN isoforms were determined using a temperature-controlled stopped-flow fluorimeter (Hi-Tech SF 61-DX-2) equipped with a 150Watt Hg-Xe lamp. The excitation was at 365 nm and emission was monitored using a 399-nm cut-off filter. Acrylodan-labeled CaM(C75) [CaM(C75)ACR] (0.1 lM) and either (0.3 lM) CaNAa or CaNAb in 25 mM Mops, pH 7.0, 150 mM KCl, 0.5 mM CaCl2 were rapidly mixed with native CaM (10 lM) in the same buffer at 20 °C. Rate constants were derived by fitting the experimental data using the Kinetasyst software supplied with the Hi-Tech stopped-flow fluorimeter. In both cases, the best fit was obtained to a double-exponential model, where each rate accounted for approximately 50% of the observed amplitude change.
RESULTS Expression and purification of CaNa and CaNb CaN heterodimers composed of the Ca2+-binding B subunit and either the a or b catalytic subunit isoform were
3542 B. A. Perrino et al. (Eur. J. Biochem. 269)
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has a slightly slower mobility in SDS/PAGE, consistent with its higher molecular mass (59 kDa) compared to CnAa (57.6 kDa). Immunoblotting the purified CaN heterodimers with isoform-specific antibodies confirm that CnAa and CnAb proteins are expressed by the appropriate recombinant CnAa and CnAb baculoviruses (Fig. 1B,C). Kinetic assays of CaNa and CaNb phosphatase activity
Fig. 1. SDS/PAGE and Western blot analysis of baculovirus expressed CaN composed of CnAa or CnAb catalytic subunit isoforms. CaN heterodimers were expressed in Sf21 cells using recombinant baculoviruses, purified as described in Experimental procedures, and analyzed by SDS/PAGE (15%) and Western blotting. Lane 1, CaN heterodimer containing the CnAa catalytic subunit isoform (5 lg); lane 2, CaN heterodimer containing the CnAb catalytic subunit isoform (3 lg). (A) Purified CaN heterodimers were separated by SDS/ PAGE and stained with Coomassie Brilliant Blue. (B) Immunostaining of purified CaN heterodimers using anti-CnAa Ig. (C) Immunostaining of purified CaN heterodimers using anti-CnAb Ig.
generated by coinfecting Sf21 cells with recombinant CnB baculovirus and either recombinant CnAa or CnAb baculoviruses. The CaN heterodimers were obtained by CaM– Sepharose chromatography as described in Experimental Procedures, and analyzed by SDS/PAGE and Western blotting. The purified CaNa and CaNb heterodimers are 90–95% pure as indicated by the Coomassie stained SDSpolyacrylamide gel shown in Fig. 1A. The CnAb subunit
Kinetic analyses of the in vitro phosphatase activities of CaNa and CaNb were carried out to determine their Km and Vmax values toward three different substrates; namely PO4-RII peptide, PO4-DARPP-32(20–38), and pNPP. The PO4-RII peptide and pNPP have been extensively used to characterize the phosphatase activity of CaN [10,11,16,17]. The PO4-DARPP-32(20–38) peptide contains amino-acid residues 20–38 of DARPP-32, which is a physiological substrate of CaN [18]. PO4-Thr34 of the DARPP-32(20–38) peptide is dephosphorylated by CaN with Km and Vmax values similar to the values obtained with native DARPP-32 [18]. In agreement with previous reports, the results in Fig. 2 show that CaN phosphatase activity is characterized by different Km and Vmax values toward different substrates [19]. However, the results also indicate that the phosphatase activities of CaN heterodimers containing the CnAa or CnAb catalytic subunit are characterized by different Km and Vmax values toward the same substrate. For each substrate tested, CaN heterodimers containing the CnAa catalytic subunit are characterized by lower Km and higher Vmax values compared to CaN heterodimers containing the CnAb catalytic subunit. For both phosphopeptide substrates, the Km values of CaN heterodimers containing the CnAb catalytic subunit are approximately threefold higher than the Km values of CaN heterodimers containing the CnAa catalytic subunit (Fig. 2A,B). With pNPP as substrate, the difference in Km values is only twofold (Fig. 2C).
Fig. 2. Kinetic analyses of CaN heterodimers composed of CnAa or CnAb catalytic subunit isoforms. (A) Dephosphorylation of PO4-RII peptide. CaNa or CaNb were each present at a final concentration of 5 nM. CaM was present at a final concentration of 15 nM. The reactions were allowed to proceed for 7 min at 30 °C. The concentrations of PO4-RII peptide used were 25 lM, 50 lM, 75 lM, 100 lM, and 150 lM. (B) Dephosphorylation of PO4-DARPP-32(20–38). CaNa or CaNb were each present at a final concentration of 50 nM. CaM was present at a final concentration of 150 nM. The reactions were allowed to proceed for 10 min at 30 °C. The concentrations of PO4-DARPP-32(20–38) used were 7 lM, 12 lM, 17 lM, and 25 lM. (C) Dephosphorylation of pNPP. CaNa or CaNb were each present at a final concentration of 50 nM. CaM was present at a final concentration of 150 nM. The reactions were allowed to proceed for 20 min at 30 °C. The concentrations of pNPP used were 10 mM, 15 mM, 20 mM, and 30 mM, 50 mM, and 100 mM. The results shown are representative of three assays performed in triplicate for each CaN heterodimer. CaNa, d; CaNb, s.
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CaN heterodimers containing the CnAa catalytic subunit are characterized by approximately twofold higher Vmax values toward the three substrates tested. Together these results indicate that CaN heterodimers used in these experiments containing the CnAa catalytic subunit are characterized by higher levels of phosphatase activity toward these three substrates. Inhibition of CaNa and CaNb phosphatase activity by CaP The CaN crystal structure shows that in the inactive state, the CaN autoinhibitory domain lies over the catalytic site [20]. The amino-acid sequences of the CnAa and CnAb catalytic domains are 90% identical, and the autoinhibitory domain amino-acid sequences are 89% identical, suggesting that CaN heterodimers containing the CnAa or CnAb catalytic subunit would be equally inhibited by CaP, which contains the autoinhibitory domain from CnAa [4,21]. However, because of the differences in Km and Vmax values obtained with the CaN heterodimers used in these experiments containing the CnAa or CnAb catalytic subunit toward the same substrate, we examined the inhibition of CaNa or CaNb phosphatase activity by CaP toward the three substrates. It has previously been reported that CaP inhibits bovine brain CaN or baculovirus-expressed rat brain CaNa with IC50 values between 12 lM and 18 lM, using 32PO4-RII peptide as substrate [10,11]. As shown in Fig. 3A, the phosphatase activities of CaN heterodimers containing the CnAa or CnAb catalytic subunit toward PO4-RII peptide are equally inhibited by CaP. The IC50 values (10 lM-12 lM) and final extent of inhibition (90% inhibition of phosphatase activity by 90 lM CaP) obtained are similar to the previously reported values using 32P-RII peptide as substrate [10,11]. Similar kinetics of inhibition were also obtained for CaP with CaN heterodimers containing the CnAa or CnAb catalytic subunit using PO4-DARPP-32(20–38) as substrate (Fig. 3B), giving IC50 values of 15 lM and 25 lM, respectively. In addition, CaN phosphatase activity is 85% inhibited by 90 lM CaP. It has
been reported that bovine brain CaN phosphatase activity is 50% inhibited by 35 lM CaP using pNPP as substrate [21]. Using CaN heterodimers containing the CnAa or CnAb catalytic subunit and pNPP as substrate, we measured IC50 values of 20 lM for CaNa and 90 lM for CaNb. Furthermore, in contrast to the results obtained using the phosphopeptide substrates, the phosphatase activities of CaNa and CaNb are only 70%, and 50% inhibited by 90 lM CaP, respectively. Inhibition of CaNa and CaNb phosphatase activity by FKBP12/FK506 or CypA/CsA The structurally unrelated immunophilin/immunosuppressant complexes of FKBP12/FK506 or CypA/CsA inhibit CaN noncompetitively by binding to the CnB-binding helix, CnB, and one side of the substrate-binding cleft of the catalytic site to alter the active-site geometry [16,17,20,22]. As the mechanism of inhibition of CaN by the immunophilin/immunosuppressant complexes is different from that of CaP, we examined the inhibition of CaN heterodimers containing the CnAa or CnAb catalytic subunit by FKBP12/FK506 or CyP/CsA. As shown in the dose– response curves of Fig. 4, using the two different phosphopeptide substrates, CaNa is more sensitive to inhibition by FKBP12/FK506 than CaNb. With PO4-RII peptide as substrate, 50% inhibition of CaNa activity was achieved with 73 nM FKBP12 (in the presence of 500 nM FK506), compared to 50% inhibition of CaNb activity by 120 nM FKBP12. As expected, the high concentration of FKBP12 (200 nM) resulted in 90% inhibition of CaNa and CaNb with PO4-RII peptide as substrate (Fig. 4A). Similarly, 50% inhibition of CaNa activity was achieved with 60 nM FKBP12, compared to 50% inhibition of CaNb activity by 117 nM FKBP12 using PO4-DARPP-32(20–38) as substrate. 200 nM FKBP12 resulted in 90% inhibition of CaNa using PO4-DARPP-32(20–38) as substrate. However, 90% inhibition of CaNb phosphatase activity was achieved by 1 lM FKBP12 using PO4-DARPP-32(20–38) as
Fig. 3. Inhibition of CaN heterodimers composed of CnAa or CnAb catalytic subunit isoforms by CaP. (A) The reactions proceeded for 10 min at 30 °C using 32 lM and 91 lM PO4-RII peptide for CaNa or CaNb, respectively. CaNa or CaNb were each present at a final concentration of 5 nM. CaM was present at a final concentration of 15 nM. (B) The reactions proceeded for 10 min at 30 °C using 6 lM and 21 lM PO4-DARPP-32(20–38) for CaNa or CaNb, respectively. CnAa or CnAb were each present at a final concentration of 50 nM. CaM was present at a final concentration of 150 nM. (C) The reactions proceeded for 20 min at 30 °C using 45 mM and 83 mM pNPP for CaNa or CaNb, respectively. CaNa or CaNb were each present at a final concentration of 50 nM. CaM was present at a final concentration of 150 nM. The results shown are the averages ± SD from three assays in triplicate for each CaN heterodimer. CaNa, d; CaNb, s.
3544 B. A. Perrino et al. (Eur. J. Biochem. 269)
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Fig. 4. Inhibition of CaN heterodimers composed of CnAa or CnAb catalytic subunit isoforms by FKBP12/FK506 or CyPA/CsA. FK506 or CsA were each present at a final concentration of 2 lM, and the FKBP12 or CyPA concentrations varied as indicated in the figure legends. The reactions proceeded for 20 min at 30 °C using 32 lM (A) and 91 lM (B) PO4-RII peptide for CaNa or CaNb, respectively. CaNa or CaNb were each present at a final concentration of 5 nM. CaM was present at a final concentration of 15 nM. The reactions proceeded for 20 min at 30 °C using 6 lM (C) and 21 lM (D) PO4-DARPP-32(20– 38) for CaNa or CaNb, respectively. CaNa or CaNb were each present at a final concentration of 5 nM. CaM was present at a final concentration of 15 nM. The results shown are the averages ± SD from three assays in triplicate for each CaN heterodimer. CaNa, d; CaNb, s.
substrate. These findings indicate that the CaNb used in these experiments is less sensitive than CaNa to inhibition by FKBP12/FK506 when PO4-DARPP-32(20–38) is used as substrate. With CyPA/CsA and PO4-RII peptide as substrate 50% inhibition of CaNa activity was achieved with 342 nM CyPA (in the presence of 2 lM CsA), compared to 50% inhibition of CaNb activity by 456 nM CyPA. A high concentration of CyPA (1000 nM) resulted in 80%-90% inhibition of CaNa and CaNb with PO4-RII peptide as substrate (Fig. 4B). Similar IC50 values were obtained for CyPA/CsA inhibition of CaNa and CaNb using PO4DARPP-32(20–38) as substrate and 80–90% inhibition of phosphatase activity was attained with 1000 nM CyPA (Fig. 4D). Using two different phosphopeptide substrates, these findings indicate that CyPA/CsA results in similar inhibition of both CaNa and CaNb. These findings also indicate that FKBP12/FK506 is a more potent inhibitor of both CaNa and CaNb than CyPA/CsA. Activation of CaNa and CaNb phosphatase activity by FKBP12/FK506 toward pNPP In contrast to the inhibition of CaN phosphatase activity toward phosphopeptide and phospho-protein substrates by FKBP12/FK506, the phosphatase activity of CaN toward the small organic compound pNPP is increased two- to fourfold by FKBP12/FK506 [16,23]. These observations are consistent with the findings that the FKBP12/FK506 complex alters the conformation of the active site [20]. To examine the possibility that FKBP12/FK506 may have
different effects on the activities of CaNa and CaNb toward pNPP, we examined the activation of CaNa and CaNb phosphatase activities toward pNPP by FKBP12/FK506. As shown in Fig. 5, a twofold increase in CaN phosphatase activity was observed with 200 nM FKBP12 and 500 nM FK506, in agreement with previous studies [16,23]. However, there was essentially no difference in the dosedependent activation of CaNa or CaNb phosphatase activities toward pNPP by FKBP12/FK506. Determination of dissociation rates between CaM(C75)ACR and CaNa or CaNb Because of the highly conserved amino-acid sequences of the CnAa and CnAb catalytic, CaM-binding, and CnBbinding domains, it was surprising to find differences in the phosphatase activities between CaNa and CaNb toward the same substrate. The results of the kinetic analyses, and inhibition studies are summarized in Table 1. Although the amino-acid sequences of the CnAa and CnAb CaMbinding domains are identical, adjacent functional domains can influence the CaM-binding properties of CaM-binding a helices [24]. Thus, stop-flow analyses of Ca2+/CaM off rates were carried out in order to determine whether there are any differences in the Ca2+/CaM-binding properties of CaNa and Canb. The dissociation rates of CaNa or CaNb from CaM(C75)ACR were determined by monitoring the rates of fluorescence decrease as CaM(C75)ACR bound to CaNa or CaNb is exchanged for excess unlabeled CaM (Fig. 6). The data were best fit by a double-exponential model and yielded two essentially identical fast and slow
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Enzymatic characteristics of calcineurin isoforms (Eur. J. Biochem. 269) 3545
Fig. 5. Stimulation of CaN phosphatase activity toward pNPP by FKBP12/FK506. FK506 was present at a final concentration of 500 nM, and the FKBP12 concentration varied as indicated in the figure legend. (A) The reactions proceeded for 45 min at 30 °C using 45 mM and 83 mM pNPP for CaNa or CaNb, respectively. CaNa or CaNb were each present at a final concentration of 5 nM. CaM was present at a final concentration of 15 nM. The results shown are the averages ± SD from three assays in triplicate for each CaN heterodimer. CaNa, d; CaNb, s.
CaM dissociation constants for both CaNa and CaNb. Fast and slow rates of 4 s)1 and 0.4 s)1, and 3.9 s)1 and 0.4 s)1 were obtained for CaNa and CaNb, respectively. These results indicate that there are essentially no differences in Ca2+/CaM-dissociation from the CnAa and CnAb catalytic subunit isoforms. The mechanistic basis for the two rate constants is not clear, although Ca2+/CaM-binding to the CaM-binding domain of CnA is modulated by Ca2+binding to CnB [25,26].
DISCUSSION Previous studies of the CaN a and b catalytic subunit isoforms have mainly focused on their tissue and subcellular distributions [5–8,27]. These reports have provided important information demonstrating regional differences in expression levels within tissues and differences in the subcellular distribution of CnAa and CnAb. Although it
has been generally assumed that CaNa and CaNb dephosphorylate the same set of substrates, the differences in CnAa and CnAb localization and expression have been proposed to reflect differences in substrate selectivity between CaNa and CaNb [2,4]. However, in the absence of information concerning enzymatic differences between these two CaN catalytic subunit isoforms, the physiological significance of their differential distribution is unclear. To address this question, we have examined the enzymatic properties of CaN heterodimers containing either the CnAa or CnAb catalytic subunit. In agreement with previous findings using purified mammalian brain CaN, we found that CaNa or CaNb heterodimers are characterized by different Km and Vmax values toward different substrates [19]. However, our results also indicate that CaNa and CaNb heterodimers exhibit differences in phosphatase activity toward the same substrate, as indicated by the different Km and Vmax values obtained. The results from the kinetic assays show that the CaN heterodimers containing the CnAa subunit have higher levels of phosphatase activity toward all three substrates tested, as indicated by lower Km and higher Vmax values (Table 1). These findings indicate that the CaN catalytic subunits are characterized by different rates of phosphatase activity towards their substrates, and suggest that the CaNa:CaNb ratio within a cell or tissue is an important determinant of CaN phosphatase activity toward specific substrates. The use of CnAa knockout mice has provided evidence that CaNa and CaNb exhibit selective phosphatase activity toward specific substrates within a cell or tissue. Tau proteins are hyperphosphorylated in the brains of CnAa –/– mice [28]. Similarly, hippocampal depotentiation is abolished while long-term depression and long-term potentiation are unaffected in CnAa –/– mice [29]. Furthermore, CnAa –/– mice have impaired antigen-specific T-cell responses in vivo [30]. Two possible interpretations of these findings are (a) CaNa selectively dephosphorylates tau proteins, and also specifically dephosphorylates substrates required for hippocampal depotentiation and antigeninduced T-cell responses, or (b) CaNa and CaNb have no substrate preferences, but the residual CaNb phosphatase activity is insufficient to dephosphorylate tau or participate in hippocampal depotentiation and antigen-induced T-cell responses. The findings that CnAb is the predominant isoform present in T-cells argues against impaired antigenspecific T-cell responses in CnAa –/– mice being due to insufficient CaN phosphatase activity [6]. Our findings that the CnAa and CnAb catalytic subunits confer differences in substrate affinity and phosphatase activity (as shown by
Table 1. Summary of kinetic parameters of CaNa and CaNb phosphatase activities toward three substrates. The Km and Vmax values, CaP IC50 values, FKBP12 IC50 and CyPA IC50 values were obtained from the inverse plots of V vs [S], the CaP dose–response experiments, and the FKBP12 and CyPA dose–response experiments, respectively.
Km Vmax (lmolÆmin)1Æmg)1) CaP IC50 (lM) FKBP12 IC50 (nM) CyPA IC50 (nM)
PO4-RII peptide (lM)
PO4-DARPP-32
pNPP
CaNa
CaNb
CaNa
CaNb
CaNa
CaNb
32 lM 4.1 10 74 342
91 lM 2.8 12 120 451
6.2 lM 0.384 15 60 303
21 lM 0.224 25 114 251
45 mM 5.8 22 – –
83 mM 3.1 90 – –
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Fig. 6. Measurement of CaM dissociation rate constants from CaNa or CaNb associated with CaM (C75)ACR using stopped-flow kinetics. The time course for CaM dissociation from CaNa (A) or CaNb (B) from CaM(C75)ACR as determined using a stopped-flow fluorimeter. CaM(C75)ACR (0.1 lM) and either (0.3 lM) CaNa (or CaNb) in 25 mM Mops, pH 7.0, 150 mM KCl, 0.5 mM CaCl2 were rapidly mixed with native CaM (10 lM) in the same buffer at 20 °C. The excitation was at 365 nm and emission was monitored using a 399-nm cut-off filter. Each curve represents the average of four exchange reactions.
different Km and Vmax values) toward the same substrate support the conclusion that CaN substrates are differentially dephosphorylated by CaNa and CaNb in vivo. The differences in phosphatase activity and sensitivity to FKBP12/FK506 or CyPA/CsA inhibition between CaNa and CaNb that we observed may be due to subtle differences in how the CnAa and CnAb catalytic subunits interact with CnB. The regulation of enzyme activity by two EF-hand Ca2+-binding proteins is unique to CaN [4]. Similar to Ca2+/CaM-dependent kinases, Ca2+/CaM-binding to CnA
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activates the enzyme by relieving inhibition due to an autoinhibitory domain, which is evidenced by an increase in Vmax [11,31]. Conversely, Ca2+-binding to CnB activates CaN primarily by affecting the affinity of the catalytic site for substrate, as evidenced by the decrease in Km [11,31]. Circular dichroism analysis has shown that CnB and CnA both undergo conformational changes upon Ca2+ binding to CnB [32]. It appears that in the absence of Ca2+ the catalytic core is in an inactive conformation and that Ca2+binding to CnB changes the conformation of the catalytic core to allow substrate binding [25,33]. The CaN crystal structure shows that the CnB-binding helix is immediately C-terminal of the b14 strand of one half of the b sandwich forming the catalytic core, providing a mechanism for transmission of Ca2+-induced conformational changes in CnB to the active site [22]. In fact, the catalytic activity of CaN is sensitive to the amino-acid composition of the region linking the CnB-binding helix to the b14 strand of the catalytic core [34,35]. Mutations S373P, H375L, and L379S decrease CaN activity, indicating the importance of this linker region to the activation of CaN by Ca2+-binding to CnB [34]. Calcium binding to CnB also influences the affinity of Ca2+/CaM for its binding domain on CnA [25,26]. CnB has two low affinity and two high affinity EFhand Ca2+-binding loops [36]. In the absence of Ca2+binding to the low affinity sites, the CaM-binding domain interacts with the exposed side of the CnB-binding helix [26]. Calcium binding to the low affinity sites on CnB disrupts the interaction between the CaM-binding domain and the CnBbinding helix and increases the affinity of the CaM-binding domain for Ca2+/CaM [25,26]. The regulation of the CaMbinding domain by CnB may partly account for the slow and fast dissociation constants we measured using stoppedflow analysis. The FKBP12/FK506 complex inhibits CaN noncompetitively using PO4-RII and PO4-DARPP-32 as substrates [20]. These findings are consistent with crystallographic data showing the active site and substrate-binding cleft are not directly blocked by the FKBP12/FK506 complex, supporting the conclusion that alterations in the active site conformation affect the substrate-binding cleft and are responsible for the inhibition by FKBP12/FK506 [20,22]. This mechanism of inhibition would account for the findings that CaN phosphatase activity toward the small organic molecule pNPP is increased by the FKBP12/FK506 complex (Fig. 5) [16,23]. However, the structural and enzymatic studies of the interactions between the FKBP12/FK506 complex and CaN have been carried out with CaN containing the CnAa catalytic subunit [16,20,22,23]. The results presented here are the first direct studies of the inhibition of CaN containing the CnAb catalytic subunit by FKBP12/FK506 or CyPA/CsA. For both immunophilin/immunosuppressant complexes, the IC50 values are higher than previously reported [23,37]. Differences in CaN preparations and methods of enzyme activity assays may account for the differences in IC50 values. For these studies the CaN activity was assayed in the presence of ascorbic acid, which results in higher activity compared to activity in the absence of antioxidants [38,39]. With both peptide substrates CaNa was more sensitive to inhibition by FKBP12/FK506 than CaNb (Fig. 4). However, CaNa and CaNb were both 90% inhibited by 200 nM FKBP12 using PO4-RII at Km concentrations. Similarly,
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Enzymatic characteristics of calcineurin isoforms (Eur. J. Biochem. 269) 3547
CaNa was 90% inhibited by 200 nM FKBP12 using PO4DARPP-32(20–38) at the Km concentration. In contrast, only 60% inhibition of CaNb by 200 nM FKBP12 was measured using PO4-DARPP-32(20–38) as substrate, and 90% inhibition required 1000 nM FKBP12. Our results using the two different phosphopeptide substrates show that CsA is a less potent inhibitor of both CaNa and CaNb than FK506. These results are consistent with the findings that in vivo, CsA and FK506 are equally effective, but FK506 is 10-fold more potent in inhibiting CaN activity and IL-2 gene activation [37]. Although FK506 and CyPA are structurally unrelated compounds, biochemical and mutational studies indicate that FKBP12/FK506 and CyPA/CsA bind to a common site on the CaN heterodimer composed of the CnB-binding helix, CnB and part of the substratebinding cleft of CaN [35,40]. However, differences in the interactions between these structurally dissimilar immunophilin/immunosupressant complexes and CaN will likely contribute to differences in their inhibitory potency. As the substrate-binding cleft geometry is affected by CnB, the interaction between CnA and CnB may affect how the FKBP12/FK506 and CyPA/CsA complexes affect the substrate-binding cleft. Thus, both the catalytic subunit and substrate may influence the degree of inhibition of CaN phosphatase activity by FKBP12/FK506 and CyPA/CsA. The differences in phosphatase activity and sensitivity to FKBP12/FK506 inhibition between CaNa and CaNb are likely not due to differences in the linker region, as the amino-acid sequences of the CnB-binding helix, and the linker region of CnAa and CnAb are identical [41]. However, proteolysis of the CnA N-terminus results in loss of CaN activity, suggesting that the CnA N-terminus is involved in enzyme activation [42]. Indeed, as shown in the crystal structure, the N-terminus of CnA interacts with the C-terminal half of CnB as part of a CnB-binding cleft, suggesting that the interaction of the CnA N-terminus with CnB is involved in Ca2+CnB-dependent activation of the enzyme [20,22]. As noted previously, the N-terminus of CnAb is different from CnAa, containing 12 Pro residues within the first 24 amino acids [41]. Information regarding the interaction of the CnAb N-terminus with CnB is lacking because only CaN containing CnAa has been crystalized [20,22]. However, the presence of 11 consecutive Pro residues in the N-terminus of CnAb suggests that the CnAa and CnAb N-termini interact with CnB differently, giving rise to the different Km and Vmax values measured for CaNa and CaNb using the same substrate. Polyproline motifs are involved in protein–protein interactions [43]. Molecular modeling indicates that an 11residue type II polyproline helix exactly spans the length of the central helix of CaM, and led to the proposal that the 11 consecutive Pro residues of CnAb may modulate the interaction of CaM with the CaM-binding domain of CnAb [41]. However, as noted previously, the crystal structure subsequently showed that the N-terminus of CnAa forms extensive contacts with CnB. Interestingly, the central helix of CnB differs in length from the central helix of CaM by only one amino-acid residue [44]. These findings suggest that the 11 Pro residues within the first 24 amino-acid residues of CnAb may instead interact with the central helix of CnB and affect its interaction with CnAb. As Ca2+binding to CnB regulates the catalytic activity of CnA, this proposal provides a potential mechanistic basis for the
differences in Km and Vmax values between CaNa and CaNb obtained using the same substrate.
ACKNOWLEDGEMENTS This work was supported by National Institutes of Health Grants NS36318, DK-57168 (B.A.P), and The Medical Research Council, UK (G117/440) (L.H.C). L.H.C. is a MRC Senior Fellow in Basic Science. We thank M. Neal Waxham (University of Texas Medical School at Houston) for the generous gift of CaM (C75), and Michael M. Lai and Solomon H. Snyder (The Johns Hopkins University School of Medicine) for providing the human CnAb cDNA.
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