Dissection of the Functional Differences betweenSarco(endo)plasmic Reticulum Ca2�-ATPase (SERCA) 1 and 2Isoforms and Characterization of Darier Disease (SERCA2)Mutants by Steady-state and Transient Kinetic Analyses*□S

Received for publication, June 25, 2003, and in revised form, September 4, 2003Published, JBC Papers in Press, September 15, 2003, DOI 10.1074/jbc.M306784200

Leonard Dode‡§, Jens Peter Andersen¶�, Natalie Leslie‡, Jittima Dhitavat‡, Bente Vilsen¶,and Alain Hovnanian**‡‡

From ‡The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UnitedKingdom, the ¶Department of Physiology, University of Aarhus, Ole Worms Alle 160, DK-8000 Aarhus C, Denmark, and**INSERM U563, Pavillon Lefebvre, Purpan Hospital, Place de Dr Baylac, 31059 Toulouse Cedex 3, France

Steady-state and rapid kinetic studies were con-ducted to functionally characterize the overall and par-tial reactions of the Ca2� transport cycle mediated bythe human sarco(endo)plasmic reticulum Ca2�-ATPase2 (SERCA2) isoforms, SERCA2a and SERCA2b, and 10Darier disease (DD) mutants upon heterologous expres-sion in HEK-293 cells. SERCA2b displayed a 10-fold de-crease in the rate of Ca2� dissociation from E1Ca2 rela-tive to SERCA2a (i.e. SERCA2b enzyme manifests truehigh affinity at cytosolic Ca2� sites) and a lower rate ofdephosphorylation. These fundamental kinetic differ-ences explain the increased apparent affinity for activa-tion by cytosolic Ca2� and the reduced catalytic turn-over rate in SERCA2b. Relative to SERCA1a, bothSERCA2 isoforms displayed a 2-fold decrease of the rateof E2 to E1Ca2 transition. Furthermore, seven DD mu-tants were expressed at similar levels as wild type. Theexpression level was 2-fold reduced for Gly233 Glu andSer920 3 Tyr and 10-fold reduced for Gly749 3 Arg. Un-coupling between Ca2� translocation and ATP hydroly-sis and/or changes in the rates of partial reactions ac-count for lack of function for 7 of 10 mutants: Gly23 3Glu (uncoupling), Ser186 3 Phe, Pro602 3 Leu, andAsp702 3 Asn (block of E1�P(Ca2) to E2-P transition),Cys3183 Arg (uncoupling and 3-fold reduction of E2-P toE2 transition rate), and Thr357 3 Lys and Gly769 3 Arg(lack of phosphorylation). A 2-fold decrease in theE1�P(Ca2) to E2-P transition rate is responsible for the2-fold decrease in activity for Pro8953 Leu. Ser9203 Tyris a unique DD mutant showing an enhanced molecularCa2� transport activity relative to wild-type SERCA2b.

In this case, the disease may be a consequence of the lowexpression level and/or reduction of Ca2� affinity andsensitivity to inhibition by lumenal Ca2�.

Sarco(endo)plasmic reticulum Ca2�-ATPases (SERCAs)1 aresingle-subunit integral membrane P-type ATPases that medi-ate the ATP-driven transport of cytosolic Ca2� against a con-centration gradient into the lumen of intracellular Ca2�-releas-able stores such as sarcoplasmic and endoplasmic reticulum(1–4). SERCAs belong to the P-type ATPase family distin-guished by the obligatory formation of an aspartyl-phospho-rylated intermediate as part of their catalytic cycle. The en-zyme cycles reversibly between several states (Scheme 1), ofwhich at least E1Ca2, E1�P(Ca2), E2-P, and E2 can be experi-mentally distinguished. The transfer of the �-phosphoryl groupof ATP to the aspartate (Asp351) of the phosphorylation domain,leading to the ADP-sensitive high energy phosphoenzyme in-termediate E1�P(Ca2), is activated by conformational changesassociated with the binding of two calcium ions in exchange forprotons (E2 to E1Ca2 transition). The conversion of this inter-mediate to the ADP-insensitive low-energy E2-P phospho-enzyme intermediate constitutes a crucial rate-limiting step inCa2� translocation (1–3). High resolution models for the atomicstructure, generated by x-ray crystallography of crystals inE1Ca2 (5) and Ca2�-free E2 (6) forms, and extensive mutationalstudies (1, 7–11) of the 110-kDa SERCA1a enzyme (994 aminoacids) have shown that Ca2� translocation and ATP utilizationare coupled through long range intramolecular interactionsbetween the 10-helix membrane-spanning domain, harboringthe Ca2� binding sites, and the large cytosolic head consistingof actuator (A), phosphorylation (P), and nucleotide-binding (N)domains (Fig. 1).

The three human SERCA genes (ATP2A1, ATP2A2, andATP2A3) encode up to a total of 10 isoforms as a result of thealternative splicing of their pre-mRNA (12–16). Mutations inSERCA genes have been recently detected in human diseasessuch as Brody disease (muscle disorder) for SERCA1 (17) and

* This work was supported in part by The Wellcome Trust, UnitedKingdom project Grant 057945/Z/99/Z (to A. H.), La Fondation pour laRecherche Medicale Grant 2000474/3 (to A. H.), and grants from theDanish Medical Research Council, Novo Nordisk Foundation, LundbeckFoundation, and Research Foundation of Aarhus University, Denmark(to J. P. A. and B. V.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org)contains Figs. A–H and additional discussions.

§ To whom correspondence may be addressed: The Wellcome TrustCentre for Human Genetics, University of Oxford, Roosevelt Drive,Oxford, OX3 7BN, United Kingdom. Tel.: 44-1865-287536; Fax: 44-1865-287533; E-mail: leonard.dode@well.ox.ac.uk.

� To whom correspondence may be addressed: Dept. of Physiology,University of Aarhus, Ole Worms Alle 160, DK-8000 Aarhus C, Den-mark. Tel.: 45-8942-2814; Fax: 45-8612-9065; E-mail: jpa@fi.au.dk.

‡‡ Supported by a Wellcome Trust Senior Clinical Fellowship.

1 The abbreviations used are: SERCA, sarco(endo)plasmic reticulumCa2�-ATPase; DD, Darier disease; M1–M11, transmembrane segmentsnumbered from the N terminus of the Ca2�-ATPase; MES, 2-(N-mor-pholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonicacid; E1, enzyme form with cytoplasmically facing high affinity Ca2�

sites; E2, enzyme form with low affinity for Ca2�; E1�P(Ca2), phos-phoenzyme with high energy phosphoryl group (transferable to ADP)and occluded calcium ions; E2-P, phosphoenzyme with low energy phos-phoryl group and lumenally facing low affinity Ca2� sites.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 48, Issue of November 28, pp. 47877–47889, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 47877

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non-insulin dependent type-II diabetes mellitus for SERCA3(18). Previous clinical and genetical investigations have estab-lished that the SERCA2 gene (ATP2A2) represents the genedefective in Darier disease (DD; OMIM 124200), a severe au-tosomal dominant skin disorder characterized by loss of adhe-sion between epidermal cells (acantholysis) and by abnormalkeratinization (dyskeratosis) and association with a wide rangeof neuropsychiatric problems, such as epilepsy and depression(19). It has been proposed that mutations in the SERCA2 geneproduce a dominant DD phenotype through haploinsufficiency,i.e. mutation in one allele would result in complete or partialloss of function in the mutated pumps (19). Over 100 nonsenseand missense mutations and in-frame deletions spanning allregions of the SERCA2 gene have been detected in DD patients(19, 20–26), but no clear genotype-phenotype correlations havebeen drawn. We recently documented that acrokeratosis ver-ruciformis of Hopf (localized disorder of keratinization affectingthe distal extremities) is also caused by mutation in theSERCA2 gene, and, hence, it is allelic to DD (27). The SERCA2gene encodes the cardiac muscle SERCA2a protein (997 aminoacids) as well as the ubiquitously expressed SERCA2b (1042amino acids), but no cardiac manifestations have been observedin DD patients. As a result of the alternative splicing, theSERCA2a-specific C terminus (AILE997) is replaced by a vari-ant sequence of 49 amino acids in SERCA2b (Fig. 1) and by a6-amino acid stretch (VLSSEL999) in SERCA2c, a very recentlyreported SERCA2 splice variant (16). The extended tail of theSERCA2b isoform probably contains an additional transmem-brane domain (M11) as part of a SERCA2b-specific 11-helixtransmembrane structure with the extreme C terminus pro-truding into the endoplasmic reticulum lumen (28). A tentativeplanar model is shown in Fig. 1, in which the indicated second-ary structures correspond to the model for the atomic structureof SERCA1a in the E1Ca2 state (5). The divergence in theC-terminal part is responsible for the known functional differ-ences between SERCA2a and SERCA2b: SERCA2b has a 2-foldhigher apparent affinity for Ca2� and a 2-fold lower turnoverrate for Ca2�-uptake relative to SERCA2a (29, 30). Upon cellstimulation, the SERCA enzymes play a critical role in restor-ing the cytosolic Ca2� concentration to its resting levels.SERCA2b is the major SERCA isoform in non-muscle cells,including epidermal cells, and the specific functional propertiesof SERCA2b have direct consequences for modulation of thefrequency and amplitude of the generated Ca2� waves (31, 32).Comparison of Ca2� wave properties of oocytes overexpressingeither SERCA2a or SERCA2b has demonstrated that both thewidth and the period of the Ca2� waves are larger for SERCA2bthan for SERCA2a, and, consistent with a higher Ca2� affinityof SERCA2b, the wave fronts are more sharply delineated forSERCA2b (31). Replenishment of the agonist-releasable endo-

plasmic reticulum Ca2� stores is, furthermore, required forproper translation and folding of resident and secreted pro-teins (33, 34), and lumenal Ca2� appears to modulate cellsensitivity to apoptosis (35). It is possible that both in Darierdisease and in the related Hailey-Hailey disease caused bymutations in the gene encoding the Golgi Ca2�-ATPase (36,37), the major pathogenic mechanism is the presence of a lowlumenal Ca2� concentration in endoplasmic reticulum andGolgi, which could result in defective processing of the newlysynthesized proteins required for normal adhesion betweenepithelial cells in the adult skin. A very recent study, ana-lyzing the effect of 12 mutations associated with DD, hasdocumented that some mutants inhibited the activity of theco-expressed wild-type through protein interactions betweenwild-type and mutant SERCA2b monomers (38). So far, thefunctional effects of DD mutations on overall and partialreactions of the catalytic cycle mediated by mutant SERCA2bproteins have not been documented in any detail. In addition,the partial reaction steps responsible for the SERCA2b-specific slow catalytic cycle (29, 30) have not yet been iden-tified. The present study addresses these issues by investi-gating the overall and partial reactions catalyzed by wild-typeSERCA2a and SERCA2b isoforms as well as 10 DD SERCA2bmutants (Fig. 1). Because this represents the first detailed studyof the partial reaction steps of SERCA2 isoforms, a comparisonwith the well characterized SERCA1a is also made.

EXPERIMENTAL PROCEDURES

The cDNA clones used encode the human SERCA2a and SERCA2bisoforms (13), and rabbit SERCA1a (39). All SERCA2b mutants weregenerated using the QuikChangeTM site-directed mutagenesis kit fromStratagene (La Jolla, CA). Transfection of HEK-293 cells with thecDNA in expression vector pMT2 or pcDNA3.1(�) (Invitrogen) wasperformed using the calcium phosphate precipitation method (40). Themicrosomal fraction containing expressed wild-type or mutant Ca2�-ATPase was isolated by differential centrifugation (41). Protein concen-tration determination, denaturing gel electrophoresis, semi-dry blot-ting, and blot immunostaining were performed as reported earlier (42,43). All methods used for the analysis of the catalytic cycle in steady-state and transient-kinetic conditions have been previously establishedfrom studies with SERCA1a mutants (7–10, 44–46) and used veryrecently to characterize several SERCA3 isoforms (47). These proce-dures including the quench-flow methodology (8, 10), which allowsrapid kinetic measurements to be performed on a millisecond scale,were directly applicable to expressed wild-type and mutant SERCA2enzymes. Further details of the functional assays are given in the figurelegends. All data presented are average values corresponding to two toseven experiments and standard errors larger than the symbols areshown as error bars in the figures. Experimental data were fitted bynonlinear regression analysis using the SigmaPlot program (SPSS Inc.)or by means of the kinetic simulation software SimZyme developed inthe Department of Physiology, University of Aarhus, Denmark, asdescribed (8). All values extracted for K0.5, Hill coefficient, and differentrate constants are listed in Tables I and II. Generally, the best fits areshown as lines in the figures only for non-overlapping curves. Severalfigures, additional explanations, and discussion are deposited as Sup-plemental Materials.

RESULTS

Expression and Phosphorylation Levels—In the presentstudy, the human wild-type SERCA2a and SERCA2b isoformsas well as SERCA2b mutants Gly23 3 Glu, Ser186 3 Phe,Cys318 3 Arg, Thr357 3 Lys, Pro602 3 Leu, Asp702 3 Asn,Gly749 3 Arg, Gly769 3 Arg, Pro895 3 Leu, and Ser920 3 Tyrwere examined. These SERCA2b mutations have been previ-ously documented in DD patients (19–21, 23, 27), except forPro8953 Leu, which is a novel DD mutation.2 For comparison,the well characterized rabbit wild-type SERCA1a isoform was

2 L. Dode, N. Leslie, J. Dhitavat, and A. Hovnanian, unpublisheddata.

SCHEME 1. Ca2�-ATPase reaction cycle. E1, enzyme form withcytoplasmically facing high affinity Ca2� sites; E2, enzyme form withlow affinity for Ca2�; E1�P(Ca2), phosphoenzyme with high energyphosphoryl group (transferable to ADP) and occluded calcium ions(shown in parentheses); E2-P, phosphoenzyme with low energy phos-phoryl group and lumenally facing low affinity Ca2� sites.

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included in some of the experiments of the present study.Furthermore, the mutation Ser920 3 Tyr was also introducedin SERCA2a, to examine its effect in the presence of the shorterC terminus (cf. Fig. 1). Below, the two Ser920 3 Tyr mutantswill be denoted Ser920 3 Tyr-2a and Ser920 3 Tyr-2b. Fig. 2Acompares the expression levels of wild-type SERCA2b and itscorresponding DD mutants by Western blot analysis of micro-somal proteins from transfected HEK-293 cells, using a poly-clonal antibody raised against an epitope corresponding to thelast 12 amino acids of the pig SERCA2b C terminus (42). In theHEK-293 cells, most mutants appeared to be expressed atlevels similar to wild-type SERCA2b, but a consistent reduc-tion in expression was found for Gly23 3 Glu, Gly749 3 Arg,and Ser920 3 Tyr-2b in several experiments. This was further

confirmed by the functional approach illustrated in Fig. 2B,showing results of measurement of phosphorylation from[�-32P]ATP in the presence of Ca2� (forward reaction 3 inScheme 1), or from 32Pi in the absence of Ca2� (reverse reaction6 in Scheme 1). Maximal phosphorylation levels of 150–250pmol of Ca2�-ATPase/mg of protein were measured for wild-type SERCA2b as well as for the SERCA1a and SERCA2aisoforms, i.e. several hundred-fold higher than that of endoge-nous HEK-293 cell SERCA2b protein (0.5 pmol of enzyme/mg ofprotein). Similar levels were obtained for the DD mutantsSer1863 Phe, Cys3183 Arg, Pro6023 Leu, Asp7023 Asn, andPro895 3 Leu. Relative to wild-type SERCA2b, the maximumphosphorylation level for Gly23 3 Glu, Gly749 3 Arg, andSer9203 Tyr-2b, was reduced to 54, 12, and 57%, respectively,

FIG. 1. Schematic presentation of the primary and putative secondary structure of the human SERCA2 isoforms highlighting theDarier disease mutants studied. The model is based on the E1Ca2 crystal structure of SERCA1a (5). Each circle corresponds to an amino acidresidue indicated by the single-letter code inside the circle, �-helical structures are shown as stacked diagonal rows of three or four residues,�-strands are displayed as ladder-type residue arrangements, and linear sections represent loops. M1–M10 denote the membrane-spanning helicesin SERCA2a. SERCA2a and SERCA2b isoforms are identical in the first 993 amino acid residues (Pro993 represents the divergence point), but differin the C terminus: the SERCA2a-specific sequence AILE997 (purple circles with white letters) is replaced in SERCA2b by a 49-amino acid tail (greencircles with white letters), which could form an additional transmembrane segment (M11). Amino acid substitutions in Darier disease patientsanalyzed in the present study are highlighted (red circles with white letters) and include Gly233 Glu, Ser1863 Phe, Cys3183 Arg, Thr3573 Lys,Pro602 3 Leu, Asp702 3 Asn, Gly749 3 Arg, Gly769 3 Arg, Pro895 3 Leu, and Ser920 3 Tyr; in this study, Ser920 3 Tyr-2a and Ser920 3 Tyr-2brepresent substitution Ser920 3 Tyr in SERCA2a and SERCA2b, respectively. Asp351 (highlighted by inverted color) represents the phosphoryl-ation site. Essential amino acid residues participating in Ca2� liganding are shown as blue circles with white letters in M4, M5, M6, and M8. Adomain, actuator domain (azure circles); P domain, phosphorylation domain (inverted color circles); N domain, nucleotide-binding domain (orangecircles); C, cytosol; M, membrane; L, lumen.

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which seems to correspond to the relative expression levelsseen by Western blot analysis in Fig. 2A. Both Thr357 3 Lysand Gly769 3 Arg are well expressed, but unable to phospho-rylate with either [�-32P]ATP or 32Pi. The protein expressionlevel and phosphorylation properties of Ser920 3 Tyr-2a wereindistinguishable from those of Ser920 3 Tyr-2b (data notshown).

Ca2� Transport Activity—The ability to transport Ca2� ac-tively in the presence of ATP was assessed by measurement ofoxalate-supported 45Ca2� accumulation in the microsomal ves-icles. Mutants Pro8953 Leu and Ser9203 Tyr-2b were able toaccumulate Ca2�, but the remaining DD mutants completelylacked Ca2� uptake activity (Fig. 2C and Table I). In Table I,the amount of Ca2� accumulated per second during a 10-minincubation at 27 °C at saturating Ca2� concentration is shownrelative to the maximum phosphorylation level from Fig. 2B,thus providing an estimate of the maximum rate of Ca2� up-take per enzyme molecule (“molecular Ca2� transport activity”or “turnover rate”). SERCA2a, like SERCA1a, showed a 2-foldhigher maximum molecular Ca2� transport activity relative toSERCA2b, in good agreement with previous studies (29, 30).Interestingly, Ser920 3 Tyr-2b also displayed 2-fold highermaximum molecular Ca2� transport activity relative to wild-type SERCA2b. Ser9203 Tyr-2a showed a maximum molecularCa2� transport activity close to that of Ser920 3 Tyr-2b andwild-type SERCA2a. Pro895 3 Leu displayed a decrease to

about half of wild-type SERCA2b. Analysis of the Ca2� depend-ence of Ca2� transport (Table I and Fig. A of SupplementalMaterials) showed a 1.6-fold increase of the apparent affinityfor Ca2� (decrease of K0.5) for SERCA2b relative to SERCA2a.Ser920 3 Tyr-2b showed a 3-fold lower apparent affinity forCa2� (increase of K0.5) compared with wild-type SERCA2b. ForSer920 3 Tyr-2a, there was likewise a substantial 3.5-foldlowering of the apparent affinity for Ca2� relative to the cor-responding wild-type SERCA2a.

ATPase Activity—Steady-state ATPase activity was deter-mined for SERCA1a, SERCA2a, SERCA2b, and 7 of the 10 DDmutants (the low expression level of Gly749 3 Arg and lack ofphosphorylation of Thr357 3 Lys and Gly769 3 Arg precludedthis measurement). For Ser186 3 Phe, Pro602 3 Leu, andAsp702 3 Asn, the level of ATPase activity was indistinguish-able from background under all conditions tested (Table I). Asignificant ATPase activity was found for each of the SERCA2isoforms and the remaining 4 DD mutants, and Ca2� titrationdata obtained in the presence and absence of the calcium iono-phore A23187 are shown in Fig. B of Supplemental Materialsand summarized in Table I. Consistent with the Ca2� transportdata presented above, SERCA2b displayed higher apparentaffinity for Ca2� at the activating sites than SERCA2a. Theaddition of calcium ionophore relieves the “back inhibition” ofthe E1�P(Ca2) to E2-P transition (steps 4 through 5 in Scheme1) brought about by binding of accumulated Ca2� at lumenal

FIG. 2. Protein expression (A), comparative phosphorylation from [�-32P]ATP and 32Pi (B), and Ca2� transport activity (C). A,Western blot of microsomes (10 �g of total membrane protein/lane) isolated from HEK-293 cells transfected with pMT2 expression vector cDNAwithout insert (control cells) or with expression vector containing either human SERCA2b or any of the indicated mutant cDNA, and immuno-assayed with a polyclonal anti-SERCA2b antibody. B, phosphorylation was performed with 5 �M [�-32P]ATP in a medium containing 40 mM

MOPS/Tris, pH 7.0, 80 mM KCl, 5 mM MgCl2, and 100 �M CaCl2 at 0 °C for 15 s (black bars), and with 0.5 mM 32Pi in a medium containing 100mM MES/Tris, pH 6.0, 10 mM MgCl2, 2 mM EGTA, 30% (v/v) dimethyl sulfoxide at 25 °C for 10 min (white bars). Phosphorylation levels are shownas picomoles of enzyme per mg of total microsomal protein. Depending on the kinetics of phosphorylation and dephosphorylation, the data obtainedwith [�-32P]ATP at 0 °C can in most cases be assumed to represent the “active site concentration.” The error bars on the columns indicate standarderrors. C, time course of 45Ca2� uptake was measured at 37 °C in medium containing 20 mM MOPS, pH 6.8, 100 mM KCl, 5.5 mM MgCl2, 5 mM ATP,0.5 mM EGTA, 5 mM potassium oxalate (to trap Ca2� inside the vesicles), and 0.450 mM 45CaCl2 (free Ca2� concentration of 3.6 �M). Ca2� uptakeactivity is shown as micromoles of Ca2� transported per mg of total microsomal protein.

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low affinity sites, because the ionophore allows passive efflux ofthe Ca2� actively transported into the microsomal vesicles,thereby maintaining the Ca2� concentration inside the micro-somes at a relatively low level. In this non-inhibited state,SERCA2b showed a 2-fold lower catalytic turnover rate thanSERCA2a (compare 35 s�1 with 70 s�1 in Table I). Surpris-ingly, the maximal catalytic turnover rate measured with mu-tant Gly23 3 Glu was similar to that of wild-type SERCA2b,even though the Ca2� accumulation was below the detectionlimit for this mutant (cf. Fig. 2 and Table I). Consistent withthe lack of significant Ca2� accumulation, there was littlestimulation of the maximal rate of ATP hydrolysis upon iono-phore addition in mutant Gly23 3 Glu. Therefore, it appearsthat the ATP hydrolysis to a large extent is uncoupled fromCa2� translocation in mutant Gly23 3 Glu. The apparent af-finity for Ca2� displayed by this mutant was, furthermore,2-fold lower (K0.5 higher) than that of wild-type SERCA2b.Cys3183 Arg is another DD mutant showing noticeable uncou-pling properties. Compared with wild-type SERCA2b, the max-imum catalytic turnover rate reached with Cys318 3 Arg wasreduced by 37%, but again no Ca2� uptake could be measured,even though in this case a significant, although reduced, iono-phore stimulation of ATPase activity was noted (Table I). Thismutant also showed a 2-fold lower apparent affinity for Ca2�

compared with wild-type SERCA2b. For mutant Pro895 3Leu, a reduced catalytic turnover rate and similar apparentaffinity for Ca2� were observed in comparison withSERCA2b, which corresponds with the Ca2� transport datashown above. For mutant Ser920 3 Tyr-2b, a 2-fold increasein the maximal catalytic turnover rate relative to wild-typeSERCA2b was seen (but similar to that of SERCA2a), anddisappearance of the activating effect of calcium ionophore.Like Ser920 3 Tyr-2b, Ser920 3 Tyr-2a was insensitive toionophore, and its turnover rate was similar to that of wild-type SERCA2a (Table I).

Ligand Dependence of Phosphorylation Levels—The phos-phoryl transfer from [�-32P]ATP requires the binding of twocalcium ions at the cytoplasmically facing high affinity sites.For the wild-type enzymes and mutants capable of forming aphosphoenzyme by reaction with [�-32P]ATP (cf. Fig. 2B), Ca2�

titration of the phosphorylation reaction was carried out (Fig. Cin Supplemental Materials). Table II shows a summary of thedata. SERCA2b displayed an almost 2-fold higher apparentCa2� affinity than both SERCA2a and SERCA1a isoforms.Mutants Gly233Glu, Cys3183 Arg, and Asp7023 Asn showeda somewhat reduced apparent affinity for Ca2� relative towild-type SERCA2b. Mutants Ser920 3 Tyr-2b and Ser920 3Tyr-2a showed as much as 3-fold reduction in apparent Ca2�

affinity compared with their corresponding wild-type isoforms.Ser1863 Phe, Pro6023 Leu, and Pro8953 Leu were wild-type-like in this respect. Table II also shows the results of titratingthe Ca2� inhibition of 32Pi phosphorylation (Fig. D in Supple-mental Materials). Again, SERCA2b showed a significantly(2-fold) higher apparent affinity for Ca2� as compared withSERCA2a, and the apparent Ca2� affinities displayed by mu-tants Ser920 3 Tyr-2a and Ser920 3 Tyr-2b in this assay were2- and 3-fold reduced relative to wild-type SERCA2a andSERCA2b, respectively. The other mutants were SERCA2b-like in this respect. It is, furthermore, shown in Table II thatthe apparent affinity for 32Pi displayed by SERCA2a andSERCA2b was similar to that of SERCA1a (Fig. E in Supple-mental Materials). Wild-type-like apparent 32Pi affinity wasobserved for mutants Cys318 3 Arg, Pro895 3 Leu, andSer9203 Tyr, but significant 3-, 7-, 3-, and �10-fold reductionswere found for mutants Gly23 3 Glu, Ser186 3 Phe, Pro602 3Leu, and Asp702 3 Asn, respectively.

Dephosphorylation of Phosphoenzyme Intermediates formedfrom ATP and Pi—The decay of phosphoenzyme formed with[�-32P]ATP was examined as illustrated in Fig. 3A and Fig. F ofSupplemental Materials, and a summary of the data is given inTable II. As previously described, the phosphorylation condi-tions applied (0 °C, pH 7.0, and presence of K�) result inaccumulation of the ADP-sensitive E1�P(Ca2) intermediate forSERCA1a and SERCA3 isoforms (44, 47). As illustrated by thedotted line in Fig. 3A (showing a very rapid dephosphorylationupon ADP addition), this was also the case for wild-typeSERCA2 isoforms and mutant SERCA2 proteins other thanCys318 3 Arg. Addition of excess EGTA (Fig. 3A) or non-radioactive ATP (Fig. F of Supplemental Materials) to theaccumulated E1�P(Ca2) intermediate prevented further phos-

TABLE ISummary of biochemical parameters characterizing the overall reactions (Ca2� -transport and ATPase activities) of the catalytic cycle for

wild-type SERCA1 and SERCA2 isoforms and for SERCA2 (Darier disease) mutants

Wild-type SERCAisoforms and

SERCA2 mutants

Maximum turnover rateIonophore effect onthe turnover ratec

K0.5 for Ca2� activation

Ca2� uptakeactivitya

ATPaseactivityb

Ca2� uptakeactivityd

ATPaseactivitye

s�1 �M � S.E.

SERCA1a 18 130 3.20 0.31 � 0.02 0.31 � 0.01SERCA2a 20 70 2.50 0.21 � 0.01 0.20 � 0.01SERCA2b 9.1 35 2.00 0.13 � 0.01 0.11 � 0.01Gly23 3 Gluf 0 31 1.19 N.D.A.g 0.22 � 0.02Ser186 3 Phef 0 0 N.D.A. N.D.ACys318 3 Argf 0 22 1.46 N.D.A. 0.20 � 0.01Pro602 3 Leuf 0 0 N.D.A. N.D.A.Asp702 3 Asnf 0 0 N.D.A. N.D.A.Pro895 3 Leuf 4.4 13.3 1.39 0.13 � 0.01 0.13 � 0.01Ser920 3 Tyr-2bf 21 73 1.02 0.42 � 0.05 0.16 � 0.01Ser920 3 Tyr-2ah 15 70 1.00 0.70 � 0.08 NDi

a Molecular Ca2� transport activity at 27 °C (Fig. A of Supplemental Materials) measured in the presence of potassium oxalate at pH 6.8 for 10min (phosphoenzyme intermediate level determined at 0 °C by phosphorylation with [�-32P]ATP, cf. Fig. 2B, was used as an estimate ofenzyme concentration).

b Turnover rate for ATP hydrolysis at 37 °C in the presence of calcium ionophore A23187 (Fig. B of Supplemental Materials).c The ratio between the maximum ATPase activities with and without calcium ionophore at pH 7.0 (Fig. B of Supplemental Materials).d The Ca2� concentration giving half-maximum activation of Ca2� transport activity at 27 °C (Fig. A of Supplemental Materials).e The Ca2� concentration giving half-maximum activation of ATPase activity at 37 °C in presence of the calcium ionophore A23187 (Fig. B of

Supplemental Materials).f Amino acid substitution in SERCA2b isoform.g N.D.A., no detectable activity above background.h Amino acid substitution in SERCA2a isoform.i ND, not determined.

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phorylation by [�-32P]ATP and allowed observation of dephos-phorylation in the forward direction (reactions 4–6 in Scheme1). The differences observed between the various isoforms andmutants were similar for EGTA chase and nonradioactive ATPchase (Table II). SERCA2b displayed half the dephosphoryl-ation rate of SERCA2a, the latter being similar to that ofSERCA1a. Mutant Pro895 3 Leu showed a reduction of thedephosphorylation rate relative to wild-type SERCA2b. De-phosphorylation was almost completely blocked in mutantsSer186 3 Phe, Pro602 3 Leu, and Asp702 3 Asn. By contrast,the dephosphorylation rate was wild-type-like for Gly233 Glu.For Ser920 3 Tyr-2b, the dephosphorylation rate was actuallyenhanced relative to wild-type SERCA2b. Likewise, the de-phosphorylation rate of Ser920 3 Tyr-2a was significantlyhigher than that of its corresponding wild-type SERCA2a (Ta-ble II). These variations of the dephosphorylation rate explainto a large extent the observed differences in the maximal cat-

alytic turnover rate described above. For the remaining mu-tant, Cys318 3 Arg, the dephosphorylation rate was signifi-cantly reduced both in the forward direction as observed uponchase with EGTA or nonradioactive ATP, as well as in thereverse direction upon addition of ADP (more than 80% of theinitial phosphoenzyme was left after 5 s of chase with ADP, Fig.3A, solid hexagon). The lack of ADP sensitivity indicates thatduring the phosphorylation period the E2-P intermediate actu-ally accumulated, suggesting a slow E2-P 3 E2 reaction. Thedecay of the E2-P phosphoenzyme intermediate was furtherexamined (Fig. 3B and Table II) by diluting E2-P formed “back-wards” from 32Pi in the dephosphorylation medium. The decayrate of SERCA2b was 3-fold reduced relative to SERCA2a. Fig.3B, furthermore, demonstrates that the E2-P decay rate forCys318 3 Arg was 3-fold reduced relative to wild-typeSERCA2b, thus confirming that a slow E2-P 3 E2 reactionconstitutes the major reason for the low dephosphorylation rateobserved for this mutant (cf. Fig. 3A). A wild-type-like E2-Pdecay was observed for Pro895 3 Leu. Mutants Gly23 3 Glu,Ser186 3 Phe, Pro602 3 Leu, and Asp702 3 Asn were charac-terized by increased rates of dephosphorylation of E2-P relativeto wild-type SERCA2b, as much as 30-fold for Ser186 3 Phe.Hence, the data in Fig. 3B support the conclusion that thereduced E1�P(Ca2) phosphoenzyme turnover observed forSer1863 Phe, Pro6023 Leu, Asp7023 Asn, and Pro8953 Leuin Fig. 3A is caused by inhibition of the E1�P(Ca2) 3 E2-Ptransition. Ser920 3 Tyr-2b displayed an increased rate of theE2-P decay relative to wild-type SERCA2b, but similar to thatof Ser920 3 Tyr-2a and its corresponding wild-type SERCA2a(Fig. 3B and Table II).

Rapid Kinetic Analysis of Phosphorylation and Ca2� Bind-ing—For SERCA2 isoforms and DD mutants, rapid kineticmeasurements were performed at 25 °C as previously reportedfor SERCA1a and SERCA3 isoforms (8, 47). The results, someof which are summarized in the last three columns of TableII, provide information on the Ca2� binding transition (reac-tions 1 and 2 in Scheme 1), phosphorylation (reaction 3 inScheme 1), Ca2� dissociation from E1Ca2 (reverse reaction 2in Scheme 1), and dephosphorylation (reactions 4 – 6 inScheme 1) at 25 °C.

When the time course of phosphorylation was studied afterthe simultaneous addition of [�-32P]ATP and excess Ca2� toCa2�-deprived enzyme (Fig. 4, examination of the sequenceconsisting of reactions 1–3 in Scheme 1), the observed rateconstants for SERCA2a and SERCA2b were, respectively, 1.7-and 2.2-fold lower than that of SERCA1a (Table II). Rate con-stants similar to wild-type SERCA2b were obtained for mu-tants Gly23 3 Glu, Ser186 3 Phe, Pro602 3 Leu, and Ser920 3Tyr-2b. Relative to wild-type SERCA2b, the rate constant formutant Cys3183 Arg was more than 2-fold enhanced, whereasthat of Asp702 3 Asn was 2-fold reduced.

When the time course of phosphorylation was monitoredwith the enzyme initially present in the Ca2�-saturated E1Ca2

form (Fig. 5), the rise of the phosphorylation level occurred withsimilar rates in the wild-type SERCA2 isoforms as inSERCA1a, but for the wild-type SERCA2 isoforms there was alarger overshoot, most pronounced for SERCA2a. The over-shoot is indicative of rapid dephosphorylation and/or a rela-tively slow step intervening between the dephosphorylationand rephosphorylation reactions. As indicated by the lines inFig. 5, the experimental data could be reproduced rather accu-rately by simulation of a simplified reaction cycle (Scheme 1 inRef. 8) using the SimZyme program as described (8). The sim-plified cycle consisted of three reactions: phosphorylation (re-action 3 in Scheme 1) with the rate constant kA, dephosphoryl-ation (reactions 4–6 in Scheme 1, combined in one step) with

FIG. 3. Dephosphorylation at 0 °C of the phosphoenzymeformed from [�-32P]ATP (A) and 32Pi (B). A, phosphorylation wascarried out for 15 s at 0 °C in medium containing 2 �M [�-32P]ATP, 40mM MOPS/Tris, pH 7.0, 80 mM KCl, 5 mM MgCl2, 0.1 mM CaCl2, and 1�M calcium ionophore A23187. To measure dephosphorylation, thephosphoenzyme was chased with 10 mM EGTA without (full lines) orwith (dotted line and solid hexagon) 1 mM ADP, followed by acid quench-ing at the indicated serial time intervals. The phosphorylation levelsare shown relative to the level determined by acid quenching directlyafter phosphorylation for 15 s. The lines show the best fits of a mono-exponential decay function and the extracted rate constants (s�1 � S.E.)for the chase with 1 mM EGTA alone are shown in Table II. The solidhexagon represents the ADP chase for Cys3183 Arg, whereas the dottedlines represent ADP chases for wild-type SERCA2 isoforms and mutantSERCA2 proteins other than Cys318 3 Arg. B, phosphorylation wasperformed at 25 °C for 10 min in the presence of 100 mM MES/Tris, pH6.0, 10 mM MgCl2, 2 mM EGTA, 30% (v/v) dimethyl sulfoxide, and 0.5mM 32Pi. Following cooling of the sample to 0 °C, the phosphoenzymewas chased by a 19-fold dilution of an aliquot into a medium (kept at0 °C) containing 100 mM MOPS/Tris, pH 7.0, 10 mM MgCl2, 2 mM EGTA,and 0.5 mM non-radioactive Pi, and acid quenching was performed atthe indicated serial time intervals. The phosphorylation levels areshown relative to the initial level determined after 10 min phosphoryl-ation without dephosphorylation. The lines represent the best fits of amonoexponential decay function and the extracted rate constants(s�1 � S.E.) are shown in Table II.

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the rate constant kB, and the Ca2� binding transition (com-bined reactions 1 and 2 in Scheme 1) with the rate constant kC.Consistent with previous reports (8, 47), a good fit forSERCA1a was obtained using the rate constants kA � 35 s�1,kB � 5 s�1, and kC � 25 s�1, but for SERCA2a it was necessaryto reduce kC to 13 s�1 and increase kB slightly to 6 s�1. Toobtain a good fit for SERCA2b, both kB and kC had to bereduced to 3 s�1 and 10 s�1, respectively. The SERCA1a-like kB

value for SERCA2a and the much lower kB value for SERCA2b,as well as the reduced kC values are in good agreement with thedata presented above for the dephosphorylation of E1�P(Ca2)and E2-P (Fig. 3) and the phosphorylation starting from the

Ca2�-deprived enzyme (Fig. 4), kC supposedly being rate-lim-iting for the latter reaction sequence (8, 47). To fit the muchlarger overshoot obtained with mutant Ser920 3 Tyr-2b (Fig.5), it was necessary to increase kB from 3 s�1 (wild-typeSERCA2b value) to 6 s�1, i.e. a 2-fold increase in the dephos-phorylation rate, making it similar to that of SERCA2a. This isalso in perfect agreement with the dephosphorylation data

FIG. 4. Time course of phosphorylation by [�-32P]ATP of en-zyme preincubated in the absence of Ca2�. Quench-flow experi-ments were carried out at 25 °C using the QFM-5 module as illustratedin the diagram above the panels, by mixing microsomes preincubated ina medium containing 40 mM MOPS/Tris, pH 7.0, 80 mM KCl, and 2 mM

EGTA with an equal volume of 40 mM MOPS/Tris, pH 7.0, 80 mM KCl,10 mM MgCl2, 2.2 mM CaCl2, and 10 �M [�-32P]ATP, followed by acidquenching at the indicated time intervals. In each panel, the phospho-rylation is shown relative to the maximum level reached. The solid linesshow the best fits of a monoexponential function, giving the rate con-stants (s�1) listed in Table II (“Rate of E2 to E1�P(Ca2)”). For directcomparison, the dotted lines in the wild-type SERCA2 panels reproducethe line corresponding to SERCA1a, whereas the dashed lines in mu-tant panels reproduce the line for wild-type SERCA2b.

FIG. 5. Time course of phosphorylation by [�-32P]ATP of en-zyme preincubated with Ca2�. Quench-flow experiments were car-ried out at 25 °C using the QFM-5 module as illustrated in the diagramabove the panels, by mixing microsomes preincubated in a mediumcontaining 40 mM MOPS/Tris, pH 7.0, 80 mM KCl, 5 mM MgCl2, and 100�M CaCl2 with an equal volume of the same medium containing inaddition 10 �M [�-32P]ATP, followed by acid quenching at the indicatedtime intervals. In each panel, the phosphorylation is shown relative tothe maximum level reached. For direct comparison, the dotted lines inthe wild-type SERCA2 panels reproduce the line corresponding toSERCA1a and the dashed lines in mutant panels reproduce the linecorresponding to wild-type SERCA2b. The lines were generated bycomputer simulation as described (8), using the following rate constantsfor SERCA1a (kA � 35 s�1, kB � 5 s�1, kC � 25 s�1), SERCA2a (kA � 35s�1, kB � 6 s�1, kC � 13 s�1), SERCA2b (kA � 35 s�1, kB � 3 s�1, kC �10 s�1), and S920Y-2b (kA � 35 s�1, kB � 6 s�1, kC � 8 s�1). For mutantsG23E, S186F, C318R, P602L, and D702N, the solid lines show the bestfits of a monoexponential function giving the following rate constants:11.4, 34, 33, 19, and 8 s�1.

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described above in relation to Fig. 3. The experimental dataobtained for the other DD mutants studied could be fitted by amonoexponential function, the extracted rate constant beingquite similar to kA for wild-type SERCA2b in the case ofSer186 3 Phe and Cys318 3 Arg, but lower for Gly23 3 Glu,Pro602 3 Leu, and Asp702 3 Asn (Fig. 5 and Table II). ForSer186 3 Phe, the absence of an overshoot is explained by thelow dephosphorylation rate (Fig. 3), corresponding to a mark-edly reduced value for kB. For Cys318 3 Arg, the dephospho-rylation was also found markedly reduced, and in addition theCa2� binding E2 3 E1Ca2 transition seems to be enhanced,corresponding to an increased kC value, as indicated by theenhanced phosphorylation rate seen for this mutant in Fig. 4under conditions where the E2 3 E1Ca2 transition is rate-limiting in the wild-type. The latter effect should contribute toremove the overshoot. For Gly233Glu, the lack of an overshootis clearly because of the reduced rate of phosphorylation (kA).Fig. 5, furthermore, indicates a reduced phosphorylation ratefor Pro602 3 Leu and, in particular, Asp702 3 Asn (kA only 8s�1, see Table II). The latter mutants also showed a very lowrate of the E1�P(Ca2) to E2-P transition (Fig. 3). These effectscause the absence of an overshoot. Because of the low rate ofphosphorylation of Gly23 3 Glu and Asp702 3 Asn, this reac-tion is probably also rate-limiting for the reaction sequencestudied in Fig. 4, which proceeded at the same rate as thephosphorylation studied in Fig. 5.

By adding an excess of EGTA together with [�-32P]ATP inexperiments otherwise similar to those illustrated in Fig. 5, itis possible to monitor a single turnover cycle, because theremoval of Ca2� with EGTA prevents rephosphorylation afterthe dephosphorylation has occurred. The results of such exper-iments are shown in Fig. G of Supplemental Materials. Thisanalysis confirms the conclusions derived from the data in Figs.3–5. It is particularly noteworthy that this precise determina-tion of kB at 25 °C shows that phosphoenzyme processing (i)occurs with similar rates in SERCA1a and SERCA2a, (ii) isslower for SERCA2b, (iii) is slower for Cys3183 Arg, relative towild-type SERCA2b, and faster for Ser920 3 Tyr-2b, the latterbehaving very similar to SERCA2a, and (iv) is completelyblocked for Ser186 3 Phe and Pro602 3 Leu.

To obtain direct information on the properties of the cyto-plasmically facing Ca2� sites for the wild-type SERCA2 iso-forms and selected mutants, we determined the rate of Ca2�

dissociation from E1Ca2 at 25 °C by the previously describedprocedure (8–10, 47), which compares the amount of phos-phoenzyme measured 34 ms after the simultaneous addition of[�-32P]ATP and excess EGTA to Ca2�-saturated enzyme(“EPATP�EGTA”) with the amount of phosphoenzyme measuredafter 34 ms incubation with [�-32P]ATP and Ca2� (“EPATP”).The data are shown in Fig. 6, and the derived rate constants forCa2� dissociation are listed in the last column of Table II.Importantly, Ca2� dissociation was about 10-fold slower inSERCA2b relative to SERCA1a and SERCA2a, thus explainingthe high affinity of SERCA2b for Ca2� seen in the above de-scribed measurements. Both Ser186 3 Phe and Cys318 3 Argdisplayed a Ca2� dissociation rate similar to that of wild-typeSERCA2b or perhaps even lower (in this range the method doesnot allow a very accurate differentiation). On the other hand,the Ca2� dissociation rate was about 8-fold increased inSer920 3 Tyr-2b relative to wild-type SERCA2b, whereas inGly23 3 Glu it was as much as 13-fold increased.

DISCUSSION

In the present study, we investigated the overall and partialreactions of the catalytic cycle mediated by the humanSERCA2a and SERCA2b isoforms, as well as 10 SERCA2bmutants, each containing a missense mutation detected in

patients with DD. The importance of the results, which havebeen summarized in Tables I and II, is 3-fold: (i) the partialreaction steps responsible for the long known differences in theCa2� activation at the cytosolic sites and catalytic turnoverrates between SERCA1a and SERCA2 isoforms are now iden-tified; (ii) abnormal enzymatic behavior and Ca2� transportproperties of DD mutants are demonstrated, underscoring thecrucial role played by the wild-type SERCA2b isoform as part ofthe cellular Ca2� signaling machinery involved in maintainingepidermal integrity; (iii) the analysis of the overall and partialreactions of DD mutants provides new insight regarding struc-ture-function relationships of SERCA enzymes. First we willdiscuss the new information about the wild-type SERCA2enzymes.

SERCA2a and SERCA2b Isoforms—Contrary to SERCA2a,SERCA2b is characterized by the inclusion of an additional49-amino acid stretch (amino acids 994 to 1042) containing ahydrophobic sequence, which most likely represents an extratransmembrane domain (M11 in Fig. 1) (28, 48, 49). The ex-tended tail in SERCA2b must be responsible for the observeddifferences between the partial reaction steps of SERCA2a andSERCA2b: (i) a �10-fold lower rate of Ca2� dissociation fromE1Ca2 in SERCA2b; (ii) a �2-fold lower rate of conversion of theADP-sensitive E1�P(Ca2) phosphoenzyme intermediate toADP-insensitive E2-P in SERCA2b; and (iii) a 3-fold lower rateof dephosphorylation of E2-P to E2 in SERCA2b. SERCA2 iso-forms showed no difference from SERCA1a with respect to therate of the phosphorylation reaction E1Ca23 E1�P(Ca2). BothSERCA2a and SERCA2b isoforms displayed a �2-fold reducedrate of the E2 to E1Ca2 transition relative to SERCA1a. Thelatter effect must be ascribed to some (or only one) of the �160amino acid differences between the SERCA1 and SERCA2

FIG. 6. Rate of Ca2� dissociation. Rate constants for Ca2� dissoci-ation (k�Ca, shown at right and in Table II for the indicated wild-typeand mutant SERCA proteins) were determined at 25 °C as previouslydescribed (8) in quench-flow experiments measuring the EPATP�EGTA/EPATP ratios (represented by columns). To determine the EPATP�EGTAlevel, the enzyme preincubated in a medium containing 40 mM MOPS/Tris, pH 7.0, 80 mM KCl, 5 mM MgCl2, and 100 �M CaCl2 was mixedwith an equal volume of 40 mM MOPS/Tris, pH 7.0, 80 mM KCl, 5 mM

MgCl2, 4 mM EGTA, and 10 �M [�-32P]ATP, followed by acid quenching34 ms later, as illustrated in the diagram above the panel. To determinethe EPATP level, the enzyme preincubated in the same medium as forthe EPATP�EGTA determination was mixed with an equal volume of thisCa2� containing medium to which 10 �M [�-32P]ATP had been added,followed by acid quenching 34 ms later.

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enzymes (50). It is noteworthy in this connection that the35-residue lumenal loop connecting M7 and M8 contains asmuch as 15 amino acid differences between SERCA1and SERCA2.

The observed differences among the isoforms with respect tothe partial reaction steps can account for the characteristicisoform differences described here and in previous studies (29,30) with respect to the apparent affinity for Ca2� and theturnover rate of the overall reaction. Relative to SERCA2a, an�2-fold increase in apparent Ca2� affinity was observed forSERCA2b by Ca2� titration of Ca2� transport activity andATPase activity at steady state (Table I), as well as phospho-rylation from ATP (Table II). The affinity for Ca2� inhibition ofphosphorylation from Pi was also higher in SERCA2b relativeto SERCA2a (Table II). By Ca2� titration of Ca2� transport andATPase activity, similar observations were made for SERCA2bfrom rabbit and human (29), as well as pig (30). The majorreason for the increased apparent affinity of SERCA2b seemsto be the �10-fold reduction of the rate of Ca2� dissociationfrom E1Ca2 (Table II and Fig. 6). This represents a uniquefinding, indicating that the “true” affinity of the E1 form forCa2� is enhanced because of the presence of the C-terminalextension in SERCA2b. In addition, the slow phosphoenzymeprocessing might contribute to the increased apparent Ca2�

affinity of SERCA2b, as more phosphoenzyme accumulates atlow Ca2� concentrations when phosphoenzyme turnover isslow, thereby shifting the Ca2� activation curve to higherapparent affinity. Such a “kinetic effect” on apparent Ca2�

affinity can be demonstrated by computer simulation of thereaction cycle (10). The slow phosphoenzyme processing def-initely constitutes a major reason for the lower overall ratesof Ca2� transport and ATP hydrolysis in SERCA2b relative toSERCA2a (Table I). Furthermore, the reduced rate of thereaction sequence E2 3 E1Ca2 in both SERCA2a andSERCA2b, relative to SERCA1a, contributes further to slow-ing of the SERCA2b cycle and explains why in some condi-tions even SERCA2a showed a reduced catalytic turnoverrate relative to SERCA1a (Table I, “ATPase activity”). Thereduced rate of E2 3 E1Ca2 in SERCA2a and SERCA2b,furthermore, explains the increased sensitivity to inhibitionby vanadate (see Supplemental Materials, Fig. H, as well asRef. 29).

Site-directed mutagenesis analysis and the crystal struc-ture of SERCA1a in the E1Ca2 form have revealed that theCa2� liganding side chains in the high affinity Ca2� binding/occlusion sites are donated by residues Glu309 in M4, Asn768

and Glu771 in M5, Asn796, Thr799, and Asn800 in M6, andGlu908 in M8 (1, 5, 7). These residues are conserved betweenSERCA1a and SERCA2 isoforms (Fig. 1), although the num-bering in SERCA2 differs by one residue from that ofSERCA1a in M5, M6, and M8, because of the deletion of asingle amino acid corresponding to Gly509 in SERCA1a.Therefore, the reduced rate of Ca2� dissociation from theE1Ca2 form of SERCA2b (and implicitly its high affinity forCa2�) is not brought about by replacement of the Ca2� li-gands themselves, but more indirectly by a fine tuning of theproperties of the binding pocket through interaction involv-ing the C-terminal extension. The two crystal structures ofSERCA1a show that the transition from E1Ca2 to E2 occur-ring in relation to Ca2� dissociation is accompanied by dra-matic rearrangements of the first six transmembrane do-mains, M1–M6 (5, 6). It is possible that because of structuralconstraints imposed by the presence of the C-terminal exten-sion, the transmembrane region is less flexible in SERCA2brelative to SERCA1a and SERCA2a. This could explain theslowing in SERCA2b of conformational changes involved in

dissociation of Ca2� from E1Ca2 and E1�P(Ca2) 3 E2-P andE2-P 3 E2 transitions. Because deletion of the last 12 aminoacid residues of SERCA2b, thought to be lumenal, or mu-tagenesis of one of these, Asn1036 in rat SERCA2b (corre-sponding to Asn1035 in human SERCA2b, cf. Fig. 1), resultedin SERCA2a-like behavior with respect to the apparent Ca2�

affinity in the overall reaction and Ca2� wave properties atthe cellular level (30, 31), it is likely that the SERCA2b-specific Ca2� binding properties owe particularly to interac-tions of the lumenal part of the tail, possibly with lumenalproteins such as calreticulin and calnexin (31, 51), therebyreducing the conformational flexibility of the transmembranepart of SERCA.

DD Mutant Ser920 3 Tyr—The introduction of mutationSer920 3 Tyr in SERCA2b accelerated Ca2� dissociation fromE1Ca2 as well as E1�P(Ca2)3 E2-P and E2-P3 E2 transitions,leading to rates similar to those of SERCA2a. As a result, theapparent Ca2� affinity and catalytic turnover rate of the over-all reaction of this SERCA2b mutant were quite SERCA2a-like.Because this raised the question whether the Ser920 3 Tyrmutation acts by interference with the effect of the C-terminalextension in SERCA2b, we tested the mutation in SERCA2a aswell. When Ser920 3 Tyr was introduced in SERCA2a, theresulting apparent Ca2� affinity at the cytoplasmically facingsites determined by Ca2� titration of Ca2� uptake and phos-phorylation from ATP was considerably lower than that of thewild-type SERCA2a or Ser9203 Tyr-2b (Tables I and II), show-ing that mutation and removal of the C-terminal extensionexerts additive and independent effects on Ca2� affinity. BothSer9203 Tyr mutants were characterized by complete absenceof the ionophore-induced activation of ATPase activity, i.e. the“back inhibition” by lumenal Ca2� was relieved by the muta-tion. Because the Ser9203 Tyr mutants showed Ca2� transportactivity, the insensitivity to ionophore cannot be ascribed tolack of Ca2� accumulation in the microsomal vesicles. It is,however, possible that the sensitivity to inhibition by the ac-cumulated lumenal Ca2� is reduced in the Ser920 3 Tyr mu-tants. This could be brought about by a reduction of the trueaffinity of the lumenally facing Ca2� release sites on the E2-Pform by increasing the rate of Ca2� dissociation from E2-PCa2,in line with the effect on the Ca2� sites in their cytoplasmicallyfacing configuration. In the crystal structures of SERCA1a, theserine corresponding to Ser920 of SERCA2 isoforms is located atthe cytoplasmic surface, in the loop connecting M8 and M9 (Fig.1). Its side chain is rather close to residues at the C-terminalend of M10, and it is likely that the replacement of the serineside chain with a bulky tyrosine causes a steric clash andthereby changes the tilt of M10. This would probably affect thelumenal loop between M9 and M10 and, in turn, M9 andpossibly other lumenal loops and transmembrane segments aswell, including also M11 in SERCA2b. This might increase theconformational flexibility of the transmembrane region, ex-plaining the enhanced rates of the conformational changesassociated with Ca2� dissociation, E1�P(Ca2) 3 E2-P, andE2-P 3 E2 in the mutants.

DD Mutant Gly23 3 Glu—Gly23 is conserved in all knownSERCAs and secretory pathway/Golgi Ca2�-ATPases, as wellas Na�,K�- and H�,K�-ATPases, but has not been previouslystudied by mutagenesis. Remarkably, the Gly233 Glu mutantdisplayed Ca2�-activated ATPase activity with a maximumcatalytic turnover rate similar to that of wild-type SERCA2b,but without any measurable Ca2� uptake in the microsomes.There was little activation of ATP hydrolysis when the vesicleswere made permeable to Ca2� with ionophore, thus confirmingthat Ca2� uptake is insignificant for this mutant (Table I). Thepartial reactions of the Gly233 Glu mutant differed from those

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of wild-type SERCA2b in several respects (Table II): the rate ofthe phosphorylation reaction E1Ca23 E1�P(Ca2) was reduced;the rate of dephosphorylation (E2-P3 E2) was 3-fold enhanced(resulting in a 3-fold reduced apparent affinity for Pi); and therate of Ca2� dissociation from E1Ca2 was 13-fold enhanced,relative to wild-type SERCA2b, resulting in a reduced apparentaffinity for Ca2�. The defects in Ca2� binding and in the Ca2�-activated phosphorylation from ATP may reflect changes to theCa2� sites related to the inability to couple ATP hydrolysiswith Ca2� transport. The highly conserved Gly23 is located in aloop that connects the two cytoplasmic �-helices before M1 nearthe N terminus (Fig. 1). These helices are part of domain A,which undergoes a large rearrangement in relation to the tran-sition from E2 to E1Ca2 (6). It is, therefore, possible that theflexibility of the loop introduced by the presence of the glycineis required for a proper domain movement and long rangeinteraction with the Ca2� sites. Similar uncoupling of ATPaseactivity from Ca2� transport induced by mutagenesis has pre-viously only been observed for a SERCA1a mutant where atyrosine located much closer to the Ca2� binding structure(corresponding to Tyr762 in Fig. 1) had been exchanged forglycine (46).

DD Mutant Cys318 3 Arg—Mutant Cys318 3 Arg was alsofound more deficient with respect to Ca2� accumulation thanATPase activity. Even though Ca2� accumulation was belowthe detection limit, a significant, although reduced, ionophorestimulation of ATPase activity was noted. The rate of dephos-phorylation of the ADP-insensitive phosphoenzyme intermedi-ate was found 3-fold reduced relative to wild-type SERCA2b,with resulting accumulation of this intermediate at steadystate. Because lumenal Ca2� binds to sites on E2-P, accumula-tion of E2-P is expected to increase the sensitivity to inhibitionby lumenal Ca2�. The functional effects of mutation Cys318 3Arg are probably caused by the presence of the bulky andhighly charged arginine side chain, because the substitution ofCys318 in SERCA1a with alanine causes little effect on the rateof Ca2� transport (52). Cys318 is located in transmembranesegment M4 near the cytoplasmic boundary, and analysis ofthe crystal structures of SERCA1a shows that the replacementwith arginine is likely to lead to steric clash with residueslocated in M5 and M6 near the Ca2� binding sites. This couldinterfere with conformational changes involving these trans-membrane segments (“a stick in the wheel” effect), therebypreventing the opening of the Ca2� binding pocket toward thelumenal side. In fact, it is possible that the E2-P form accumu-lated during the enzymatic cycle of mutant Cys318 3 Argcontains bound Ca2� (i.e. the E2-PCa2 intermediate indicatedin Scheme 1). Hence, a common explanation of the increasedsensitivity to inhibition by lumenal Ca2� and the reduced de-phosphorylation rate could be a reduced rate of lumenal disso-ciation of Ca2� from E2-PCa2.

DD Mutant Ser186 3 Phe—The major defect in mutantSer186 3 Phe seems to be a reduced rate of the E1�P(Ca2) 3E2-P transition, accounting for the lack of significant Ca2�

transport and ATPase activity. In addition, a 30-fold increase ofthe rate of E2-P dephosphorylation was observed, suggestingthat the underlying cause of both effects could be a destabili-zation of the E2-P state. Ser186 is located close to the highlyconserved TGES segment of domain A, which according to theSERCA1a crystal structures moves from an isolated position inE1Ca2 into close contact with domain P in the E2 conformation(6), a rearrangement that also seems to occur during theE1�P(Ca2) 3 E2-P transition (53–55). It is conceivable thatreplacement of the serine with the bulky phenylalanine couldinterfere profoundly with the interactions at the interface be-tween domains A and P in the E2P conformation, thereby

creating instability of this form.DD Mutants Pro602 3 Leu, Asp702 3 Asn, and Pro895 3

Leu—Mutants Pro602 3 Leu and Asp702 3 Asn also showed aconspicuous reduction of the rate of the E1�P(Ca2) 3 E2-Ptransition, explaining the lack of significant Ca2� transportand ATPase activity. For mutant Pro8953 Leu, the rate of theE1�P(Ca2) 3 E2-P transition was �2-fold reduced, consistentwith more moderately reduced molecular Ca2� transport andATPase activities. Pro602 is located at the “hinge” betweendomains N and P, whereas Asp702 is located in domain P, nearthe phosphorylated Asp351, and Pro895 is located far away atthe lumenal end of transmembrane helix M8 (Fig. 1). Theeffects on the E1�P(Ca2) 3 E2-P transition of mutations ofthese residues, as well as mutation Ser186 3 Phe in domain Adiscussed above, support a global nature of this conformationalchange, involving almost every region of the protein, similar tothe transition between the E1Ca2 and E1 forms for which theSERCA1a atomic structures are known (6). The importance ofPro602 and Asp702 for the E1�P(Ca2) 3 E2-P transition inSERCA2b demonstrated here is consistent with previousfindings for the corresponding mutations, Pro603 3 Leu andAsp703 3 Asn, in SERCA1a (56), whereas Pro895 has not beenpreviously studied by mutagenesis. The reduced rate of phos-phorylation of E1Ca2 to E1�P(Ca2) observed for Pro602 3 Leuand Asp702 3 Asn (Fig. 5) has not yet been reported for thecorresponding SERCA1a mutants, but the formation of phos-phoenzyme was completely abolished in SERCA1a mutantsPro603 3 Gly and Asp703 3 Ala (56, 57), thus indicating thecrucial roles of these residues for the phosphorylation reaction.Pro602 (SERCA2 numbering) is probably critical for a hingebending movement involved in the approach of domains N andP in connection with the transfer of the �-phosphoryl groupfrom ATP to Asp351. Asp702 is conserved among all members ofthe haloacid dehalogenase superfamily to which P-type AT-Pases belong (58). As the corresponding aspartate in phospho-serine phosphatase, Asp702, through its two side chain oxygenatoms, could contribute to ligation of Mg2� required as co-factor for catalysis of the phosphorylation reaction (59, 60).

DD Mutants Thr357 3 Lys, Gly749 3 Arg, and Gly769 3Arg—The lack of phosphorylation of Thr357 3 Lys andGly769 3 Arg from either ATP or Pi is in agreement withprevious findings for SERCA1a mutants Thr357 3 Ala andGly7703 Val (61, 62). Thr357 is located in domain P close to thephosphorylated Asp351 and has been shown to be crucial to ATPbinding in SERCA1a (63). Gly769 is located in transmembranehelix M5 adjacent to a glutamate participating in Ca2� ligation(Glu770 in Fig. 1). It is, therefore, not surprising that substitu-tion of the glycine with arginine interferes with the Ca2� acti-vated phosphorylation from ATP. The abolishment of phospho-rylation from Pi is, however, not a consequence of defectiveCa2� binding, as the latter reaction requires a Ca2�-free en-zyme (cf. Scheme 1). In fact, mutation of the Ca2� ligatingglutamate in M5 leads to increased stability of the E2-P phos-phoenzyme intermediate (64, 65). It may be envisioned that theflexibility introduced by the presence of the glycine is impor-tant for conformational changes involved in the long rangeinteraction between the transmembrane region and the phos-phorylation site occurring in connection with formation of E2-P.Finally, mutant Gly7493 Arg was expressed only at a very lowlevel. The corresponding glycine in SERCA1a has been re-placed by alanine without loss of expression, but substitutionwith leucine reduced enzyme expression (9). For SERCA1a, ithas, furthermore, been shown that the adjacent arginine (cor-responding to Arg750 in SERCA2) is crucial both to the struc-tural and functional integrity of the enzyme (9), probably be-cause it participates in a network of hydrogen bonds and van

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der Waals interactions with residues in the loop between trans-membrane segments M6 and M7 (5). This function would prob-ably be disturbed by the insertion of another arginine insteadof the adjacent glycine, and the low expression level of theGly749 3 Arg mutant may be explained by structural instabil-ity leading to enhanced proteasome degradation.

Why Do These Mutations Cause Disease?—Eight of the 10DD mutants studied here showed no significant Ca2� transportactivity. The change in Ca2� homeostasis leading to DD can,thus, be explained by the hypothesis of haploinsufficiency, i.e.because one of the two alleles encodes a Ca2� pump withimpaired Ca2� transport activity or no activity at all, the totalcellular capacity for Ca2� accumulation in internal stores ismarkedly reduced. Ser9203 Tyr and Pro8953 Leu are the firstDD mutants reported to show significant Ca2� transport activ-ity. For Pro895 3 Leu the molecular Ca2� transport activitywas reduced, in line with the hypothesis of haploinsufficiency.For Ser9203 Tyr, the molecular Ca2� transport activity was infact increased relative to wild-type SERCA2b (Table I). Thedominant DD phenotype produced by mutant Ser920 3 Tyrcould, nevertheless, be caused by haploinsufficiency, becausethe expression level of mutant Ser920 3 Tyr in HEK-293 wassignificantly lower than its corresponding wild-type protein(Fig. 2, A and B) and its contribution to the total cellularcapacity for Ca2� accumulation was markedly reduced (cf. Fig.2C), despite its high intrinsic molecular Ca2� transport activ-ity. The marked reduction in the protein level for Ser9203 Tyrin HEK-293 cells, which could be because of enhanced cellularproteasome-mediated degradation, may also reflect the situa-tion in epidermal cells. It has earlier been proposed that Ca2�

transport activity of the wild-type enzyme encoded by one ofthe alleles is inhibited through protein-protein interactionswith mutant enzyme (38). In addition, it is possible that thewild-type enzyme present in dimeric complexes with mutantSer9203 Tyr becomes more susceptible to cellular degradationthan in its monomeric state. Furthermore, the observed reduc-tion of Ca2� affinity for Ser9203 Tyr to a value similar to thatof SERCA2a may contribute to an abnormal Ca2� homeostasis,thus implying that wild-type SERCA2b with its higher Ca2�

affinity is crucial for normal functioning of epidermal cells.Finally, the observed reduced sensitivity of Ser920 3 Tyr toinhibition by Ca2� accumulated in the lumen appears impor-tant and could contribute to abnormal Ca2� homeostasis andregulation in Darier disease.

Acknowledgments—We thank Dr. D. H. MacLennan (Banting andBest Department of Medical Research, C. H. Best Institute, University ofToronto, Ontario, Canada) for the gift of human SERCA2a and SERCA2bcDNA clones, Dr. F. Wuytack (Laboratory for Physiology, Catholic Uni-versity of Leuven, Belgium) for kindly supplying the SERCA2b-specificpolyclonal antibody, and Dr. R. J. Kaufmann (Genetics Institute, Boston,MA) for providing the expression vector pMT2. We also appreciate theskillful technical assistance of Lene Jacobsen and Karin Kracht (Depart-ment of Physiology, University of Aarhus, Denmark).

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Alain HovnanianLeonard Dode, Jens Peter Andersen, Natalie Leslie, Jittima Dhitavat, Bente Vilsen and

(SERCA2) Mutants by Steady-state and Transient Kinetic Analyses-ATPase (SERCA) 1 and 2 Isoforms and Characterization of Darier Disease2+

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doi: 10.1074/jbc.M306784200 originally published online September 15, 20032003, 278:47877-47889.J. Biol. Chem. 

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