Amino terminus conserved H1 domain is important for calcium ...

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A Domain with Homology to Neuronal Calcium Sensors is Required forCalcium-Dependent Activation of Diacylglycerol Kinase αααα∗

Ying Jiang, Weijun Qian†, John W. Hawes and James P. Walsh‡

Departments of Medicine and Biochemistry and Molecular Biology, Indiana University School ofMedicine, Indianapolis Indiana

*This work was done while Dr. James P. Walsh was a Pfizer Scholar. Additional support wasprovided by a grant from the US Veterans Administration to Dr. James P. Walsh.

†Deceased

‡To whom correspondence should be addressed:Section of Endocrinology and Metabolism (111-E), Roudebush VA Medical Center, 1481 WestTenth Street, Indianapolis, Indiana, 46202.Phone: 317/554-0000 ext. 3073,Fax: 317/554-0004E. mail: [email protected]

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on July 28, 2000 as Manuscript M004914200 by guest on February 12, 2018

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ABSTRACT:

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol produced during stimulus-induced

phosphoinositide turnover and attenuate protein kinase C activation. Diacylglycerol kinase α is

an 82 kDa DGK isoform which is activated in vitro by Ca2+. The DGKα regulatory region

includes tandem C1 protein kinase C homology domains and Ca2+-binding EF hand motifs. It

also contains an N-terminal RVH (recoverin homology) domain which is related to the N-termini

of the recoverin family of neuronal calcium sensors. To probe the structural basis of Ca2+

regulation, we expressed a series of DGKα deletions spanning its regulatory domain in COS-1

cells. Deletion of the RVH domain resulted in loss of Ca2+-dependent activation. Further

deletion of the EF hands resulted in a constitutively active enzyme, suggesting that sequences in

or near the EF hands are sufficient for autoinhibition. Binding of Ca2+ to the EF hands protected

sites within both the RVH domain and EF hands from trypsin cleavage and increased the phenyl-

Sepharose binding of a recombinant DGKα fragment which included both the RVH domain and

EF hands. These observations suggested that Ca2+ elicits a concerted conformational change of

these two domains. A cationic amphiphile, octadecyltrimethylammonium chloride, also

activated DGKα. As with Ca2+, this activation required the RVH domain. However, this agent

did not protect the EF hands and RVH domain from trypsin cleavage. These findings indicate

that the EF-hands and RVH domain act as a functional unit during Ca2+-induced DGKα

activation.

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Introduction

Hydrolysis of phosphatidylinositol 4,5-bisphosphate is a common mechanism of stimulus

transduction (1). Diacylglycerol (DAG) released in this reaction activates protein kinase C

(PKC) and is then rapidly metabolized back to phosphatidylinositol in a series of reactions

initiated by a diacylglycerol kinase (DGK). As such, DGKs attenuate DAG-mediated PKC

activation (2). Recent studies indicate that DGKs are also activated by mechanisms independent

of phosphoinositide turnover (3,4). Diacylglycerol kinases catalyze the ATP-dependent

phosphorylation of sn-1,2-diacylglycerol to form phosphatidic acid (PA), which is also a lipid

mediator (5,6). Several DGK isoforms have been cloned (7). All these sequences share a

homologous catalytic domain and two or three C1 protein kinase C homology domains (7-9).

Some DGKs contain EF hands, which are Ca2+-binding sites (7). These DGKs also have a

domain at their N-termini with homology to the recoverin family of neuronal calcium sensors

(Figure 1). We term this the RVH (recoverin homology) domain. In S-modulin, the frog

orthologue of recoverin, this domain associates with the EF hands to mediate Ca2+-dependent

inhibition of rhodopsin kinase (10).

The varied structures of DGK regulatory domains suggest divergent mechanisms of regulation.

Several studies have shown variation among DGKs with regard to activation by phospholipids,

sphingosine, or Ca2+ (11-15). Kanoh and coworkers have studied DGKα, a Ca2+-activated

isoform highly expressed in oligodendrocytes and thymocytes (16-18). They have shown that

Ca2+ binds the EF hand region of the enzyme and that deletion of the EF hands results in

constitutive enzyme activation (19,20). We have now examined a series of DGKα mutants in

which the RVH and EF hand domains are sequentially deleted. Our results indicate that the


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N-terminal RVH domain is required for Ca2+ to activate this enzyme. In contrast to the

constitutive activation seen with deletion of the EF hands, DGKs with deletions involving only

the RVH domain expressed activity similar to that of wild-type enzyme in the absence of Ca2+.

Sites within both the EF hands and RVH domain were protected from trypsin proteolysis by

Ca2+, indicating that both domains participate in a Ca2+-induced conformational change. A

cationic amphiphile, octadecyltrimethylammonium chloride, markedly stimulated DGKα activity

in vitro. This effect, like Ca2+-dependent activation, was dependent on the RVH domain. The

DGKα RVH domain does not itself bind Ca2+. However it does appear function together with

the EF hands to couple Ca2+-binding to release of EF hand-mediated autoinhibition of DGKα.


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Experimental Procedures:

Materials—Restriction and DNA modifying enzymes were from Promega or Gibco-BRL. A

dideoxy sequencing kit (Sequenase version 2.0) was from US Biochemical. [γ-32P]ATP and

[α-35S]dATP were from DuPont-New England Nuclear. sn-1-Palmitoyl-2-oleoyl

phosphatidylserine (PS) and sn-1-palmitoyl-2-oleoyl phosphatidic acid (PA) were from Avanti

Polar Lipids, Birmingham, AL. sn-1-Palmitoyl-2-oleoyl glycerol (16:0-18:1 DAG) was prepared

by digestion of the corresponding phosphatidylcholine (Avanti) with Bacillus cereus

phospholipase C (21). Octyl-β-D-glucopyranoside (octylglucoside), sodium deoxycholate, N-

hexadecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (C16 sulfobetaine, C16SB), Triton X-

100, Triton X-114, dihexadecylphosphate, diethylenetriaminepentaacetic acid (DTPA), EDTA,

EGTA, leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), pepstatin A, and sorbitan

trioleate were from Sigma. Glutathione agarose beads, thrombin and p-aminobenzamidine-

agarose were from Sigma. Protein A/G agarose beads were from Santa Cruz Biotechnology.

Trypsin (sequencing grade) was from Promega. 2,6-Di-tert-butyl-4-methylphenol (BHT) was

from Aldrich. Octadecyltrimethylammonium bromide was purchased from Aldrich and

recrystallized twice from ethanol/ethyl acetate (1:1, v/v). Octadecyltrimethylammonium chloride

(OTAC) was prepared from the bromide salt by ion exchange over Dowex AG 1-X8 (Bio-Rad).

A chemiluminescent Western blotting detection system was from Pierce. Anti-Flag M2 antibody

was from Eastman Kodak. Reagents for protein assay and electrophoresis were from Bio-Rad.

Diethylaminoethyl cellulose (DE52) ion exchange resin was from Whatman. Q-sepharose Fast

Flow and SP-Sepharose Fast Flow ion exchange resins were from Pharmacia. Silica gel 60

plates were from E. Merck. Oligonucleotides were synthesized by our campus biotechnology

facility.


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Construction of DGKα deletion mutants

DGKα-pBluescript II (SK+), pSRE-DGKα, and pSRE-DGKα ∆196 were provided by Drs. F.

Sakane and H. Kanoh (19,20). To prepare the DGKα ∆40 and ∆87 mutants, PCR amplification

was performed using DGK α-pBluescript II (SK+) as a template. Sense primers (d40,

5’-GCGAATTCCACCATGGCTGAATATCTCCAAGGA-3’ and d87, 5’-GCGAATTCCACC-

ATGGTAAAAAGAGATGTGGT-3’, EcoRI sites underlined) were designed based on the

peptide sequences MAEYLNG (positions 39-45) and TVKRDVV (positions 87-93). Nucleotide

substitutions were introduced to create the initiation codon and Kozak consensus sequence. An

antisense primer (a1, 5’-CTCACTATAGGGCGAATTGG-3’) was from pBluescript II SK+.

Amplification reactions were carried out using d40 plus a1 or d87 plus a1 under the following

condition: 94ºC for 1 min, 55ºC for 1 min, 72ºC for 3 min for 25 cycles and a final elongation

step at 72ºC for 10 min. Amplified fragments were digested with EcoRI and cloned into pUC19.

Expression of diacylglycerol kinases in COS-1 cells

Fragments containing DGKα and the mutant sequences were subcloned into pCDNA3

(Invitrogen). To facilitate quantitation of expression, a FLAG-epitope tag was introduced at

C-terminus of DGKα. Primers (s-nhe1, 5’-CGAATTCTGGACATCGGCTAGCCAAGT-3’,

NheI site underlined; and a-flag1, 5’-GGCTGCAGTCAGCCTGGTCCCTTGTCGTCATCGTC-

TTTGTAGTCCCCTCCGCACAGAAGCCAAAG-3’, stop codon underlined) were designed to

amplify a FLAG-tagged DGKα fragment. PCR amplification was performed at: 94ºC for 45 sec,

58ºC for 1 min and 72ºC for 1 min for a total of 25 cycles with a final elongation step at 72ºC for

10 min. The amplified fragment was cloned into pBluescript KS+. This construct was digested


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with Nhe I and Xho I and the NheI-XhoI fragment inserted into DGKα-pCDNA3 in place of a

460 bp NheI-XhoI fragment at the DGK C-terminus, positions 2031-2491. The FLAG-epitope

was similarly inserted into the truncated DGKα constructs.

COS 1 cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (Gibco-BRL)

supplemented with 10% fetal bovine serum (hyclone), 100 U/ml penicillin, 100 µg/ml

streptomycin, 250 ng/ml amphotericin B. Transfection of COS-1 cells was performed by the

calcium-phosphate method (22). After 48 hours, cells were harvested and lysed by sonication in

ice-cold 20 mM Tris∙HCl, pH 7.4, 250 mM sucrose, 100 mM NaCl, 2.5 mM EGTA, 1 mM

MgCl2, 1 mM DTT, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, 50 µM ATP, and

0.02% Triton X-100. After removal of undisrupted cells by brief centrifugation, the lysates were

centrifuged at 100,000 x g (Beckmann TL-100) for 20 min at 4ºC to pellet membranes. The

resultant supernatants were rapidly frozen in a dry ice/ethanol bath and stored at -70ºC until

assayed. For immunodetection, 5−10 µg of the 100,000 x g supernatants of lysates from COS-1

cells transiently expressing DGKα or the truncation mutants was applied to SDS-PAGE.

Proteins were electrophoretically transferred to nitrocellulose membranes (Schleicher and

Schuell). After blocking in TBS-T buffer containing 5% non-fat dry milk for 1 hour, the

membrane was incubated with anti-Flag M2 antibody (1:2000 in TBS-T) for 1 hour. Membranes

were washed with TBS-T and incubated with HRP-conjugated sheep anti-mouse IgG (1:5000

dilution in TBS-T) for 30 minutes. After washing 4 times in TBS-T buffer, the HRP conjugates

were detected by chemiluminescence.

Expression of DGKα regulatory domain sequences:


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Sequences corresponding to conserved domains in the DGKα regulatory region were prepared as

GST fusions in E. coli. DNA fragments expressing the selected sequences were prepared by

PCR amplification using DGKα-pCDNA3 as a template. Amplification reactions were run at

94ºC for 45 seconds, 49ºC for 45 seconds, and 72ºC for 150 seconds for a total of 28 cycles with

a final elongation step at 72ºC for 10 minutes. Primers GSTN-1 (5’-TGAATTCCAAGGAGAG

GGGGCTG-3’) and GSTC-1 (5’-ACTCGAGTCATTTGTCTTCTGGCCGGCCG-3’) amplified

DGKα:2-110, which encompasses the RVH domain. Primers GSTN-2 (5’-TGAATTCCTGCTA

CTTCTCCCTTC-3’) and GSTC-2 (5’-ACTCGAGTCAATTGTCCTTCAAGGTC-3’) amplified

DGKα:99-202, which encompasses the EF hands. Primers GSTN-3 (5’-AGAATTCCTTGAAG

GACAATGGGCA-3’) and GSTC-3 (5’-TCTCGAGTCAACTGGGATAGATGGAAG-3’)

amplified DGKα:198-336, which encompasses the C1 domains. Primers GSTN-1/GSTC-2,

GSTN-1/GSTC-3, and GSTN-2/GSTC-3 were used to ampilfy DGKα:2-202, DGKα:2-336, and

DGKα:99-336, respectively. The DNA fragments were inserted into the PCRblunt vector and

sequenced. They were then digested with EcoRI and XhoI and inserted into pGEX-4T-3

(Pharmacia). All insertions were in frame with GST.

The GST-fused DGKα-pGEX-4T-3 constructs were expressed in E. coli strain BL21(DE3).

Tranformed cells were grown at 37ºC in LB medium supplemented with 100 µg/ml ampicillin to

an OD at 600 nm of 0.8 and induced with 0.2 mM IPTG for 4 hours at 37ºC or overnight at 25ºC.

Cells were harvested and lysed in 20 mM Tris∙HCl, pH 7.5, 250 mM sucrose, 100 mM NaCl,

1 mM EDTA, 1 mM DTT, 0.5% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml aprotinin and

1 mM PMSF by two passes through a French Press. The lysates were clarified by centrifugation


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at 10,000 x g for 20 minutes. The GST fusions were then purified to apparent homogeniety by

glutathione-agarose affinity chromatogtaphy. When removal of the GST moiety was desired, the

recombinant protein adhering to the glutathione-agarose beads was incubated with 2 IU thrombin

per mg protein at 4ºC overnight. The supernatant was then collected and incubated with

p-aminobenzamidine-agarose at 1:100, v/v ratio for 1 hour to remove the thrombin. The

resultant supernatant was dialyzed against 50 mM Tris∙HCl, pH 7.5, 100 mM NaCl, 250 mM

sucrose and 1 mM DTT. Aliquots were rapidly frozen and stored at -80ºC.

Diacylglycerol kinase assays

The standard DGK assay contained, in volume of 200 µl: 1 mM sodium deoxycholate, 50 mM

triethanolamine∙HCl, pH 7.5, 100 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 0.1 mM (γ-32P) ATP

(100 cpm/pmol), 20 µM sn-1-palmitoyl-2-oleoyl-glycerol (16:0, 18:1 DAG), 1 mM DTT, and

enzyme (11,23). In a typical reaction, an appropriate volume of DAG stock solution (10-20 mM

in CHCl3) was evaporated under a stream of nitrogen in a 16 x 100 mm glass test tube. To the

DAG droplet were added: 50 µl of 4 x assay buffer, 50 µl of 4 x detergent, DTT, water, and

enzyme to a final volume of 180 µl. Stock solutions of 4 x sodium deoxycholate, 10 x

[γ-32P]ATP and 4 x aqueous buffer were as described previously (21). Reactions were initiated

by adding 20 µl of 1 mM [γ-32P]ATP. Reactions were allowed to proceed for 10 min at 25ºC and

terminated by the addition of 3.0 ml of CHCl3/ethanol (2:1 v/v) containing 1.0 mg

dihexadecylphosphate and 1.0 mg sorbitan trioleate. The organic phase was washed 3 times with

2.0 ml of 1.0% HClO4 and 0.1% H3PO4 in H2O/ethanol (4:1, v/v). The volume of the final

organic phase was 2.25 ml. Cerenkov counting 1.2 ml of this organic phase determined

incorporation of 32P into PA. For some assays, mixed micelles of octylglucoside and


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phosphatidylserine (PS) were employed instead of deoxycholate. In these assays, the total

concentration of micelle components, octylglucoside + PS + DAG, was maintained at 25 mM.

Total octylglucoside added to the assays was the sum of micellar and monomeric octylglucoside,

which was calculated as described (24). For purposes of these calculations, the critical micelle

concentration of octylglucoside was assumed to be 25 mM. The DAG concentration in these

assays was 0.5 mM (2 mol%). Other assay components were unchanged. When Ca2+ was added

to assays, the buffer contained 1 mM EDTA instead of EGTA, and the free Mg2+ was maintained

at 1 mM. The total Mg2+ and Ca2+ added were calculated using published stability constants to

give the desired levels of free cations (25). Other assays employing Triton and OTAC or Triton

and C16SB instead of deoxycholate have been described previously (23). All data reported are

averages of at least duplicate determinations which agreed within 10% in all cases. Moreover, all

results are representative of two or more independent experiments performed with completely

independent enzyme preparations.

Ca2+ overlay analyses

Calcium binding was assessed by the 45Ca overlay method of Maruyama et al. (26). 1 µg of

recombinant proteins or immunoprecipitated DGKα truncation mutants were separated by SDS-

PAGE and transferred to nitrocellulose. The membrane was washed free of Ca2+ in 75 ml of 5

mM EGTA, pH 7.0. It was rinsed three times with 10 mM imidazole∙HCl, pH 6.8, 60 mM KCl,

and 5 mM MgCl2, and then incubated for 15 minutes at 25ºC in 30 ml of the same buffer

supplemented with 250 nM 45CaCl2 (1 µCi/ml). The membrane was then rinsed twice with 45%

ethanol, blotted dry, and exposed to X-ray film. The nitrocellulose was stained with amido black

to verify protein transfer. To prepare immunoprecipitates of the DGKα truncation mutants for


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these experiments, COS-1 cells transfected with DGKα or the truncation mutants were extracted

with buffer containing 20 mM Tris∙HCl, pH 7.4, 250 mM sucrose, 100 mM NaCl, 5 mM EGTA,

1 mM NaF, 1 mM MgCl2, 1 mM DTT, 50 µM ATP, 1% Triton X-100, 10 µg/ml leupeptin, 10

µg/ml aprotinin and 1 mM PMSF. The extract was precleared with protein A/G agarose beads to

eliminate nonspecific binding. Anti-FLAG antibody was then added to the together with fresh

protein A/G agarose beads and the mixture incubated at 4°C overnight. The immune complexes

were collected by centrifugation. The precipitate was washed with the above buffer containing

only 0.1% Triton X-100 and the bound proteins eluted into SDS reducing buffer.

Limited proteolysis of DGKα:2-202

Recombinant GST-DGKα:2-202, which includes the RVH and EF hand domains, was expressed

in E. coli and the GST moiety removed as described above. Limited trypsin proteolysis was

performed as described by Rudnicka-Nawrot et al. (27). The reaction contained, in a volume

150 µl, 0.6 mg/ml (90 µg) of DGKα:2-202, 50 mM Tris∙HCl, pH 7.5, 100 mM NaCl, 1 mM

DTT, and either 0.1 mM CaCl2 or 0.1 mM EGTA. The reaction was initiated by adding 1 µg of

trypsin and incubating at 37ºC. Samples (18µl) were withdrawn at various times and quenched

by adding 2 µl of 1 mM Nα-p-tosyl-L-lysine chloromethylketone (TLCK). The products were

analyzed by SDS-PAGE. To examine the effect of OTAC on proteolysis, the reaction mixture

was supplemented with 0.4 mM Triton X-100, 0.4 mM Triton X-114 and 0.2 mM OTAC

(20 mol%) and the trypsin concentration was halved. The Triton mixture was added together

with the OTAC to simulate the conditions under which maximal activity stimulation is observed

(11,23). Triton X-100 and Triton X-114 were also included in the control reactions.


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Peptides from selected time points of the trypsin digests were analyzed by capillary LC-MS using

an ABI 140D solvent delivery system. Samples (5 µl, 2.7 µg protein) were injected directly into

300 µm id fused silica capillaries packed with C18 resin (Vydac). The columns were eluted with

a 60 minute, 2 to 95% gradient of acetonitrile in H2O containing 0.2% isopropanol, 0.1% acetic

acid, and 0.001% TFA. The solvent flow rate was 7 µl/min. The column was eluted directly into

the electrospray ionization source of a Finnigan LCQ mass spectrometer. Nitrogen was used as

the sheath gas at 35 psi and no auxiliary gas was used. Electrospray ionization was conducted

with a spray voltage of 4.8 kV, a capillary voltage of 26 V, and a capillary temperature of 200°C.

Spectra were scanned over an m/z range of 200 to 2000. Base peak ions were trapped using the

quadrupole ion trap and further analyzed both with a high resolution scan performed at an

isolation width of 3 m/z and with collision induced dissociation (CID) scans at a collision energy

of 40.0. Sequences of all eluting peptides were confirmed on the CID scans. Ion currents at m/z

ratios corresponding to the peptide fragments were integrated over the entire peak using Finnigan

Bioworks software provided with the mass spectrometer.

Other Methods

Calcium-dependent binding of recombinant DGKα:2-202 (RVH domain + EF hands, GST

cleaved off) and DGKα:99-202 (EF hands only, GST cleaved off) to phenyl-Sepharose was

assayed by a modification of the procedure of Landar et al. (28). Briefly, 240 µg of recombinant

polypeptide in a total volume of 0.5 ml was loaded onto 1 ml phenyl-Sepharose columns in

50 mM Tris!HCl, pH 7.5, 500 mM NaCl, 1 mM DTT and either 10 mM CaCl2 or 10 mM EGTA.

The columns were eluted with 4 ml of the same buffer and 0.5 ml fractions were collected.


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Purification of DGK activites from testis cytosol, salivary cytosol, NIH 3T3 cells (two activities),

thymus cytosol, and testis membranes has been described (23). Calcium stimulated the activities

from testis cytosol, salivary cytosol, and one of the 3T3 activities (23). Methods used for

expression of DGK activities in Saccharomyces cerevisiae have also been described (23).

Protein concentrations of enzyme preparations were determined by the Bradford method (29).

Thin layer chromatography of DAG and PA was as described previously (21). Concentrations of

phospholipids used in DGK assays were confirmed by determination of organic phosphate (30).

Protein sequences with homology to the DGKα N-terminus were identified by PSI BLAST

searches and aligned using ClustalX (31,32). Structural features of proteins with homology to

DGKs were visualized using RasMol 2.6 (33).


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Results:

Expression of DGKα and its truncation mutants in COS-1 cells

Regulatory domains of Ca2+-activated DGKs contain several conserved regions. These include

EF hands and tandem C1 PKC homology domains (8,9). EF hand-containing DGKs also contain

a 70 amino acid conserved sequence at their N-termini, (34,35). Motif searches revealed that this

domain is related to the recoverin family of neuronal calcium sensors (Figure 1). We thus refer

to this region as the RVH (recoverin homology) domain. The homology between DGKs and

neuronal calcium sensors extends through the EF hands (Figure 1).

To investigate the role of the RVH domain in DGKα regulation, a series of NH2-terminal

deletion mutants was prepared (Figure 2). DGKα ∆40 lacks the first half of the RVH domain

and DGKα ∆87 lacks the entire RVH region. DGKα ∆196 lacks both the RVH domain and the

EF hands. A FLAG epitope attached to the C-termini facilitated detection and quantification of

protein expression. These mutants were expressed in COS-1 cells. Cytosol from COS-1 cells

expressing DGKα or the deletion mutants showed a marked increase in DGK activity as

compared to control cells transfected with vector only. The mutant activities were stable in cell

lysaytes, but were only partially recoverable from DEAE cellulose or mono Q columns. The

100,000 x g supernatants of COS-1 lysates were thus used for all studies. The presence of

protein products with the expected molecular masses was verified by immunoblotting (Figure 2).

Densitometry of the immmunoblots indicated that DGKα ∆40, DGKα ∆87 and DGKα ∆196

were expressed at 1/4, 1/4, and 1/8 the level of wild-type DGKα. COS-1 cells express an

endogenous DGK activity. Transfected cells all expressed activities at least 20-fold over this

background. After subtraction of the COS-1 background and normalization for expression, the


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specific activities of DGKα ∆40 and DGKα ∆87, which lack RVH domains, were not

significantly different from wild-type enzyme (Table 1). However, deletion of the EF hands

(DGKα ∆196) resulted in an 18-fold increase in specific activity. Similar results were obtained

in both the DOC and OG/PS assays. All of the constructs were activated 5 to 10-fold as the

surface concentration of PS in octylglucoside micelles was increased from 10 to 20 mol%

(Table 1). As discussed below, the ∆40, ∆87, and ∆196 mutant activities were also activated by

OTAC and C16SB similarly to several Ca2+-independent DGKs. To obtain further evidence that

the mutants express DGK activity, we also expressed the truncated DGKs in Saccharomyces

cerevisiae. All of the mutants expressed DGK activity in the yeast and, in all cases, the

phosphatidate product comigrated with authentic PA on thin layer chromatograms (data not

shown). As yeast do not possess an endogenous DGK, expression of activity in this background

provides additional confirmation that the mutant DGKs are catalytically competent (23). These

results indicate that all the truncated DGKs express a functional catalytic domain. Activities

expressed by full length DGKα and DGKα ∆196 with the native C-termini were identical to

those expressed by the corresponding epitope tagged constructs, indicating that the FLAG tag

does not appreciably alter DGKα activity.

Activation of DGKα truncation mutants by Ca2+

We examined Ca2+ activation of full-length DGKα and the deletion mutants. Wild-type enzyme

showed significant stimulation by Ca2+, close to maximum activity being achieved with 5 µM

cation (Figure 3). However, none of the mutants, including DGKα ∆40 and ∆87, which retain

the EF hands, showed any Ca2+-dependent activation. To exclude the possibility that deletion of


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RVH domain disrupted the folding of EF hands, we immunoprecipitated DGKα ∆40 and ∆87

with anti-FLAG antibody and performed Ca2+ overlay assays on these immunoprecipitates

(Figure 4). Our results clearly showed that deletion of RVH domain didn’t affect Ca2+ binding.

As described below, deletion of the RVH domain also had no effect on Ca2+ binding to

regulatory domain sequences expressed in E coli. Thus, while Ca2+ binding occurs at the EF

hands, the RVH domains are also required for Ca2+-dependent activation. The activities of the

∆40 and ∆87 mutants were similar to that of wild-type DGKα in the absence of Ca2+. Additional

deletion of the EF hands increased the activity to a level equal to that of fully Ca2+-activated

wild-type enzyme (Figure 3). This suggests that sequences in or near the EF hands inhibit the

catalytic domain and that in full-length DGKα, and that Ca2+ relieves this inhibition.

A Ca2+-induced conformational change involves both the EF hands and the RVH domain

Binding of Ca2+ to recoverin elicits a 45° rotation of the region corresponding to the DGKα RVH

domain relative to the C-terminal EF hands (36). The loss of Ca2+-dependent activation in the

RVH-deleted mutants may reflect loss of a similar conformational change during DGKα

activation. We expressed the RVH domain, EF hands and C1 domains of DGKα as recombinant

proteins in E. coli (Figure 5). Overlay assays indicated that DGKα:2-202, DGKα:99-202,

DGKα:2-336 and DGKα:99-336, all of which include the EF hands, bind Ca2+ (Figure 5). The

RVH domain alone did not bind Ca2+ and was not required for Ca2+ binding (Figure 5). To probe

for a Ca2+-induced conformational change, we performed limited trypsin proteolysis of

DGKα:2-202 (DGKα-RVH+EF) with and without Ca2+. Aliquots of the reaction were stopped

at various times and analyzed by SDS-PAGE (Figure 6). Calcium protected DGKα:2-202 from


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proteolysis (Figure 6). In the absence of Ca2+ (0.1 mM EGTA), the polypeptide was completely

digested within 30 minutes, while in 0.1 mM Ca2+, appreciable full-length polypeptide remained

after 4 hours. These results are not due to an effect of Ca2+ on trypsin. High concentrations of

Ca2+ (> 1 mM) protect trypsin from autolysis and modestly stimulate its activity, but these effects

should be negligible in the 0.1 mM Ca2+ used for these experiments (37,38). Moreover, if Ca2+

were stimulating trypsin activity, the protection we observed would be even more significant. To

identify those sites particularly susceptible to trypsin, peptide fragments in the 5 minute and 4

hour digests were analyzed by capillary LC-MS. Table 2 shows integrated ion currents of the

peptides identified in each digest. In the 5-minute digests, Ca2+ had only small effects on the

appearance of most peptides derived from sequences between the N-terminus and K58.

However, cleavage at K25 was inhibited by Ca2+. Calcium markedly inhibited cleavage at sites

from K89/R90 to the C-terminus. In the 4 hour digests, Ca2+ reduced the level of most

fragments, which is consistent with the fact that appreciable full-length polypeptide was still

present on SDS-PAGE. As was seen in the 5-minute digests, the levels of peptides derived from

cleavage at K89/R90 and beyond were much more dramatically reduced by Ca2+. However, Ca2+

significantly increased the levels of three peptides in this region. One of these, L120-N202,

includes both EF hands, while N126-N202 and M144-N202 encompass the second EF hand only.

Protection of these fragments from further digestion by Ca2+ suggests that both EF hands of

DGKα coordinate Ca2+, as previously concluded by Yamada et al. (39). Overall, Ca2+ inhibited

tryptic cleavage of sites within the RVH domain (K25), in the loop between the RVH domain

and the EF hands (K89/R90), and within the EF hands (multiple sites). Calcium-induced

conformational changes of EF hand proteins, including neuronal calcium sensors, can result in

altered binding to hydrophobic resins (28,40,41). We thus examined whether Ca2+ modulated the


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binding of DGKα-RVH+EF to phenyl-Sepharose. As shown in Figure 7, inclusion of Ca2+

caused DGKα-RVH+EF to be retained by the resin. The peak elution in EGTA was between 1

and 1.5 ml, while in Ca2+ it was between 2 and 3 ml. The EF hands alone (DGKα:99-202) bound

tightly to phenyl-Sepharose, both with and without Ca2+, and were not eluted under the

conditions used for this experiment (data not shown). This suggests exposure of a hydrophobic

surface upon loss of the RVH domain. Overall, these observations are consistent with a Ca2+-

induced conformational change involving both the RVH domain and the EF hands.

Activation of DGKα by OTAC and C16SB

We have previously shown that DGKα is markedly stimulated by the cationic amphiphile, OTAC

(11). This agent stimulated three other Ca2+-dependent DGK activities from testis cytosol,

salivary cytosol, and NIH 3T3 cells to a similar degree (data not shown). Several Ca2+-

independent DGK activities, including an arachidonoyl-DGK from testis membranes, and

cytosolic activities from NIH 3T3 cells and thymus cytosol were only modestly (2- to 6-fold)

activated by OTAC (11,23). Wild type DGKα activity expressed in COS-1 cells was activated

109-fold by OTAC (Table 3). Similar stimulation was seen with the zwitterionic amphiphile,

C16SB, although 15-fold higher concentrations were required to achieve the same activation. In

lysates of COS-1 cells expressing the truncated DGK activities, OTAC and C16SB had much

smaller effects (Table 3). Activation of the mutants was comparable to that seen with Ca2+-

independent DGKs. In the presence of maximally activating concentrations of OTAC, Ca2+ does

not further activate wild-type DGKα (23). These results suggest that OTAC and C16SB have

two effects on DGKs, a nonspecific stimulation seen with all isoforms and an additional

stimulation seen only with Ca2+-activated DGKs. This latter effect, like Ca2+-dependent


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activation, required the RVH domains. OTAC and C16SB thus appear to act through RVH and

EF hand domains to mimic Ca2+-dependent activation of DGKα. We examined whether OTAC,

like Ca2+, protects DGKα:2-202 from trypsin proteolysis. In the absence of Ca2+, OTAC

modestly accelerated the proteolysis of DGKα:2-202 (data not shown). In the presence of

OTAC, Ca2+ no longer protected DGKα:2-202 (Figure 8). Triton alone slightly increased

proteolysis, but the rate was greatly accelerated by OTAC. Inclusion of OTAC/Triton or

C16SB/Triton in the binding and wash buffers of overlay assays had no effect on 45Ca2+ binding

(data not shown). Overall, these results suggest that C16SB and OTAC mimic Ca2+-dependent

DGKα activation by disrupting EF hand-mediated autoinhibition, but do not compete with Ca2+

binding or mimic the Ca2+-induced conformational change. Loss of OTAC and C16SB

stimulation of DGKα activity with deletion of the RVH domain provides independent evidence

of a role for this domain in enzyme activation.


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Discussion:

Our results indicate that Ca2+-dependent diacylglycerol kinase α activation requires an

N-terminal RVH domain with homology to the recoverin family of neuronal calcium sensors.

Several amino acids within the N-terminal region of recoverin that corresponds to the DGK RVH

domain contribute to Ca2+-dependent inhibition of rhodopsin kinase (10). Neuronal calcium

sensors derive from an ancestral 4 EF hand protein, but have a unique structure (42). In contrast

to the dumbbell shaped structure of calmodulin and troponin C, neuronal calcium sensors are

folded into compact globular structures (36,43-45). Neuronal calcium sensors also have short

helices at the N- and C-termini and between EF3 and EF4 that are not present in calmodulin

(Figure 1; positions 8-17, 147-153, and 195-200) (36,43). Guanylyl cyclase activating proteins

and calcineurin B subunits are related to neuronal calcium sensors, but do not align as well in the

regions corresponding to the RVH domain (Figure 1). Consistent with this, the N-terminal helix

of Guanylyl cyclase activating protein-2 has a different orientation than that of recoverin (44).

Alignment of the first 204 amino acids of DGKα with recoverin suggests that many of its unique

features are also present in DGKs (Figure 1). Nonpolar amino acids involved in the interaction

between the N-terminal region of recoverin and its EF hands have homologs in DGKs (Figure 1;

Y70, H73, V74, F78, I100, A101, M104, L120, Y121, and I137 and M144, numbering from the

figure). Our observation that the DGKα EF hands bind phenyl-Sepharose much more tightly

than the combined RVH+EF hand polypeptide may reflect exposure of many of these residues

upon loss of the RVH domain. These similarities suggest that the DGK N-terminal region

resembles recoverin. Neuronal calcium sensors are myristoylated at their N-temini. In Ca2+-free

recoverin, the myristoyl moiety is tucked into a hydrophobic pocket formed by several nonpolar

residues (Figure 1; L13, L27, W30, Y31, F53, I56, Y57, F60, F61, Y98, V99, L102, W116, and


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L120, numbering from the figure) (36). These positions are also nonpolar in DGKα and other EF

hand-containing DGKs, suggesting that, although DGKs are not myristoylated, this pocket is

conserved. Binding of Ca2+ to recoverin results in a 45° rotation of the region corresponding to

the DGKα RVH domain relative to EF hands 3 and 4 (36,46). The two domains pivot about

G108, which is conserved in DGKs. Calcium binding to recoverin also ejects the myristate and

N-terminal helix (36,46). DGKs lack the consensus for N-terminal myristoylation, but align with

neuronal calcium sensors in the region corresponding to the N-terminal helix (Figure 1, positions

8-17). In DGKα, Ca2+ protected K25 from trypsin cleavage. The corresponding amino acid in

recoverin is located in a loop between the first and second helices, far from the C-terminal EF

hands, and becomes less exposed as a result of the Ca2+-induced conformational transition. In

recoverin, sequences corresponding to the ancestral EF1 and EF4 are disabled and it is binding of

Ca2+ to EF3 that elicits the conformational change (46). The two EF hands of DGKs correspond

to the ancestral EF3 and EF4. Overall, these similarities suggest that Ca2+-activation of DGKα

involves a conformational transition of the N-terminal region analogous to that of recoverin.

Consistent with a coupled conformational transition, Ca2+ protected sites within both the RVH

domain and the EF hands from trypsin cleavage.

Deletion of the EF hands from DGKα resulted in a constitutively active enzyme, suggesting that

Ca2+ activation releases an autoinhibitory effect exerted by this region. The presumed

conformational change of the RVH domain and EF hands may cover a region of the EF hands

involved in autoinhibition and/or contribute to Ca2+ induced unfolding of the entire enzyme.

Surprisingly, OTAC, a cationic amphiphile, and C16SB, a zwitterionic amphiphile, also activated

DGKα. As with Ca2+-dependent activation, this effect required the presence of the RVH domain.


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However, OTAC, did not protect the RVH/EF hands polypeptide from trypsin proteolysis,

indicating that it does not elicit the same conformational change as Ca2+. This result provides

additional evidence that the RVH domain plays a critical in DGKα activation. Overall, our

results indicate that functional coupling of the DGKα EF hands to its RVH domain is required to

couple Ca2+ binding to release of the catalytic domain from EF hand-mediated autoinhibition.


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Acknowledgements:

We thank Hui Zong and Dr. Lawrence Quilliam for technical assistance and helpful discussions.

The assistance of Dr. Anna Depaoli-Roach in COS cell expression is gratefully acknowledged.

We also thank Drs. F. Sakane and H. Kanoh for providing DGK constructs and Dr. Peter Roach

for helpful comments on the manuscript.


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Footnotes:

1Abbreviations used include: BHT, 2,6-di-tert-butyl-4-methylphenol; C16SB, N-hexadecyl-N,N-

dimethyl-3-ammonio-1-propanesulfonate; CID, collision induced dissociation; DAG,

diacylglycerol; DGK, diacylglycerol kinase; DTPA, diethylenetriaminepentaacetic acid; OTAC,

octadecyltrimethylammonium chloride; PA, phosphatidic acid; PKC, protein kinase C; PMSF,

phenylmethylsulfonyl fluoride; PS, phosphatidylserine; TLCK, Nα-p-tosyl-L-lysine

chloromethylketone.


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Figure Legends

Figure 1: Alignment of DGKα NH2-termini with neuronal calcium sensors. The first 214 amino

acids of DGKα were aligned with other Ca2+-activated DGKs and with neuronal calcium sensors

using ClustalX. Numbering is according to the DGKα sequence. Neuronal calcium sensors are

subclassified into visinins (recoverins), frequenins, VILIPs, and other NCS proteins (42).

Sequences of a few other 4 EF hand proteins are also shown for comparison. Only some of the

sequences are shown for regions corresponding to the DGKα EF hands. Amino acids conserved

in both neuronal calcium sensors and DGKs are shaded. Residues 8-78 comprise the RVH

domain. EF hands conforming to the Prosite consensus are boxed. The first Zn-coordinating

amino acid of the DGKα C1a domain (H205) is indicated. Guanylate cyclase activating proteins

and calcineurin B subunits did not align as well with the RVH domain and had weaker overall

homology.

Figure 2: Structure and expression of DGKα mutant proteins. Upper Panel) Schematics of

DGKα mutants used in this work are shown. RVH, EF hand, C1, and catalytic domains are

indicated. Lower Panel) Immunoblot analysis of DGKα deletion mutants expressed in COS-1

cells. The 100,000 x g supernatants of extracts of COS-1 cells transfected with the constructs

were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. Proteins were

detected with anti-FLAG M2 monoclonal antibody as described under Experimental Procedures.

1: pCDNA3 vector only, 10µg; 2: DGKα ∆196, 10µg; 3: DGKα ∆87, 10µg; 4: DGKα ∆

40, 10µg; 5: DGKα, 5µg.


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Figure 3: Calcium activation of DGKα deletion mutants. The 100,000 x g supernatants (10µg) of

extracts of COS 1 cells transfected with the constructs were assayed for DGK activity in

octylglucoside micelles containing 20 mol% PS. Background DGK activities of COS 1 cells

transfected with vector only were determined under identical conditions and subtracted. The

activities were normalized for expression level as determined by densitometry of immunoblots.

Calcium concentrations were varied from 0 to 50 µM as described under experimental

procedures. ", DGKα; !, DGKα ∆40; #, DGKα ∆87; $, DGKα ∆196.

Figure 4: Ca2+ overlay of DGKα deletion mutants expressed in COS-1 cells. COS-1 cells

transfected with the DGKα constructs were extracted and immunoprecipitated with anti-FLAG

M2 antibody as described under “Experimental Procedures”. The immunoprecipitated proteins

were separated by SDS-PAGE and transferred to nitrocellulose. A Ca2+ overlay was performed

on the nitrocellulose and bound 45Ca detected by autoradiography. Upper Panel) Ca2+ overlay of

immunoprecipited DGKα and mutants. Lower Panel) immunoblot of immunoprecipitated DGKα

and mutants The arrows are indicate the positions of IgG heavy and light chains. Three

nonspecific bands between 90 and 120 Mr are also seen in the control lane.

Figure 5: Calcium binding to recombinant DGKα regulatory domain sequences. Polypeptides

encompassing different domains of the DGKα regulatory region were prepared as GST-fused

recombinant proteins as described under "Experimental Procedures". 1 µg of the recombinant

proteins were applied to SDS-PAGE, and transferred to nitrocellulose. Calcium binding was


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assessed by 45Ca2+ overlay as described under "Experimental Procedures". Left) Schematic of the

DGKα regulatory region polypeptides employed. The RVH domain, EF hands and C1 domains

are indicated. The N-terminal GST moiety is not shown. Upper Right) Coomassie blue stain of

expressed polypeptides. Lower Right) 45Ca2+ overlay of expressed polypeptides.

Figure 6: Effect of Ca2+ on limited trypsin proteolysis of DGKα-RVH+EF. DGKα-RVH+EF

(DGKα:2-202) was prepared as a GST fusion and the GST moiety removed as described under

"Experimental Procedures". Limited trypsin digestion was performed both with and without

Ca2+ (0.1 mM CaCl2 versus 0.1 mM EGTA). Aliquots of the reaction mixture were quenched at

various time points and the products analyzed by SDS-PAGE with Coomassie staining. Details

of the reaction conditions are given under "Experimental Procedures". Upper Panel) shows the

time course of digestion in 0.1 mM Ca2+. Lower Panel) shows the same experiment with the

Ca2+ replaced by 0.1 mM EGTA. Arrows indicate the position of uncut DGKα:2-202.

Figure 7: Phenyl-sepharose binding of DGKα-RVH+EF. Recombinant DGKα:2-202 which

includes both the RVH domain and EF hands was prepared as a GST fusion and the GST moiety

removed. It was then passed over phenylsepharose in either 10 mM CaCl2 or 10 mM EGTA

(28). Fraction 1 is part of the included volume and elution begins with fraction 2. The fractions

were analyzed by SDS-PAGE with Coomassie staining. This experiment has been repeated

twice with identical results. Upper panel: 10 mM EGTA. Lower panel: 10 mM CaCl2.

Figure 8: Effect of OTAC on limited proteolysis of DGKα-RVH+EF. DGKα-RVH+EF

(DGKα:2-202) was subjected to limited trypsin proteolysis in 0.1 mM Ca2+ and the products


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analyzed as described in the legend to Figure 6. The trypsin concentration was halved because of

rapid proteolysis induced by OTAC. Upper Panel) Trypsin digestion of DGKα:2-202 in 0.1 mM

Ca2+ only. Middle Panel) Digestion in 0.1 mM Ca2+, 0.5 mM Triton X-100 and 0.5 mM Triton

X-114. Lower Panel) Digestion in 0.1 mM Ca2+, 0.2 mM OTAC, 0.4 mM Triton X-100 and 0.4

mM Triton X-114. Arrows indicate the position of uncut DGKα:2-202.


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Table1

Activities of Wild-type DGKαααα and Deletion Mutants

Expression Specific Activity

relative to WT nmol/min/mg/relative expression

DOC assay 10 mol% PS 20 mol% PS

WT DGKαααα 1.00 4.83 .71 5.6

DGKαααα ∆∆∆∆40 0.25 5.44 .20 3.4

DGKαααα ∆∆∆∆87 0.25 13.56 .88 8.0

DGKαααα ∆∆∆∆196 0.125 87.84 20.6 77.6

Assays were performed on 100,000 x g supernatants of lysates from COS-1 cells transiently expressing

the DGK constructs. These were assayed for DGK activity by the deoxycholate method and by the

octylglucoside/phosphatidylserine method with both 10 mol% and 20 mol% PS. All activities are

corrected by subtracting the background DGK activity, determined under identical assay conditions, of a

lysate of COS-1 cells transfected in the same experiment with the pCDNA3 vector. Expression levels of

DGKα and its mutants in COS-1 cell were determined by densitometry of immunoblots using anti-FLAG

M2 antibody (Eastman Kodak). In all cases, the majority of the anti-FLAG immunoreactivity was in the

100,000 x g supernatant. Immunoreactivity associated with the pellets was estimated from immunoblots

as follows: WT DGKα, <10%; DGKα ∆40, 15%; DGKα ∆87, 15%; DGKα ∆196, 25% (data not

shown). Details of the methods are given in the Experimental Procedures section. All data are averages

of duplicate determinations which, in all cases, agreed within 10%. Similar results were observed in two

additional, independent experiments.


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Table 2

Tryptic Fragments of DGKαααα RVH/EF Hands Domain

FragmentCalculated

massObserved

mass5 minutes

EGTA5 minutes

Ca2+4 hoursEGTA

4 hoursCa2+

total ion current

4-18 1687.9 1687.8 1.370 0.950 7.310 1.500

6-18 1402.7 1402.0 7.630 7.190 140.000 28.700

19-25 920.4 920.4 0.547 10.500 4.200

19-26 1048.5 1049.4 0.522 0.117

26-32 787.5 788.0 0.570 17.200 4.910

27-32 659.4 659.8 7.460 1.270

33-58 2997.4 2997.2 5.020 4.140 108.000 33.400

59-89 3519.7 3520.0 0.740 8.450 6.140

59-90 3675.8 3676.4 2.890 0.369 30.000 9.880

90-119 3451.7 3452.0 0.046 1.740 0.420

91-119 3295.6 3296.0 1.460 23.200 1.410

91-125 4058.9 4058.4 1.030 0.210 1.900 0.950

114-119 783.4 783.4 0.250 17.500 1.470

120-125 781.4 781.4 14.400 0.743

120-136 1966.9 1966.2 13.800 11.400 1.090

120-143 2852.4 2853.6 10.600 0.380

120-202 9458.7 9459.2 9.410 3.270 1.620 7.490

126-136 1203.6 1203.4 4.270 65.500 3.300

126-143 2089.1 2090.0 4.440

126-202 8695.4 8696.1 2.140 0.186 0.570 0.980

137-143 903.5 904.0 6.540 55.800 4.950

144-164 2596.2 2596.2 55.000 0.686 662.000 24.100

144-202 6624.3 6624.4 0.615 0.294 0.800

165-181 1895.9 1896.8 2.390 0.246 207.000 4.400

182-200 1939.1 1939.8 11.600 134.000 8.350

Recombinant DGKα:2-202 was digested with trypsin as described in the legend to Figure 6. Samples of the digests

from the 5 minute and 4 hour time points were analyzed by capillary HPLC/MS/MS as described in the

"Experimental Procedures" section. The table shows the fragments identified (sequence numbering as in Figure 1),

the predicted and observed masses, and the integrated ion currents observed in full MS mode.


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Table 3

Activation of DGKαααα Mutants by OTAC and C16SB

OTAC

fold increase

C16 SB

fold increase

WT DGKαααα 109 137

DGKαααα ∆∆∆∆40 3.4 7.2

DGKαααα ∆∆∆∆40 2.0 6.2

DGKαααα ∆∆∆∆196 2.2 5.2

The 100,000 x g supernatants (10µg) of extracts of COS-1 cells transfected with the constructs were

assayed for DGK activity in Triton micelles with and without 20 mol% OTAC or C16SB as described

under "Experimental Procedures". Background DGK activities from COS-1 cells transfected with

pCDNA3 vector only were subtracted prior to determination of ratios. Activity is reported as fold

increase of activity in Triton with 20 mol% OTAC (or C16SB) versus activity in Triton only. Data are

from duplicate determinations, which agreed within 10% in all cases. Similar results were observed in

two additional, independent experiments


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Diacylglycerol Kinases

DGKαααα Pig P20192 6 G L I S P S D F A Q L Q K Y M E Y S T K K V S D V L K L F E D G G D A I G Y E G F Q Q F L K I Y L E V D S V P S H L S L A L F Q S F 78DGKββββ Rat P49621 8 A H L S P S E F S Q L Q K Y A E Y S T K K L K D V L E E F H G N N Q T I D F E G F K L F M K T F L E A E - L P D D F T A H L F M S F 87DGKγγγγ Human P49619 7 V S L T P E E F D Q L Q K Y S E Y S S K K I K D A L T E F N E G H E P I S Y D V F K L F M R A Y L E V D - L P Q P L S T H L F L A F 79DGK-1 Nematode Q03603 1 M L L S P E Q F S R L S E Y A A Y S R R K L K D M L S D F Q Q D F K T I N I D G F R A F L I D Y F G A D - L P S D L V D Q L F L S F 90

VisininsRecoverin Human P35243 7 G A L S K E I L E E L Q L N T K F S E E E L C S W Y Q S F L K D C P - - T G R I T Q Q Q F Q S I Y A K F F P D T - D P K A Y A Q H V F R S F 73S26 Bullfrog O73924 7 G A L S K E I L E D L K M N T K Y S E E E L F N W Y E S F K K Q C P - - D G K I T R P D F E K I Y A N F F P N S - D P K T Y A R H V F R S F 73Visinin Chick P22728 7 S A L S R E V L Q E L R A S T R Y T E E E L S R W Y E G F Q R Q C P - - D G R I R C D E F E R I Y G N F F P N S - E P Q G Y A R H V F R S F 73

Frequenins

NCS-1 Chick P36610 6 S K L K P E V V E E L T R K T Y F T E K E V Q Q W Y K G F I K D C P - - S G Q L D A A G F Q K I Y K Q F F P F G - D P T K F A T F V F N V F 72Freqenin Drosophila P37236 6 S K L K Q D T I D R L T T D T Y F T E K E I R Q W H K G F R K D C P - - N G L L T E Q G F I K I Y K Q F F P Q G - D P S K F A S L V F R V F 72NCS-1 S cerev. Q06389 6 S K L S K D D L T C L K Q S T Y F D R R E I Q Q W H K G F L R D C P - - S G Q L A R E D F V K I Y K Q F F P F G - S P E D F A N H L F T V F 72

VILIPSHippocalcin Human P41211 S K L R P E M L Q D L R E N T E F S E L E L Q E W Y K G F L K D C P - - T G I L N V D E F K K I Y A N F F P Y G - D A S K F A E H V F R T F 72VILIP 1 Human P42323 6 S K L A P E V M E D L V K S T E F N E H E L K Q W Y K G F L K D C P - - S G R L N L E E F Q Q L Y V K F F P Y G - D A S K F G Q H A F R T F 72NCS Homolog Drosophila P42325 6 S K L K P E V L E D L K Q N T E F T D A E I Q E W Y K G F L K D C P - - S G H L S V E E F K K I Y G N F F P Y G - D A S K F A E H V F R T F 72

Other NCS ProteinsCalsenilin Human Q9Y2W7 V R H Q P E G L D Q L Q A Q T K F T K K E L Q S L Y R G F K N E C P - - T G L V D E D T F K L I Y A Q F F P Q G - D A T T Y A H F L F N A F 138C16H3.1 Nematode Q94172 E R Q T P P S I D Y L I E I T N F S K R E I Q Q L Y R S F K E L W P - - I G T V D L E Q F Q L I Y A S I F P N G - D S K G Y A E L V F K N I 88C54E10.2 Nematode O45313 I G V Q P P S L E Q L T Q R T Q F S P K W I K Y M Y A K F K N E C P - - T G K M K E E E F R N L L A S I I A P E K A T D Q Y I S R L F T A F 158

Other Proteins

GCAP1 Human P43080 1 M G N V M E G K S V E E L S S T E C H Q W Y K K F M T E C P - - S G Q L T L Y E F R Q F F G L K N L S P - S A S Q Y V E Q M F E T F 63F30A10.1 Nematode Q93640 G V F T R E Q L D E Y Q D C T F F T R K D I I R L Y K R F Y L A I T T L T F E E V E K M - - - - - - P E L K E N P F K R R I C E V F 202Calcineurin B Human P06705 1 M G N E A S Y P L E M C S H F D A D E I K R L G K R F K K L D L D N S G S L S V E E F M S L - - - - - - P E L Q Q N P L V Q R V I D I F 62Calmodulin Human P02953 1 M A D Q L T E E Q I A E F K E A F S L F D K D G D G T I T T K E L G T V M R S L G Q N P - T E A E L Q D M I N E V - 56

DGKαααα Q T S Y C S E E T V K R D V V C L S D V S C Y F S L L E - G G R P E D K L E F T F K L Y D T D R N G I L D S S E V D R I I I Q M M R M A E Y L D W - - - - - 150DGKββββ S P A N T C F P E V I H L K D I V C Y L S L L E - G G R P E D K L E F M F R L Y D T D G N G F L D S S E L E N I I G Q M M H V A E Y L E W - - - - - 189DGKγγγγ P R S S S S E S P V V Y L K D V V C Y L S L L E - T G R P Q D K L E F M F R L Y D S S E N G L L D Q A E M D C I V N Q M L H I A Q Y L E W - - - - - 216DGK-1 V A N N D S Q E P R I P L K P L I C T L S L L E - A D T P E N K L D V V F H V Y D S D G N G F L D K S E I D G I I E Q M M N V A R Y Q Q W - - - - - 226Recoverin - - - - - - - D S N L D G T L D F K E Y V I A L H M T T - A G K T N Q K L E W A F S L Y D V D G N G T I S K N E V L E I V M A I F K M I T P E D V K L L P D 143NCS-1 - - - - - - - D E N K D G R I E F S E F I Q A L S V T S - R G T L D E K L R W A F K L Y D L D N D G Y I T R N E M I D I V D A I Y Q M V G N - - T V E L P E 140Hippocalcin - - - - - - - D T N S D G T I D F R E F I I A L S V T S - P G R L E Q K L M W A F S M Y D L D G N G Y I S R E E M L E I V Q A I Y K M V S S - - V M K M P E 140Calsenilin - - - - - - - D A D G N G A I H F E D F V V G L S I L L - R G T V H E K L K W A F N L Y D I N K D G Y I T K E E M L A I M K S I Y D M M G R - - H T Y P I L 206GCAP1 - - - - - - - D F N K D G Y I D F M E Y V A G L S L V L - K G K V E Q K L R W Y F K L Y D V D G N G C I D R D E L L T I I R - A I R T I N P C S D - - - - - 127Calcineurin B - - - - - - - D T D G N G E V D F K E F I E G V S Q F S V K G D K E Q K L R F A F R I Y D M D K D G Y I S N G E L F Q V L K - M M V G N N L K D - - - - - - 126Calmodulin - - - - - - - D A D G N G T I D F P E F L T M M A R K M K D T D S E E E I R E A F R V F D K D G N G Y I S A A E L R H V M T N L G E K L T D E E - - - - - - 121

C1a>>>DGKαααα D V S E L R P I L Q E M M K E I D Y D G S G S V S L A E W L R A G A T T V P L L V L L G L E M T - L K D N G Q H M W R P K R F P R 734DGKββββ D V T E L N P I L H E M M E E I D Y D R D G T V S L E E W I Q G G M T T I P L L V L L G L E N N - V K D D G Q H V W R L K H F N K 801DGKγγγγ D P T E L R P I L K E M L Q G M D Y D R D G F V S L Q E W V H G G M T T I P L L V L L G M D D S G S K G D G G H A W T M K H F K K 791DGK-1 D T I E L E Q V I R Q M M V D I D Y D N D G I V S F D E W R R G G L T N I P L L V L L G F D T E - M K E D G S H V W R L R H F T K 827Recoverin D E N T P E K R A E K I W K Y F G K N D D D K L T E K E F I E G T L A N K E I L R L I Q F E P Q K V K E K M K N A 200NCS-1 E E N T P E K R V D R I F A M M D K N A D G K L T L Q E F Q E G S K A D P S I V Q A L S L Y D G L V 190Hippocalcin D E S T P E K R T E K I F R Q M D T N N D G K L S L E E F I R G A K S D P S I V R L L Q C D P S S R S Q F 193Calsenilin R E D A P A E H V E R F F E K M D R N Q D G V V T I E E F L E A C Q K D E N I M S S M Q L F E N V I 256GCAP1 T T M T A E E F T D T V F S K I D V N G D G E L S L E E F I E G V Q K D Q M L L D T L T R S L D L T R I V R R L Q N G E Q D E E G A D E A A E A A G 201Calcineurin B - - T Q L Q Q I V D K T I I N A D K D G D G R I S F E E F C A V V G G L D I H K K M V V D V 170Calmodulin - - - - - - - - V D E M I R E A D I D G D G Q V N Y E E F V Q M M T A K 149

<25aa>

<7aa><15aa><8aa>

127

57

7374

6364

73

207

139

10 20 30

122

144144141

128

151190 <548 aa>

<510 aa><537 aa>

798880

217227

91<70 aa>

6

<15aa>

912271

<520 aa>

128

12011010090

190 200 210

50 60 70

15014013080

160 170 180

<69 aa>

<35 aa>

Figure 1

by guest on February 12, 2018 http://www.jbc.org/ Downloaded from

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DGKαααα WT

DGKαααα ∆ ∆ ∆ ∆40

DGKαααα ∆ ∆ ∆ ∆87

DGKαααα ∆ ∆ ∆ ∆196

Catalytic DomainC1EFHands

RVH

Figure 2


http://www.jbc.org/

calcium concentration (µµµµM)

0 10 20 30 40 50

DG

K a

ctiv

ity

nm

ol/m

in/m

g/e

xpre

ssio

n

0

20

40

60

80

100

120

140Figure 3


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204120

80

50.4

33.9

29.2

20412080

50.4

33.9

29.2

21.6

CO

NT

@ @@@19

6

WT

@ @@@40

@ @@@87

Figure 4


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DGK αααα:2-110

DGK αααα:2-202

DGK αααα:99-202

DGK αααα:99-336

DGK αααα:198-336

DGK αααα:2-336

RVHdomain

EFhands

C1 domain

97.4

66.2

45

31

21.5

14.4

21.6

29.2

33.9

50.4

80120204

GS

T-D

GK

α ααα:2

-110

GS

T -

DG

Kα ααα

:2-2

02

GS

T -

DG

Kα ααα

:2-3

36

GS

T -

DG

Kα ααα

:99-

202

GS

T -

DG

Kα ααα

:99-

336

GS

T -

DG

Kα ααα

:198

-336

Figure 5


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97.466.245

31

21.5

97.4

14.4

66.245

14.4

21.5

31

0 5 10 20 40 60 120 240

Figure 6


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EGTA

Calcium

1 2 3 4 5 6 7 8

Fraction Number

Figure 7


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45

31

21.5

14.4

14.4

21.5

31

45

21.5

45

31

14.4

0 10’5’ 30’ 60’

Figure 8


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Ying Jiang, Weijun Qian, John W Hawes and James P Walshcalcium-dependent activation of diacylglycerol kinase alpha

A domain with homology to neuronal calcium sensors is required for

published online July 28, 2000J. Biol. Chem.

10.1074/jbc.M004914200Access the most updated version of this article at doi:

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Amino terminus conserved H1 domain is important for calcium ...

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