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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
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DGKαααα WT
DGKαααα ∆ ∆ ∆ ∆40
DGKαααα ∆ ∆ ∆ ∆87
DGKαααα ∆ ∆ ∆ ∆196
Catalytic DomainC1EFHands
RVH
Figure 2
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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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