Volume 270,
Number 10,
Issue of March 10, 1995 pp. 5495-5505
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Identification of
Amino Acid Residues of Human Phospholipase C
1 Essential for
Catalysis (*)
(Received for publication, December 14, 1994; and in revised form, December 29, 1994)
Hwei-Fang
Cheng
(1),
Meei-Jyh
Jiang
(2),
Chih-Lin
Chen
(2),
Su-Min
Liu
(2),
Li-Ping
Wong
(2),
Jon
W.
Lomasney
(3),
Klim
King
(2)(§)From the
(1)Department of Health, National
Laboratories of Foods and Drugs, Executive Yuan, Taipei 115, Taiwan,
the
(2)Institute of Biomedical Sciences, Academia
Sinica, Taipei 115, Taiwan, Republic of China, and the
(3)Feinberg Cardiovascular Research Institute,
Northwestern University Medical School, Chicago, Illinois 60611-3008
ABSTRACT
- INTRODUCTION
- EXPERIMENTAL PROCEDURES
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
In vitro single point mutagenesis, inositol
phospholipid hydrolysis, and substrate protection experiments were used
to identify catalytic residues of human phosphatidylinositide-specific
phospholipase C
1 (PLC1) isolated from a human aorta cDNA
library. Invariant amino acid residues containing a functional side
chain in the highly conserved X region were changed by in vitro mutagenesis. Most of the mutant enzymes were still able to
hydrolyze inositol phospholipid with activity ranging from 10 to 100%
of levels in the wild type enzyme. Exceptions were mutants with the
conversion of Arg
to Leu (R338L), Glu
to
Gly (E341G), or His
to Leu (H356L), which made the enzyme
severely defective in hydrolyzing inositol phospholipid. Phospholipid
vesicle binding experiments showed that these three cleavage-defective
mutant forms of PLC1 could specifically bind to
phosphatidylinositol 4,5-bisphosphate (PIP
) with an
affinity similar to that of wild type enzyme. Western blotting analysis
of trypsin-treated enzyme-PIP complexes revealed that a
67-kDa major protein fragment survived trypsin digestion if the wild
type enzyme, E341G, or H356L mutant PLC1 was preincubated with 7.5
µM PIP, whereas if it was preincubated with 80
µM PIP, the size of major protein surviving
was comparable to that of intact enzyme. However, mutant enzyme R338L
was not protected from trypsin degradation by PIP binding.
These observations suggest that PLC1 can recognize PIP
through a high affinity and a low affinity binding site and that
residues Glu and His are not involved in
either high affinity or low affinity PIP binding but rather
are essential for the Ca
-dependent cleavage activity
of PLC.
INTRODUCTION
Phospholipase C hydrolyzes inositol phospholipids into
diacylglycerol and inositol 1,4,5-trisphosphate (IP
), (
)a process that constitutes a major pathway for
receptor-coupled signaling at the plasma membrane of most eukaryotic
cells. Both diacylglycerol and IP function as important
second messengers that activate protein kinase C and mobilize
intracellular Ca, respectively, thus triggering
multiple enzymatic cascades to regulate cellular functions including
cell growth and neuronal activity(1) . Phospholipase C exists
as isoenzymes (PLC
, PLC
, and PLC)(2) . PLC
is activated following phosphorylation by nonreceptor or receptor
protein tyrosine kinase activities(3, 4) , whereas
PLCs are regulated by 
subunits of G proteins (Gq family) (5, 6, 7) or by
subunits(8, 9, 10) . How PLC1 is
regulated still remains to be determined.
Although PLC isozymes
differ in the way they are regulated, they have similar enzymatic
properties(2) . All three members of the PI-PLC family are able
to recognize phosphatidylinositol (PI), phosphatidylinositol
4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate
(PIP) and to carry out the Ca-dependent
hydrolysis of these inositol phospholipids. Comparison of the amino
acid sequences of all three isoforms reveals that PI-PLCs are highly
conserved in two distinct regions designated X and Y(2) .
Structural integrity of the highly conserved X and Y region is
essential for a functional catalytic core, as partial deletion of
either the X or Y region sequence in PLC1 (11, 12) or PLC (13, 14) inactivates the enzyme. The intervening
peptide connecting the X and Y regions is not essential for the
hydrolytic properties of either PLC or PLC1, as partial
deletion of these sequences in PLC1 (14) or PLC2 (13) or trypsin cleavage of this sequence in PLC1 (11) does not inactivate the truncated enzymes. The first 60
NH-terminal residues of the PLC1 sequence are not
essential for Ca-dependent catalysis, but are
required for the enzyme to hydrolyze PIP in a processive
manner (15) . A subfamily-specific sequence of 400 amino acid
residues in the family is located between the X and Y regions and
is characterized by src homology domains (SH2 and SH3), which
are essential for the activation of PLC by tyrosine protein
kinases(3) . Partial deletion of either the NH terminus or COOH terminus of PLC does not affect its
catalytic activity, but the enzyme loses its ability to be activated by
G proteins(10, 16) . These observations strongly
indicate that residues essential for specific substrate recognition and
Ca-dependent cleavage can be identified among those
conserved residues located in the X and Y regions.
To identify
specific amino acid residues involved in catalysis, we isolated cDNA of
the smallest PLC from human aorta, expressed it in Escherichia
coli, and purified the protein. To minimize structural disturbance
of the enzyme and to facilitate genetic analysis of the role of
PLC1 in cellular function, we used single amino acid residue
substitution mutagenesis to evaluate the contribution of each conserved
residue in PI-PLC to its catalytic function. To narrow down the number
of potential residues involved in catalysis, only those residues
containing a functional side chain and which are invariant in both
prokaryotic and eukaryotic PI-PLC were subjected to base substitution
mutagenesis. The mutant enzymes were assayed for their abilities to
hydrolyze PI and PIP, to bind inositol phospholipid, and to
form trypsin-resistant enzyme-substrate complexes. We demonstrate that
His and Glu, located in the X conserved
region of PLC1, are essential for the
Ca-dependent hydrolysis of PIP rather
than for substrate binding.
EXPERIMENTAL PROCEDURES
Isolation and Sequencing of cDNA Clones
A 0.6-kb
DNA fragment spanning from nucleotide 1194 to 1841 of the rat C6 glioma
cell PLC1 gene was obtained using forward primer
5`-CAGCACGGAGGCCTACATCC-3` and reverse primer
5`-ACAAAACCATTTCCTGATTCTTG-3` to amplify cDNA of C6 glioma
cells(17) . This 0.6-kb fragment was
[
P]dCTP labeled by the random priming DNA method
and used to screen 10
phage plaques from a human aortic
gt11 cDNA library (CLONTECH). Duplicate nitrocellulose filter
papers were hybridized at 42 °C in 50% formamide, 1% dextran
sulfate and washed at 55 °C in 1 
SSC. DNA sequences were
determined by the dideoxy chain termination method, using a
primer-directed sequencing strategy and dye terminator method on
purified double-stranded gt11 DNA template.
Subcloning of PLC1
The PLC1 coding gene
was isolated from gt11 recombinant phage and subcloned into the EcoRI site of pTZ19R. EcoRI digestion of the
gt11 PLC1 cDNA clones results in 2.0- and 0.7-kb fragments
corresponding to cDNA sequences coding for the NH terminus
and COOH terminus of PLC1. These two fragments were subcloned
separately into the EcoRI site of pTZ19R to obtain constructs
pTZ19R5`PLC and pTZ19R3`PLC, respectively.
Construction of Expression Vector
To remove the
5`-untranslated region and construct a BamHI sequence 5` to
the initiation codon, a set of oligo DNA primers was used to perform
PCR using the cloned cDNA as template. The forward primer
(5`-GGGGGATCCATGGACTCGGGCCGGGACTT-3`) contained a BamHI
sequence (GGATCC) followed by a sequence identical to the PLC1
coding sequence spanning from nucleotide 95 to 114. The reverse primer
spans from nucleotide 624 to 599 (5`-TCCACCTGGATGTTGAGCTCCTTCAG-3`).
This set of primers was used to amplify a PLC1 cDNA fragment that
covers the first 529 base pairs (from nucleotide 95 to 624) of human
PLC1 cDNA coding sequence including the BglII site. The
amplified PCR product was blunt ended with T4 DNA polymerase and
inserted into the blunt-ended EcoRI site of pTZ19R to obtain
pTZ19B5`500plc. To construct pTZ19RB5`PLC which harbors the PLC1
coding sequence from the initiation codon to the end of its internal EcoRI site (nucleotide 1945) with a BamHI sequence 5`
to the initiation codon, the BglII/HindIII DNA
fragment from pTZ19R5`plc was isolated and inserted into the BglII/HindIII-digested pTZ19R600plc. To obtain the
full-length PLC1 gene without its 5`-untranslated region, the EcoRI fragment containing the remaining part of the
COOH-terminal coding sequence and its 3`-untranslated sequence was
isolated from pTZ19R3`plc and inserted into the EcoRI site of
pTZ19RB5`plc. The BamHI fragment from pTZ19RhPLC1 covers
the entire coding region, and the 3`-untranslated sequence of human
PLC1 was inserted into the BamHI site in the polylinker
region of pRSETA expression vector (Invitrogen) for expressing the
cloned PLC1 in bacteria E. coli BL21 (DE3) pLys. The
orientation of the insertion was determined by restriction mapping with EcoRI/BglII. In this vector (pRSETAplc), the
transcription of PLC1 cDNA in E. coli BL21 (DE3) pLys is
directed by the T7 promoter.
Expression of Human PLC1 in E. coli
E.
coli BL21 (DE3) pLys cells harboring pRSETAplc plasmid were grown
at 30 °C in 2 liters of LB medium containing 100 µg/ml
ampicillin. When the OD
of the culture reached 1.0, 20 ml
of 0.1 M isopropyl 1-thio--D-galactopyranoside
was added, and the culture was incubated for an additional 2 h. Cells
were harvested by centrifugation and resuspended in 10 ml of ice-cold
lysis buffer containing 50 mM sodium phosphate, pH 8.0, 0.1 M KCl, 5 mM EDTA, 5 mM EGTA, 10 µM phenylmethylsulfonyl fluoride, and 0.1% Tween 20. All subsequent
steps were performed at 4 °C. Cells were lysed by five cycles of
20-s pulses sonication with 40-s cooling intervals in between in an MSE
sonicator. The cell lysate was then cleared by centrifugation at 15,000
g for 30 min, and the supernatant fraction (10 ml of
33.8 mg/ml protein) was applied directly onto a column of 1 ml of
Ni-NAT agarose (Qiagen) equilibrated with a buffer of
50 mM sodium phosphate, pH 8.0, 0.1 M KCl, 0.1% Tween
20, and 10 µM phenylmethylsulfonyl fluoride (buffer A).
The column was washed with 40 ml of buffer A containing 15 mM imidazole, then eluted with 10 ml of the same buffer containing
100 mM imidazole; eluent was collected as 0.5 ml/fraction. The
active fractions were identified by PI hydrolysis assay and then
pooled. The partially purified PLC1 from the
Ni-NAT column was concentrated by an Amicon
Centriprep-30 concentrator, diluted 10-fold with lysis buffer, and then
applied directly to 1 ml of a heparin-Sepharose CL-6B column (Pharmacia
Biotech Inc.) preequilibrated with lysis buffer. After being washed
with 10 ml of the same buffer, PLC1 was eluted by a 50-ml linear
salt gradient from 0.1 to 0.5 M KCl in 50 mM sodium
phosphate, pH 8.0, 0.1% Tween 20, 5 mM EDTA, and 5 mM EGTA. The active fractions were identified by PI hydrolysis
activity or Western blotting and were pooled.
Protein Determination
The protein concentration
was determined by the method of Bradford(18) , and bovine serum
albumin (BSA) was used to calibrate the assays.
Immunoblotting Analysis
PLC1 protein was
separated in 10% SDS-polyacrylamide gels and then electrophoretically
transferred to nitrocellulose membrane in blotting buffer (48 mM Tris-HCl, pH 9.2, 39 mM glycine, 0.037% SDS, and 20%
methanol). The membrane was blocked with 5% skim milk in 20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 0.2% Tween 20 (TBS-T
buffer) for 1 h at room temperature and then rinsed with two changes of
TBS-T followed by one 15- and two 5-min washes in the same buffer.
Membrane was then incubated with mixed monoclonal anti-PLC1
antibody (0.2 µg/ml in TBS-T) at 4 °C for 14 h, rinsed and
washed as before, and then incubated with peroxidase-labeled sheep
anti-mouse antibody (0.055 µg/ml in TBS-T) for 1 h at room
temperature. The membrane was washed and rinsed as before, and
PLC1 proteins were detected by Amersham ECL detection system. The
quantity of PLC1 protein was determined by reading the density of
bands on a densitometer.
RNA Analyses
Multiple human tissue blots were
obtained from CLONTECH. [P]dCTP-labeled BamHI fragment coding for full-length human PLC1 was used
to probe the amount of transcripts. Hybridization was carried out as
recommended by the supplier.
Construction of Point Mutations in Human
PLC1
Site-directed point mutagenesis by a two-stage PCR
method was used(19) , with a slight modification as shown in Fig. 6. Primary PCR steps consisted of two separate PCRs for
each point mutation. One of the PCRs was initiated by using a mutagenic
primer and one of the outer primers; the other was initiated by using
an internal primer and the other outer primer. Both reactions used
pRESTAplc as template. The PCR took place in a reaction volume of 100
µl containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl,
0.01% gelatin (w/v), 1.5 mM Mg, 200
µM dNTPs, 10 pM template (pRESTAplc), 1
µM primers, and 2.5 units of Taq polymerase. The
reaction mixture was prewarmed to 55 °C for 5 min; the three-step
PCR reaction (94 °C for 40 s, 55 °C for 1 min, and 72 °C
for 2 min) for 30 cycles was then initiated by adding 0.5 µl (2.5
units) of Taq polymerase. The products of primary PCRs were
separated electrophoretically and isolated in a low melting point
agarose gel. The purified primary PCR products in low melting agarose
were incubated at 70 °C for 15 min and then diluted with distilled
HO to a final concentration of 5 µg/ml. Equal volumes
(20 µl) of two diluted primary PCR products were mixed, boiled to
100 °C for 5 min, and cooled slowly to 30 °C. The secondary PCR
reaction was carried out in a volume of 100 µl, containing 10
mM Tris-HCl, pH 8.3, 50 mM KCl, 0.01% gelatin (w/v),
1.5 mM Mg, 200 mM dNTPs, 50 ng of
template (the mixed and annealed primary PCR products), a 1 µM concentration of two outer primers, and 2.5 units of Taq polymerase. Amplified mutant DNA fragments from the secondary PCR
reaction were electrophoretically purified in low melting agarose
followed by phenol/chloroform extraction and ethanol precipitation. The
purified mutant DNA fragment was then digested with SphI and SacII restriction endonuclease and was used to replace the
corresponding restriction fragment of the wild type pRSETAplc. The
desired point mutation and sequence flanking by SphI and SacII sites were confirmed by DNA sequence analysis from 10
independently isolated pRSETAplc-containing mutant DNA fragments. For
mutagenesis of Lys
, the mutagenic PCR products from the
secondary PCR were digested with SphI and EcoRI and
cloned into the SphI/EcoRI-cleaved pRSETAplc(RI),
whose EcoRI in the polylinker region had been destroyed by T4
DNA polymerase filling in and the subsequent self-ligation.
Figure 6:
Schematic presentation of two-stage PCR
method to construct point mutations in PLC1. The first stage of
mutagenesis consists of two independent primary PCRs. One reaction uses
the mismatch primer (mutagenic primer) paired with one of the external
primers flanking one of the unique restriction sites; the other uses
the internal primer paired with another external primer flanking the
other unique restriction site. Both reactions used pRSETAplc plasmid as
template, and the PCRs were carried out as described under
``Experimental Procedures.'' The primary PCR products were
isolated and purified, mixed, and reannealed. The hybrid template was
first extended with Taq polymerase and followed by using the
same pair of external primers for the secondary stage PCR as described
under ``Experimental Procedures.'' The final mutagenic DNA
fragment was cleaved with a pair of unique restriction endonuclease (SphI/SacII or SphI/EcoRI) and used
to replace the corresponding fragment in wild type
pRSETAplc.
Measurement of PLC Activity
Hydrolysis of
PIP by PLC1 was determined exactly as described
previously (7) in an assay volume of 60 µl of 50 mM HEPES, pH 7.2, 3 mM EGTA, 0.2 mM EDTA, 0.83
mM MgCl, 20 mM NaCl, 30 mM KCl,
1 mM dithiothreitol, 0.1 mg/ml BSA, 0.16% sodium cholate, 1.5
mM CaCl containing 50 µM PIP (8,000 cpm) and 500 µM PE. The reaction was carried
out at 30 °C for 2-15 min and terminated by adding 0.2 ml of
10% ice-cold trichloroacetic acid and 0.1 ml of BSA (10 mg/ml). After
incubating on ice for 15 min, the unhydrolyzed
[H]PIP (pellet) was separated from
[H]IP (supernatant) by centrifugation
at 2,000 g for 10 min. Radioactivity in the
supernatant was measured by liquid scintillation counting. The activity
of PIP hydrolysis is expressed as µmol of
InsP/min/mg of protein; usually 0.5-10 ng of
partially purified recombinant PLC1 was used per assay.
Determination of PI hydrolysis activity was essentially the same as
described by Hofmann and Majerus(20) . The reaction was carried
out in a volume of 200 µl of 50 mM HEPES, pH 7.0, 3 mM CaCl, 1 mM EGTA, 0.1% sodium cholate
containing 30,000 cpm of [H]PI (300
µM). After incubation at 37 °C for 5-15 min, the
reaction was terminated by adding 1 ml of chloroform/methanol/HCl
(100:100:0.6) followed by 0.3 ml of 1 N HCl containing 5
mM EGTA. The aqueous and organic phases were separated by
centrifugation, and a 400-µl portion of upper aqueous phase was
counted by liquid scintillation.
Preparation of Phospholipid Vesicles
Phospholipid
vesicles PE/PC (8:2 molar ratio) containing the indicated concentration
of PI or PIP were prepared followed Mueller and Chien (21) with slight modifications. A dry phospholipid film was
formed by slowly blowing a 0.25-ml solution of chloroform/methanol
(2:1, v/v) containing mixing lipids (320 nmol of PE, 80 nmol of PC, and
the indicated amount of PIP or PI) under a stream of
nitrogen followed by freeze-drying under vacuum for 4 h. The
phospholipid film was hydrated under nitrogen with 1 ml of
nitrogen-aerated 0.18 M sucrose solution for 18 h at 4 °C
followed by mixing with an equal volume of 100 mM HEPES, pH
7.0, 200 mM KCl, and 10 mM EGTA. Vesicles were
isolated from the pellet by centrifuging the hydrated phospholipids at
1,200 g, 4 °C for 20 min. The phospholipid
vesicles were washed once with 1 ml of 50 mM HEPES, pH 7.0,
100 mM KCl, 5 mM EGTA, 200 µg/ml BSA (binding
buffer) and resuspended in 1 ml of the same buffer.
Binding Assay
The binding of PLC1 to
phospholipid vesicles was estimated by centrifugation
assay(22) . The final assay volume was 0.2 ml containing 50
mM HEPES, pH 7.0, 100 mM KCl, 5 mM EGTA, and
200 µg/ml BSA (binding buffer) plus the indicated concentration of
phospholipid vesicles and 1 µg of enzyme. To perform the assay, 1
µg of enzyme in 20 µl of binding buffer was incubated with 180
µl of mixed phospholipid vesicles in the same buffer at 30 °C
for 15 min. The free and bound PLC1 were separated by
sedimentation of the samples at 100,000 g for 30 min.
The amounts of the free PLC1 (supernatant) and the bound enzyme
(pellet) were determined by PI hydrolysis assay and Western blotting
analysis.
Trypsin Digestion of PLC1
Digestion of 0.2
µg of PLC1 with 0.1 µg of trypsin was carried out in a
reaction volume of 40 µl of 12.5 mM sodium phosphate, 37.5
mM HEPES, pH 7.0, 5 mM EGTA, 3.75 mM EDTA,
75 mM NaCl, 25 mM KCl, 0.025% Tween 20, 0.075% sodium
cholate, 375 µg/ml BSA containing 400 µM PE, and the
indicated concentration of PIP. (This is the minimal
concentration of trypsin which will degrade more than 95% of PLC1
protein within 5 min when incubated with phospholipid micelles free of
PIP.) To carry out the reaction, PLC1 was diluted to
0.02 mg/ml with the ice-cold buffer containing 50 mM sodium
phosphate, pH 7.0, 0.1 M KCl, 0.1% Tween 20, 5 mM EDTA, and 5 mM EGTA. To prepare the substrate solution,
0.44 mg of PE and various amounts of PIP in
chloroform/methanol (2:1, v/v) were first dried down under a stream of
nitrogen gas, and the phospholipids were solubilized by resuspending
the dry lipid with 0.9 ml of 50 mM HEPES, pH 7.0, 5 mM EGTA, 100 mM NaCl, and 0.1% sodium cholate. The mixture
was sonicated in a water bath sonicator for 10 min. One hundred µl
of BSA stock (5 mg/ml) in the same buffer was then added to make a
final concentration of 0.5 mg/ml. The proteolysis reaction was
initiated by mixing 10 µl of diluted PLC1 with 30 µl of 37
°C warmed trypsin buffer containing 50 mM HEPES, pH 7.0, 5
mM EGTA, 100 mM NaCl, 0.1% sodium cholate, 0.5 mg/ml
BSA, 0.1 µg of trypsin, 400 µM PE, and the indicated
concentration of PIP. The reaction was proceeded at 37
°C for an indicated period and was stopped by adding trypsin
inhibitor to a final concentration of 2.5 µg/ml. The residual
PLC1 was determined by PI hydrolysis activity and Western blotting
analysis.
RESULTS
Isolation and Sequencing of Human Aortic PLC1 cDNA
and Its Homology to Other PI-specific Phospholipase Cs
A human
aortic smooth muscle cDNA library was screened with a probe derived
from the rat C6 glioma cell PLC1 cDNA gene(17) . This
resulted in the isolation of 23 positive cDNA clones. Through direct
DNA sequence analysis using the purified recombinant gt11 phage
DNA as template, two of the clones were found to contain a 2.8-kb
insert with an open reading frame of 2.3 kb, which encoded a protein of
756 amino acids with an estimated molecular mass of 87 kDa. The human
PLC1 cDNA had 95% homology to the rat PLC1 gene at the level
of deduced amino acid sequence (Fig. 1). Like members of the
PLC subfamily, human PLC1 lacks the COOH-terminal tail
immediately after the Y domain.
Figure 1:
Nucleotide and deduced amino acid
sequence of human PLC1 cDNA. Panel A, the structure of
human PLC1 cDNA (2.6 kb) is schematically shown with the coding
region (open box) flanked by untranslated sequences (solid
line). The 2040- and 591-base pair (bp) EcoRI
fragments from the phage clone, corresponding to the sequence coding
for the NH-terminal and COOH-terminal region of PLC1,
respectively, were subcloned further into the EcoRI site of
pTZ19R to generate pTZ19R5`plc and pTZ19R3`plc. Panel B, the
nucleotide sequence of the 2.6-kb full-length human phospholipase
C1 cDNA was determined as described under ``Experimental
Procedures.'' The nucleotide residue numbers are given to the right of each line. The deduced amino acid sequence encoded by
the longest open reading frame (beginning at nucleotide 95 ATG) is
shown using the single letter amino acid code. Deduced amino acid
residues are numbered beginning with the initiation methionine, and the
residue numbers are shown to the left of each line. Amino acid
residues different from those of rat enzyme are shown. Conserved X and
Y regions of PI-PLC are underlined.
Fig. 2compares the deduced
amino acid sequence of the most conserved X and Y regions of human
PLC1 with those of the previously described PI-specific PLCs.
Within these regions, human PLC1 has 40 and 38% sequence identity
to rat PLC1, 35 and 43% sequence identity to rat PLC, 40 and
38% sequence identity to the Drosophila NorpA gene, and 38 and
32% sequence identity to yeast PLC1. PI-specific PLC from Bacillus
cereus only contains the X domain, with which the human clone
shares 30% similarity.
Figure 2:
Comparison of the primary structures of
the conserved regions of PLC isoenzymes from rat, human, Drosophila, yeast, and bacillus. Amino acid sequence
comparison of human PLC1 with the conserved X and Y regions of rat
PLC1(17) , PLC3(42) , PLC1(43) ,
PLC2(13) , human PLC2 (44) , yeast
PLC(45) , Drosophila norpA (Dro
PLC)(46) , and the X region of bacillus
PI-PLC(41) . Organisms, PI-PLC isoenzyme classes, and the
starting position in each protein sequence of the residues shown are
indicated on the far left. The boxed areas denote
positions at which amino acids in seven or more sequences are identical
or represent conservative substitutions grouped as follows: A and G; T
and S; I, L, M and V; K, H, and R; W, Y, and F; D and E; N and Q. Gaps
introduced to optimize the alignment are indicated by hyphens.
Identical residues containing functional side chain in the X region of
PI-PLC from human to bacillus are indicated by an asterisk and
were subjected to base substitution mutagenesis
study.
Tissue Distributions of PLC1 mRNA
Since the
cDNA clone of human PLC1 was initially isolated from a human aorta
cDNA library, we examined further the level of expression of PLC1
mRNA in various human tissues by Northern hybridization. Polyadenylated
RNA was prepared from various human tissues and hybridized with a P-labeled PLC1 cDNA probe. As shown in Fig. 3,
PLC1 mRNA is ubiquitously expressed with a predominant length of
3.8 kb in all human tissues we examined. However, the level of mRNA
varied significantly depending on tissues, with the 3.8-kb transcript
most abundantly expressed in lung, heart, pancreas, skeletal muscle,
and kidney. Hybridization of mRNA prepared from skeletal muscle showed
a 7.5-kb hybridizing species in addition to the 3.8-kb band.
Figure 3:
Northern analysis of RNA from various
human tissues. Human multiple tissue Northern blot obtained from
CLONTECH was hybridized with full-length human PLC1 cDNA washed
and exposed as suggested by the supplier. Lane 1, pancreas; lane 2, kidney; lane 3, skeletal muscle; lane
4, liver; lane 5, lung; lane 6, placenta; lane 7, brain; and lane 8,
heart.
Expression of Human PLC1 in E. coli
To
confirm that the human PLC1 cDNA gene encodes a protein comparable
to that of rat, we expressed the cloned human cDNA in E. coli and purified the recombinant protein by two-step column
chromatography (Table 1). The BamHI DNA fragment
containing the full coding length of PLC1 cDNA was inserted into
the BamHI site of expression vector pRSETA to obtain
pRSETAplc; upon induction with
isopropyl-1-thio--D-galactopyranoside, E. coli BL21 harboring PRSETAplc would express a fusion human PLC1
with 34 amino acids (derived from enterokinase cleavage site, phage T7
gene 10 sequences, and six consecutive histidine tags) at its NH terminus. This modification would allow us to remove most
bacterial proteins by one-step Ni-NAT affinity column
chromatography, while the hydrolysis activity was not affected. Trace
amounts of bacterial contaminants in this PLC1 preparation were
further removed by heparin-Sepharose column chromatography. The
homogeneity of the recombinant human PLC1 was examined by
SDS-polyacrylamide gel electrophoresis (Fig. 4). PLC1
expressed from E. coli was used to hydrolyze PI and
PIP, and the specific activities were determined to be 64
and 37 µmol/min/mg, respectively. When using the phospholipid
micelle substrate with a 1:10 molar ratio of PIP to PE, we
found that the specific activity of the recombinant PLC1 was
highly dependent on the free calcium concentration. As shown in Fig. 5, the specific activity increased 17-fold as the free
calcium concentration increased from 0.1 to 5.6 µM.
Figure 4:
SDS-polyacrylamide gel electrophoresis and
immunoblots of human PLC1 expressed from E. coli BL21. Panel A, 100 µg of crude extracts from E. coli BL21 carrying pRSETAplc (lane a), vector pRSET (lane
b), or 0.1 µg of heparin-Sepharose column
chromatography-purified PLC1 (lane c) were separated on
10% SDS-polyacrylamide gels and further detected with mixed monoclonal
anti-PLC1 antibodies. Panel B, 5 µg of
heparin-Sepharose column chromatography-purified PLC1 (lane
a), 100 µg of crude extracts from E. coli BL21
carrying pRSETAplc (lane b), and 10 µg of
Ni-NAT agarose column-purified PLC1 (lane
c) were separated on 10% SDS-polyacrylamide gels and stained with
Coomassie Blue.
Figure 5:
Ca dependence of
PLC1. The PIP hydrolysis activity of purified
PLC1 expressed from E. coli BL21 was assayed as described
under ``Experimental Procedures,'' and the free calcium
concentrations were calculated according to Fabiato and
Fabiato(47) . The values are expressed relative to the activity
at a free Ca concentration of 95
nM.
Amino Acid Residues Essential for Catalytic Activity of
PLC1
To identify residues potentially involved in cleavage
activity of PI-PLC, 9 amino acids with a functional side chain which
were invariant in PI-PLCs from human to bacteria (Fig. 2) were
subjected to residue substitution mutagenesis. Since the prokaryotic
enzyme lacks the conserved Y region, we believe that these residues
located in the X region of eukaryotic enzymes play a critical role in
the catalysis of PI-PLC. To test this possibility, these 9 residues
were individually substituted by in vitro mutagenesis of the
PLC1 cDNA. A mutant restriction DNA fragment containing a base
substitution at the corresponding amino acid codon was generated by PCR (Fig. 6) and used to replace the corresponding restriction
fragment in the native pRSETAplc expression construct. Crude extract
derived from the E. coli BL21 strain harboring a mutant
expression construct was passed through Ni-NAT
agarose followed by heparin-Sepharose column chromatography, and the
homogeneity of mutant protein was examined by SDS-polyacrylamide gel
electrophoresis and Western blotting analysis (data not shown). The
purified mutant was tested for the characteristic inositol phospholipid
hydrolysis using PI or PIP as substrate. This analysis
allowed us to categorize the present mutants into at least three
classes (Table 2). In the first class, the mutant enzymes bear no
detectable PIP or PI hydrolysis activity, e.g. R338L, E341G, and H356L; the second class of mutants is partially
active in inositol phospholipid hydrolysis, e.g. S381A, K434Q,
and K441Q; the inositol phospholipid hydrolysis activities of the third
group are similar to that of wild type, e.g. E327G, S388A, and
K440Q. Only the first class of mutant enzymes (R338L, E341G, and H356L)
were subjected to further characterization in the present report.
Binding of PLC1 to Phospholipid Vesicle
Since
no PI or PIP hydrolysis activity was detectable by mutant
enzymes R338L, E341G, and H356L at free Ca concentrations of 0.1 µM to 3 mM and at
PIP concentrations of 50-500 µM (data
not shown), these mutant enzymes might be defective either in cleavage
or in substrate binding. To distinguish between these possibilities and
to identify the role of residues Arg, Glu,
and His in the catalysis, equilibrium binding of the wild
type and mutant forms of PLC1 to phospholipid vesicles was
examined by centrifugation assay. As shown in Fig. 7A,
wild type and all three cleavage-defective mutant forms of PLC1
were unable to bind vesicles of PE/PC and bound weakly (45% were bound)
to PE/PC/PI vesicles (molar ratio 4:1:2.5). In contrast, more than 90%
of the wild type and mutant enzymes were bound to PE/PC vesicles
containing 5% (molar ratio) PIP. The binding of PLC1
to PIP phospholipid vesicles was highly dependent on
vesicle concentration and could be saturated as shown in Fig. 7B. Fifty percent of the wild type protein was
bound when incubated with 1.25 µM PIP, and the
binding was saturated when the PIP reached a concentration
of 10 µM. Although mutant enzymes R338L, E341G, and H356L
are not able to hydrolyze PI or PIP, all of these three
cleavage-defective mutant forms of PLC1 can bind phospholipid
vesicles containing 5% (molar ratio) PIP in a
concentration-dependent and saturable fashion with affinities
comparable to that of the wild type enzyme.
Figure 7:
Centrifugation assay of PLC1 binding
to lipid vesicles. Panel A, binding of wild type PLC1 and
cleavage-defective mutants to phospholipid vesicles of different lipid
content: 400 µM phospholipid vesicles with a PE/PC molar
ratio of 4:1 (
), 600 µM phospholipid vesicles of
PE/PC/PI with a molar ratio of 4:1:2.5 (&cjs2108;), and 420 µM () phospholipid vesicles of PE/PC/PIP with a
molar ratio of 4:1:0.25 (
). Panel B, dose-dependent
binding of PE/PC/PIP (4:1:0.25) lipid vesicles to wild type
and cleavage-defective mutants. The concentration of PIP (µM) was a fraction of phospholipid vesicle
containing PE/PS/PIP with a molar ratio of 4:1:0.25. All of
the centrifugation assays were carried out in a 0.2-ml total volume
using a Beckman TL-100 table top ultracentrifuge and TLA-100 rotor (see
``Experimental Procedures''). The unbound enzyme fractions
(supernatant) were quantified by PI hydrolysis assay and immunoblotting
using mixed monoclonal antibodies (for cleavage-defective mutant). The
bound enzyme fractions (pellets) were dissolved in 0.05 ml of
phosphate-buffered saline buffer then quantified by Western blotting
analysis.
PIP Protection of PLC1 from Trypsin
Digestion
To study further specific protein-PIP interaction and the structure of the enzyme-PIP complex, we compared the sensitivity of free enzyme and
enzyme-phospholipid complexes to trypsin digestion. As shown in Fig. 8A, digestion of PLC1 (0.2 µg) with
trypsin (0.005 µg) gave one major 80-kDa proteolytic fragment
concomitant with two minor degraded fragments of about 60 kDa. When the
trypsin concentration was increased to 0.1 µg, more than 90% of
PLC1 protein (0.2 µg) was degraded within 5 min. The rapid
degradation of PLC1 protein by trypsin correlated with loss of PI
hydrolysis activity, as shown in Fig. 8B; the specific
activity of the PLC1 was reduced to 50% within less than 1 min at
37 °C by trypsin and was reduced to 50% in less than 2 min when the
enzyme was preincubated with 400 µM PE or PI. In contrast,
this rapid inactivation of PLC1 by trypsin was prevented by
preincubating the enzyme with its specific substrate PIP.
As shown in Fig. 8B, the half-life of PLC1 was
prolonged to 7.8 min when the enzyme was preincubated with 200
µM specific substrate (PIP) prior to trypsin
digestion. The loss of PI hydrolysis activity by PLC1 as a result
of trypsin digestion was prevented by PIP in a substrate
concentration-dependent and saturable manner (Fig. 8C).
The activity of PLC1 was protected by as much as 65% as the
PIP concentration increased from 20 to 500 µM.
The saturation concentration of PIP for protecting PI
hydrolysis activity from the action of trypsin was 80 µM,
and half-maximal protection occurred at 34 µM PIP. Although PE also protected PLC1 activity
from the action of trypsin in a concentration-dependent manner, this
nonspecific phospholipid did not protect as efficiently as
PIP, and the protection was not saturable even when the
concentration was increased to 500 µM.
Figure 8:
Effect of PIP on the trypsin
cleavage of PLC1. Panel A, Western blotting analysis of
PLC1 (0.2 µg) digested with 0 (lane 1), 0.005 (lane 2), 0.01 (lane 3), 0.05 (lane 4), and
0.1 µg (lane 5) of trypsin for 5 min in the presence of
400 µM PE. Panel B, kinetic analysis of PLC1
inactivation by the proteolytic action of trypsin. 0.2 µg of the
PLC1 was preincubated with PE/PIP micelles containing
200 µM PIP and 400 µM PE
(
); PE micelles containing 600 µM PE (
) or in
the absence of phospholipid (). At each indicated time point,
an aliquot of incubation mixture was diluted and used to determine the
residual PI hydrolysis activity. Panel C, the residual
activity of PLC1 after a 5-min trypsin digestion in the presence
of PE/PIP micelles containing 400 µM PE and
the indicated concentration of PIP () or PE only
().
The ability of
PIP to protect PLC1 against trypsin-inflicted loss of
PI hydrolysis activity was correlated to the extent of degradation of
PLC1 by trypsin. As shown in Fig. 9A, incubating
PLC1 with PE/PIP micelles prior to trypsin digestion
markedly reduced the rate of trypsin cleavage; at least 50% of the
PLC1 protein still remained intact after a 5-min trypsin
digestion, and this was reduced to less than 10% as the
enzyme-PIP complexes were digested further with trypsin for
a total period of 15 min. However, 90% of the enzyme was degraded by
trypsin within 5 min when the enzyme was preincubated with lipid
micelles containing PE only. The pattern and extent of digestion of
PLC1PIP complexes by trypsin were highly
dependent on the concentration of PIP used to form the
specific complexes. As the concentration of PIP was
increased to 7.5 and 35 µM, the major protein species
surviving trypsin cleavage was a 67-kDa fragment. As the PIP concentration increased to 200 µM, the size of
protein surviving trypsin cleavage was similar to that of the intact
enzyme. The proportion of the 67-kDa fragment and intact protein
surviving the action of trypsin was dependent on the concentration of
PIP. Trypsin digestion of PLC1 preincubated with low
concentrations of PIP yielded a major 67-kDa proteolytic
fragment. As the concentration of PIP increased, the size
of the major protein species surviving the trypsin digestion was 89
kDa. We were not able to detect the 80-kDa fragment, which was the
major proteolytic product when free PLC1 was digested with the
reduced level of trypsin, suggesting that both the sensitivity and
specificity of the enzyme to trypsin cleavage were changed as a result
of specific binding of PIP. This observation shows that the
site of enzyme-PIP complex and its sensitivity to trypsin
cleavage were highly dependent on the concentration of PIP used to form the specific enzyme-PIP complex.
Figure 9:
Western blotting analysis of trypsin
cleavage of PLC1. Panel A, trypsin (0.1 µg) cleavage
of PLC1 for 0, 5, and 15 min in the presence of 200 µM or absence of PIP (control). Panel B, trypsin
(0.1 µg) cleavage of PLC1 (0.2 µg) for 5 min in the
presence of PE/PIP micelles containing the indicated
concentration of PIP. All of the trypsin cleavage reactions
were carried out at 37 °C in 40 µl of 12.5 mM sodium
phosphate, 37.5 mM HEPES, pH 7.0, 5 mM EGTA, 3.75
mM EDTA, 75 mM NaCl, 25 mM KCl, 0.025% Tween
20, 0.075% sodium cholate, 375 µg/ml BSA containing 400 µM PE and the indicated concentration of
PIP.
Interaction of E341G, H356L, and R338L with
PIP
To determine whether these three
cleavage-defective mutant PLC1s could interact with PIP like the wild type enzyme does, we compared the sensitivity and
specificity of trypsin cleavage of these mutants preincubated with
PIP. The rates of degradation of H356L and E341G mutants by
trypsin were significantly reduced if the mutant enzymes were allowed
to preform enzyme-substrate complexes by preincubating them with 200
µM PIP (Fig. 10A). In
contrast, PIP did not affect the rate of trypsin cleavage
of mutant PLC1 which has Arg replaced by Leu; 90% of
this mutant enzyme was degraded by trypsin within 5 min whether or not
it was preincubated with 200 µM PIP.
Figure 10:
Analysis of the trypsin sensitivity of
mutant forms of PLC1 in the presence or absence of
PIP. Panel A, trypsin (0.1 µg) cleavage of 0.2
µg of mutant PLC1 E341G, H356L, and R338L proteins in the
presence of 200 µM PIP or in the absence of
PIP (control) for 0, 5, 10, and 15 min. Panel B,
trypsin (0.1 µg) cleavage of 0.2 µg of mutant PLC1 E341G,
H356L, and R338L proteins for 5 min in the presence of the indicated
concentration of PIP. All of the trypsin cleavage reactions
were carried out in a buffer (40 µl) containing 12.5 mM sodium phosphate, 37.5 mM HEPES, pH 7.0, 5 mM EGTA, 3.75 mM EDTA, 75 mM NaCl, 25 mM KCl, 0.025% Tween 20, 0.075% sodium cholate, 375 µg/ml BSA
containing 400 µM PE and the indicated concentrations of
PIP. At the indicated time point, the reactions were
stopped and analyzed by Western blotting as described under
``Experimental Procedures.''
The
cleavage site(s) of preformed substrate-enzyme complexes by trypsin was
also highly dependent on the PIP concentration. For mutants
H356L and E341G, enzyme-substrate complexes formed at a low
concentration of PIP (7.5 µM) were easily
cleaved to a 67-kDa fragment by trypsin, whereas enzyme-substrate
complexes formed at a saturating concentration of PIP (200
µM) were relatively resistant to trypsin cleavage (Fig. 10B). Preincubating mutant R338L with increasing
concentrations of PIP from 7.5 to 200 µM did
not protect it from trypsin cleavage (Fig. 10B). The
saturation concentration of PIP required to protect H356L
and E341G from trypsin digestion was estimated to be 100
µM, and the half-maximal protection concentration of
PIP was estimated to be 35 µM. This result
demonstrated that according to their sensitivity to trypsin cleavage,
mutant E341G and H356L are able to form at least two types of complex
with PIP in a way similar to that of the wild type enzyme.
This indicates that Glu and His may play a
role in the step of cleavage rather than in the steps of substrate
binding.
DISCUSSION
At least four isoforms of PLC have been identified and
cloned: 1, 2, 3(23, 24) , and
4(25) . Judged by the slight deviation of its amino acid
sequence from those of rat and bovine isoforms, the present PLC
isoform we isolated from a human aorta library is classified as 1
type. Patterns of expression of PLC1 from various tissues of rats
and bovines examined by immunoanalysis (26) and Northern
analysis (27) reveal that although the level of expression is
relatively lower than for other isoforms of PLC, PLC1 is
widespread among most tissues. We found that transcripts of PLC1
are also present in almost all human tissues, implying that the enzyme
may play a role in some fundamental cellular process. Several lines of
evidence agree with this implication. First, in an attempt to select
mutants from mutagenized Chinese hamster lung fibroblasts defective in
thrombin-induced mitogenesis, several of these mutants were later shown
to lack PLC1 protein(28) . Second, disruption of a
PLC-like (PLC1) gene in yeast resulted in slow growth or
death of the cells, which could be rescued by exogenous expression from
a cloned rat PLC1 cDNA(29) . Third, a mutation in
PLC1 leading to an 8-fold increase in specific activity has been
identified in the spontaneous hypertensive
rat(30, 31) . Finally, our laboratory has recently
found that suppression of PLC1 in a rat cell line arrests cell
growth and diminishes mitogen-induced intracellular calcium
mobilization. ()
In at least two structural aspects, the
present recombinant version of PLC1 differs from purified forms.
First, a 34-amino acid region including 6 consecutive histidine
residues was fused to the NH terminus of the recombinant
enzyme. Second, post-translational modification of PLC1 expressed
in E. coli may be quite different from that of PLC1
expressed in mammalian cells. Since we have demonstrated here that
recombinant human PLC1 expressed from E. coli displays PI
and PIP hydrolyzing activity similar to that of the
purified forms(7, 32) , this version of recombinant
human PLC1 should be suitable for in vitro structure-function analyses.
Centrifugation binding assays show
that recombinant human PLC1 expressed from E. coli can
form specific complexes with PIP vesicles, with an affinity
comparable to that of the purified form(22, 33) . The
structure of the enzyme-substrate complex differs from that of the free
enzyme, as the enzyme-PIP complex is much more resistant to
trypsin cleavage than its free form. Moreover, the site and sensitivity
of the enzyme-PIP complex to trypsin cleavage are highly
dependent on the concentrations of PIP used to bind
PLC1. One of the cleavage-defective mutants PLC1 (R338L)
bound PIP vesicles as tightly as the wild type enzyme did,
but its complex was cleaved by trypsin as readily as the free protein.
Therefore, these changes in the sites and sensitivities to trypsin
cleavage are not caused by hindrance of the phospholipid moiety but
rather reflect a temporal rearrangement of potential trypsin-sensitive
sites during binding.
At least two types of specific
PLC1PIP complexes were demonstrated in these
experiments; complexes formed at a low concentration of PIP were readily degraded to a 67-kDa fragment, whereas specific
complexes formed at a higher concentration of PIP micelles
were relatively resistant to trypsin cleavage. The simplest
interpretation is that there are at least two types of PIP binding sites in PLC1: a high affinity site and a low
affinity site. When the PIP substrate concentration is low,
PLC1 will bind to PIP through the high affinity site
to form a complex that is cleaved readily by trypsin to give a 67-kDa
fragment. As the concentration of PIP increases, another
binding site with lower affinity will be saturated and cause formation
of a complex much more resistant to trypsin digestion. Because the
67-kDa fragment was retained in a Ni-NAT gel (data
not shown), the present results also suggest that the COOH-terminal
sequences of PLC1, although required for
catalysis(11, 12) , are not involved in the high
affinity binding of PIP. Two lines of evidence are
consistent with this interpretation that at least two PIP binding sites exist in PLC1. Deletion of the first 60 amino
acids of bovine PLC1 does not eliminate its PIP hydrolysis activity but dramatically reduces the binding of the
truncated enzyme to PIP lipid vesicles(15) .
Furthermore, a peptide corresponding to residues 30-43 of
PLC1 binds to IP(12) . These observations
indicate that in addition to the PIP cleavage site,
PLC1 can bind to PIP via a noncatalytic PIP binding site in the NH terminus of the protein.
Indeed, residues 16-134 in the NH terminus of
PLC1 have been found to share homologous sequences with pleckstrin
(PH domain)(34, 35, 36) , which can
specifically bind to PIP(37) . The binding of
PIP to the noncleavage site of PLC1 can either allow
the enzyme to hydrolyze the phospholipid in a processive manner (15) or may be required for other regulatory
process(38, 39) .
All the eukaryotic PI-PLC
identified so far can be structurally divided into conserved X and Y
regions(2, 17, 40) . Deletion mutagenesis
studies have shown that both X and Y region sequences are essential for
the catalytic
activity(11, 12, 13, 14) .
Consistent with this observation, conversion of several highly
conserved amino acid residues in the Y region causes the enzyme to lose
its catalytic functions (data not shown). In particular, conversion of
Arg
to Gly (R549G) makes the enzyme selectively defective
in the cleavage of PIP, whereas this mutant enzyme can
retain 20% of the ability to hydrolyze PI. We are investigating further
the contribution of these conserved residues to the catalytic function
of PLC1.
Prokaryotic PI-PLC does not contain a Y
conserved region(41) . Its catalytic activity is independent of
Ca and does not recognize PIP or PIP.
However, structural comparison of the X region sequences between
eukaryotic and prokaryotic PI-PLC reveals a high degree of similarity.
This supports the hypothesis that catalytic sites of PLCs might reside
in the X region. Within this region, 9 amino acid residues are
invariant from prokaryotic to human PLCs. Our preliminary in vitro mutagenesis and enzymatic characterization of the mutant human
PLC1 show that side chain switching of most of these residues does
not cause significant loss of catalytic activity of the mutant enzyme.
It is unlikely that switching their functional side chains does not
alter catalytic function of PLC1. We would rather believe that
these mutant enzymes might be defective in other functions not detected
in the present in vitro enzymatic assay.
However, in
agreement with the hypothesis that catalytic residues could be
localized in the X region, we found that conversion of Glu to Gly, His to Leu, or Arg to Leu
totally eliminated the catalytic activity of PLC1. Although these
three cleavage-defective mutant enzymes can bind to PIP vesicles as tightly as the wild type enzyme does, our substrate
protection experiments demonstrated that the structures of specific
PIP-protein complexes of these three mutants are not quite
the same. Both the sites of and sensitivity to trypsin digestion of the
specific PIP-protein complexes of H356L and E341G are
similar to those of the specific complexes of the wild type enzyme,
whereas binding of R338L to PIP does not affect its site or
sensitivity to trypsin digestion. The simplest interpretation is that
upon sequential binding of PIP, both mutant PLC1 E341G
and H356L can adopt structural adjustments similar to those of the wild
type enzyme, thus Glu and His are not
involved in either high affinity or low affinity interactions with
PIP. Although R338L is still able to bind tightly to
PIP vesicles, its structural adjustment upon binding to
PIP is distinct from that of the wild type enzyme.
In
conclusion, this study shows that Glu and His are not involved in specific interaction with PIP.
Since Ca is not essential for the PIP binding activity of PLC but is required for cleavage, this
implies that Glu and His may be involved
either in the binding of Ca or in the cleavage step.
However, His and Glu are also invariant in
prokaryotic PI-PLC, whose activity is independent of
Ca. Therefore, these two residues are most likely
involved in the hydrolysis of PIP once the specific
substrate-enzyme complex has been formed.
FOOTNOTES
- *
- This
work was supported by the National Sciences Council of the Republic of
China Grants NSC83-0203-B-001-006. The costs of publication of this
article were defrayed in part by the payment of page charges. This
article must therefore by hereby marked
``advertisement'' in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
U09117[GenBank].
- §
- To
whom correspondence should be addressed. Fax: 886-2-785-3569.
- ()
- The abbreviations used are: IP,
inositol 1,4,5-trisphosphate; PLC, phospholipase C; PI-PLC,
phosphoinositide-specific phospholipase C; PIP,
phosphatidylinositol 4,5-bisphosphate; PIP, phosphatidylinositol
4-phosphate; PI, phosphatidylinositol; PE, phosphatidylethanolamine;
PC, phosphatidylcholine; BSA, bovine serum albumin; PCR, polymerase
chain reaction; kb, kilobase(s).
- ()
- H.-F. Cheng,
M.-J. Jiang, C.-L. Chen, S.-M. Liu, L.-P. Wong, J. W. Lomasney, and K.
King, manuscript in preparation.
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