From the Department of Life Science, Faculty of
Science, Himeji Institute of Technology, Harima Science Garden City,
Hyogo 678-12, Japan, the ¶ Cancer Research Campaign Centre for
Cell and Molecular Biology, Chester Beatty Laboratories, Fulham Road,
London SW3 6JB, United Kingdom, the
Department of Applied
Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790, Japan, the ** Department of Biochemistry, Faculty of Dentistry, Kyushu
University, Fukuoka 812, Japan, and the
Medical Research Council Laboratory of
Molecular Biology, Hills Road,
Cambridge CB2 2QH, United Kingdom
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ABSTRACT |
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The pleckstrin homology (PH) domain of
phosphatidylinositol-specific phospholipase C-1 (PLC-
1) binds
to both D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and phosphatidylinositol
4,5-bisphosphate (PtdIns(4,5)P2) with high affinities. We
have previously identified a region rich in basic amino acids within
the PH domain critical for ligand binding (Yagisawa, H., Hirata, M.,
Kanematsu, T., Watanabe, Y., Ozaki, S., Sakuma, K., Tanaka, H., Yabuta,
N., Kamata, H., Hirata, H., and Nojima, H. (1994) J. Biol.
Chem. 269, 20179-20188; Hirata, M., Kanematsu, T., Sakuma, K.,
Koga, T., Watanabe, Y., Ozaki, S., and Yagisawa, H. (1994)
Biochem. Biophys. Res. Commun. 205, 1563-1571). To
investigate the role of these basic residues, we have performed
site-directed mutagenesis replacing each of the basic amino acid in the
N-terminal 60 residues of PLC-
1 (Lys24,
Lys30, Lys32, Arg37,
Arg38, Arg40, Lys43,
Lys49, Arg56, Lys57, and
Arg60) with a neutral or an acidic amino acid. The effects
of these mutations on the PH domain ligand binding properties and their consequence for substrate hydrolysis and membrane interactions of
PLC-
1 were analyzed using several assay systems. Analysis of
[3H]-Ins(1,4,5)P3 binding, measurement of the
binding affinities, and measurements of phospholipase activity using
PtdIns(4,5)P2-containing phospholipid vesicles,
demonstrated that residues Lys30, Lys32,
Arg37, Arg38, Arg40, and
Lys57 were required for these PLC-
1 functions; in
comparison, other mutations resulted in a moderate reduction. A subset
of selected mutations was further analyzed for the enzyme activity
toward substrate present in cellular membranes of permeabilized cells and for interaction with the plasma membrane after microinjection. These experiments demonstrated that mutations affecting ligand binding
and PtdIns(4,5)P2 hydrolysis in phospholipid vesicles also
resulted in reduction in the hydrolysis of cellular
polyphosphoinositides and loss of membrane attachment. All residues
(with the exception of the K43E substitution) found to be critical for
the analyzed PLC-
1 functions are present at the surface of the
PH domain shown to contain the Ins(1,4,5)P3 binding
pocket.
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INTRODUCTION |
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The pleckstrin homology
(PH)1 domain has been
initially identified as a region of sequence similarity of about 120 amino acid residues (3, 4). At the last count, more than 100 proteins have been reported to have this sequence motif; many of these proteins
are involved in cellular signaling and cytoskeletal functions (5-8).
Studies of several PH domains using x-ray crystallography or NMR
(9-12) revealed a conserved structural module containing a
seven-stranded -sandwich formed by two orthogonal antiparallel
-sheets and a C-terminal amphiphilic
-helix. The loops between the
-strands, particularly the
1/
2,
3/
4, and
6/
7,
differ greatly in length and sequence. Each PH domain is
electrostatically polarized, and the most variable loops coincide with
the positively charged face.
By analogy with other conserved structural modules (e.g.
SH2 and SH3 domains), it has been proposed that the PH domain
could be involved in signaling by mediating intermolecular
interactions. Consequently, a great effort has been made to identify
ligand(s) for this domain. Although there are examples of PH domains
involved in protein-protein interactions (e.g. binding of
G by
-adrenergic receptor kinase PH domain (13) or recognition
of phosphotyrosine by Sch PH/PTB domain (14)) there is an increasing
evidence that many PH domains interact with different inositol lipids
and inositol phosphates (15, 16). In this respect, the PH domain of
phospholipase C-
1 (PLC-
1) has been studied most extensively.
Determination of association constants for different inositol lipids
and their head groups (1, 2, 17), and relative abundance of these phospholipids in the cell identified PtdIns(4,5)P2 as a
potentially important physiological ligand (18, 19).
Ins(1,4,5)P3 can bind to the same binding pocket as the
head group of PtdIns(4,5)P2 with even higher affinity (1,
20). Studies of PH domain interactions also identified inositol lipids
other than PtdIns(4,5)P2 as possible ligands for several PH
domains. Findings that some of the phosphoinositides generated by
activation of phosphatidylinositol 3-kinase (PtdIns(3)P, PtdIns(3,4)P2, and PtdIns(3,4,5)P3) bind to the
PH domains of the serine-threonine kinase PKB/RAC/Akt (21-24), protein
GAP1 (25), and protein GRP1/ARNO/cytohesin-1 (22, 26, 27) suggested that these proteins could be signaling targets of phosphatidylinositol 3-kinase. Bruton's tyrosine kinase (Btk) PH domain has also been reported to bind PtdIns(3,4,5)P3 (28) and several inositol
polyphosphates (1,3,4,5-tetrakisphosphate, 1,3,4,5,6-pentakisphosphate,
and 1,2,3,4,5,6-hexakisphosphate) (29). Other examples of high affinity
binding of different inositol phosphates to PH domains include binding
of inositol 1,4,5,6-tetrakisphosphate to p130 inositol
phosphate-binding protein (30).
In this article, we focus on the PLC-1 PH domain located at the N
terminus of the molecule. Although it is not clear how the PH domain
interacts with the three-domain core structure of PLC-
1 (containing
the EF-hand, catalytic, and C2 domain), a flexible, surface-exposed
link has been suggested (31). Previous studies indicating that the PH
domain could play a role in the regulation and catalysis of PLC-
1
demonstrated its requirement for a high rate of substrate hydrolysis
(32) and membrane interactions (33). The structure of the PLC-
1 PH
domain has been obtained in a complex with Ins(1,4,5)P3,
revealing not only structural elements but also identified residues
within the Ins(1,4,5)P3 binding pocket (12). Based on
detailed structural information, availability of different functional
assays, and our previous studies, we performed extensive
structure/function analysis. It was demonstrated that single basic
amino acid replacements in the N-terminal part of the PH domain, when
microinjected into cells, could cause a significant reduction in the
Ins(1,4,5)P3/PtdIns(4,5)P2 binding, the
catalytic activity, and the interaction with the plasma membrane.
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EXPERIMENTAL PROCEDURES |
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Materials--
[3H]Ins(1,4,5)P3
(specific radioactivity: 777 GBq/mmol) was obtained from NEN Life
Science Products. Non-radioactive Ins(1,4,5)P3 and
[inositol-2-3H]PtdIns(4,5)P2
(specific activity: 37 GBq/mmol) was from Amersham Life Science.
Monoclonal antibodies against PLC-1 were obtained from Upstate
Biotechnology, Inc.
Expression Vector and Site-directed Mutagenesis--
The
cDNA encoding rat aortic PLC-1 (34) was subcloned into a
pGEX-2T-based bacterial expression vector (pGEX-2T from Pharmacia Biotech Inc.), designated as pGST3 (1). Individual point mutations were
introduced to the plasmid pGST3/PLC-
1 encoding the wild-type enzyme
by a T4 DNA polymerase-based mutagenesis using a TransformerTM site-directed mutagenesis kit (CLONTECH) with a
selection primer (5
-GGTTTCTTAGTCGACAGGTGGCAC-3
), which converts the
AatII site (3502-3525) of pGST/PLC-
1 into a
SalI site, and each of the following mutagenic primers:
K24A, 5
-AGGCCCTTCTGGCGGGCAGCCAGCTT-3
(1003-1028); K30E,
5
-CCAGCTTCTGGAGGTGAAGTCCA-3
(1022-1044); K30L,
5
-CCAGCTTCTGCTGGTGAAGTCCA-3
(1022-1044); K32E,
5
-TCTGAAGGTGGAGTCCAGCTCGT-3
(1028-1050); K32L,
5
-TCTGAAGGTGCTGTCCAGCTCGT-3
(1028-1050); R37D,
5
-CAGCTCGTGGGATAGGGAACGCTT-3
(1043-1066); R37L,
5
-CAGCTCGTGGCTTAGGGAACGCTT-3
(1043-1066); R38E,
5
-CTCGTGGCGTGAGGAACGCTTCTA-3
(1046-1069); R38V,
5
-CTCGTGGCGTGTGGAACGCTTCTA-3
(1046-1069); R37D/R38E,
5
-CAGCTCGTGGGATGAGGAACGCTTC-3
(1043-1067); R40D,
5
-GGCGTAGGGAAGACTTCTACAAGC-3
(1051-1074); R40A,
5
-GCGTAGGGAAGCCTTCTACAAGC-3
(1052-1074); K43E,
5
-ACGCTTCTACGAGCTACAGGAGG-3
(1061-1083); K43L,
5
-ACGCTTCTACCTGCTACAGGAGG-3
(1061-1083); K49A,
5
-AGGAGGACTGCGCGACCATCTGG-3
(1078-1100); R56A,
5
-GGCAGGAATCTGCAAAGGTCATG-3
(1099-1121); K57A,
5
-GGAATCTCGAGCGGTCATGAGG-3
(1103-1124); R60A,
5
-AAAGGTCATGGCGTCCCCGGAGT-3
(1112-1134). The desired
point mutation and sequence flanking the mutagenic primer-annealing
site was confirmed by DNA sequence analysis. Deletion mutants
1-223
(1) and
1-135 (33) have been described previously.
Protein Expression and Purification--
The wild-type and
mutated PLC-1 proteins were expressed as glutathione
S-transferase (GST) fusion proteins in Escerichia coli (PR745) as described (1). In brief, cells grown in LB medium
were incubated in the presence of 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside (3 h at 37 °C
or 15 h at 25 °C). After centrifugation, resulting cell pellets
were resuspended in a buffer containing a mixture of protease
inhibitors (20 mM Tris-HCl, pH 7.5, 1 mM EDTA,
1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 50 units/ml aprotinin, 2 µg/ml
leupeptin, 2 µg/ml pepstatin A, 0.1 mM benzamidine) and
subjected to sonication using a Bioruptor (Tosho Co.). The cell debris
was removed by centrifugation (10,000 × g for 5 min), and the resultant supernatant was used as an E. coli lysate.
Quantification of the GST fusion proteins in lysates was carried out by
measurements of GST activity using the GST substrate
1-chloro-2,4-dinitrobenzene and purified recombinant GST as a standard.
Purification of PLC-
1 fusion proteins to near homogeneity was
achieved using glutathione-Sepharose 4B (Pharmacia) affinity
chromatography. The purity of the samples used for the enzyme assay and
the kinetic measurement was at least 90%, as judged by Coomassie
Brilliant Blue staining of SDS-polyacrylamide gel electrophoresis
(PAGE) gels. For microinjection experiments and experiments using
permeabilized cells, proteins were further purified and concentrated on
a Mono Q column (HR5/5) (Pharmacia) using conditions described
previously (33).
Assay of [3H]Ins(1,4,5)P3
Binding--
[3H]Ins(1,4,5)P3 binding assays
were carried out as described elsewhere (35, 36). In brief, the assay
mixture (0.45 ml) contained 50 mM Tris-HCl buffer (pH8.3),
1 mM EDTA, 1.07 nM
[3H]Ins(1,4,5)P3 and a protein sample. After
incubation of the mixture on ice for 15 min, the
[3H]Ins(1,4,5)P3-protein complex was
precipitated with 0.5 ml of polyethylene glycol 6000 (30%, w/v)
together with 50 µl of bovine -globulin (10 mg/ml) as a carrier
protein. The pellet after centrifugation was dissolved in 0.3 ml of 0.1 N NaOH and then subjected to liquid scintillation counting.
The specific binding was obtained after subtracting the radioactivity
of samples in the presence of 1 µM non-radiolabeled
Ins(1,4,5)P3.
Measurement of Binding Constants Using an Optical Evanescent
Resonant Biosensor--
An optical evanescence resonant mirror cuvette
system (IAsysTM, Affinity Sensors), whose principle is based on a
quantum mechanical phenomenon (37) similar to that of a surface plasmon
resonant biosensor (38, 39), was used to measure interaction kinetics between an immobilized Ins(1,4,5)P3 analog and PLC-1
mutants.
Assay of PLC Activity-- PLC activity was measured using PtdIns(4,5)P2 as a substrate. The assay mixture (50 µl) contained the protein sample, 2.5 µg of PtdIns(4,5)P2, 25 µg of phosphatidylethanolamine containing 370 Bq (0.01 µCi) of [3H]PtdIns(4,5)P2, 20 mM Hepes/NaOH (pH 7.2), 0.83 mM MgCl2, 1 mM dithiothreitol, 2 mM EGTA, 0.2 mM EDTA, 30 mM KCl, and 2 mM CaCl2. Incubation was carried out for 10 min at 37 °C, then terminated by transfer to 0 °C. Extraction of water-soluble hydrolysis products was carried out by addition of 1 ml of chloroform/methanol/HCl (100:100:0.6) and 0.3 ml of 1 N HCl, 5 mM EGTA. After vortexing the sample, the radioactivity in the aqueous phase was measured in a liquid scintillation counter. For assays using detergent mixed micelles as substrates, 0.5% (w/v) sodium cholate was added to the reaction mixture as described previously (45).
Microinjection and Immunofluorescence Analysis--
Subcellular
localization of PLC-1 was analyzed according the method described
previously (33). MDCK cells grown to confluence were subjected to
microinjection on a Zeiss microinjection workstation (Carl Zeiss).
Samples of purified proteins (2 mg/ml) were microinjected intracytoplasmically. PBS was used as a standard diluent and the microinjected volume estimated at approximately 10 pl/cell.
Immunofluorescence staining was carried out as described by Cowley
et al. (46). Briefly, 4 h after microinjection, cells
were fixed (4% formaldehyde for 15 min), washed (several changes of
PBS for 30 min), permeabilized with 0.2% Triton X-100 for 10 min, and
quenched by 100 mM glycine for 15 min followed by 10%
fetal calf serum for 15 min. Incubation with a pool of mouse
anti-PLC-
1 antibodies (primary antibody) diluted 1:150 was for
1 h at room temperature. After a 15-min wash in PBS, the cells
were incubated with fluorescein isothiocyanate-labeled anti-mouse
conjugate for 1 h at room temperature. Labeled preparations were
mounted under glass coverslips, and examined with a Bio-Rad MRC 600 confocal imaging system in conjunction with a Nikon Diaphoto epifluorescence microscope.
Inositol Lipid Hydrolysis in Permeabilized
Cells--
Permeabilization and reconstitution of HL60 cells with
PLC-1 was performed according to the method of Cockcroft et
al. (47). In brief, HL60 cells were labeled with
[3H]inositol (2 µCi/ml) over 48 h. Cells were then
washed in buffer A (20 mM PIPES, 137 mM NaCl,
2.7 mM KCl, 1 mg/ml bovine serum albumin, 1 mg/ml glucose,
and 2 mM MgCl2), and permeabilized in the same
buffer with the addition of 0.4 IU/ml streptolysin O and 1 mM Mg-ATP over 40 min at 37 °C. Following
permeabilization, aliquots of cells were incubated in buffer A with 1 mM Mg-ATP, 10 mM LiCl, Ca2+/EGTA
buffer (pCa 5), and 2 µg/ml PLC-
1. PLC-
1
preparations for this study were purified GST fusion proteins of the
wild-type PLC-
1, a deletion mutant lacking the PH domain
(
1-135), or full-length protein containing the selected point
mutations within the PH domain. After a 20-min incubation at 37 °C,
reactions were quenched with 1:1 CHCl3:MeOH and phases
separated by centrifugation. The aqueous phase was then applied to
Dowex columns and inositol phosphates eluted with 1 M
ammonium formate/0.1 M formic acid. Radioactivity in the
samples was determined by scintillation counting.
Other Methods--
Protein assays were carried out by the method
of Bradford (48) with bovine -globulin as a standard. SDS-PAGE was
performed according to Laemmli (49).
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RESULTS |
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Site-directed Mutagenesis of the Basic Amino Acids within the
N-terminal Part of the PLC-1 PH Domain--
Our previous studies
(1, 2) have shown that the N-terminal 60 residues of PLC-
1 are
essential for high affinity Ins(1,4,5)P3 and
PtdIns(4,5)P2 binding and that the basic residues within
this region may play a crucial role in the recognition of the
negatively charged partner, the phosphate groups of the inositol ring.
Elucidation of the three-dimensional structure of the PLC-
1 PH
domain (residues 17-132) in complex with Ins(1,4,5)P3 (12)
revealed putative residues that could form direct interactions with
Ins(1,4,5)P3; five of these residues are basic amino acids
from a region identified by our studies. To obtain further insight into
the relative importance of each of the basic residues within the
N-terminal part of the PH domain, we performed site-directed
mutagenesis of these residues.
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Determination of Basic Amino Residues Critical for
[3H]Ins(1,4,5)P3 Binding--
Analysis of
GST/PLC-1 fusion proteins using
[3H]Ins(1,4,5)P3 binding demonstrated its
stereospecific, high affinity (Kd ~0.01
µM and Kd ~0.1 µM,
respectively) binding to PLC-
1 or to its PH domain (1, 50). The
specific [3H]Ins(1,4,5)P3 binding activity of
the PH domain mutants was compared with that of the wild-type using
preparations normalized for their expression levels. Results of this
analysis are shown in Fig. 2.
Substitutions at Lys30, Lys32,
Arg37, Arg38, Arg40, and
Lys57 resulted in the complete loss of the binding or a
large reduction, with activity remaining below 10% of the wild-type
PLC-
1 binding activity. In addition to residues implicated in
Ins(1,4,5)P3 binding by structural studies
(Lys30, Lys32, Arg40, and
Lys57) residues Arg37 and Arg38 are
also essential for binding. The only double mutant analyzed in this
study with substitutions by negative charges, R37D/R38E, was completely
inactive in ligand binding.
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Determination of Kinetic Parameters for the Ins(1,4,5)P3 Binding Using an Evanescent Resonance Biosensor-- Simple binding experiments using [3H]Ins(1,4,5)P3 as a ligand provides equilibrium rate constants, but do not usually give kinetic constants such as the association rate constant (ka) and the dissociation rate constant (kd). To estimate these constants and the dissociation constant for the Ins(1,4,5)P3 binding of each site-directed mutant, we have introduced a real-time kinetic assay using an optical evanescent wave biosensor, IAsysTM.
An Ins(1,4,5)P3 analog, 204BTN3, was immobilized to the cuvette surface of the biosensor. Addition of GST alone to the cuvette did not show a response, whereas the wild-type GST/PLC-Substrate Hydrolyzing Activities of the Site-directed
Mutants--
To examine the effect of point mutations within the
PLC-1 PH domain on substrate hydrolysis by the enzyme, several assay systems have been used. When substrate was presented as 0.5% sodium cholate/PtdIns(4,5)P2 mixed micelles, wild-type PLC-
1
and all the mutations examined had similar specific activities (about 800-1000 units/mg). This is consistent with our previous observation (51) that deletion of the PH domain did not reduced the specific activity of the enzyme in this assay. In assays with reduced
concentrations of sodium cholate (0.08%), some differences between the
mutants and the wild-type enzyme could be seen (data not shown). The
differences, however, could be more clearly detected and measured when
phospholipid vesicles containing PtdIns(4,5)P2 were used as
a substrate instead of mixed micelles (Fig.
3). For these measurements, crude
preparations of the mutants (normalized for their expression) were
used. The activity was greatly reduced in mutants K30L, K30E, K32L,
K32E, R37D, R38E, R37D/R38E, R40A, R40D, K43E, and K57A. Although
the charge-reversed mutation almost completely abolished the
activity, the proteins with neutral substitutions R37L, R38V, and, in
particular, K43L retained some activity. Decreased but still
significant activities were shown for mutants K24A, K49A, K56A, and
R60A.
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Subcellular Localization of Site-directed Mutants--
We have
demonstrated previously (33) that a portion of PLC-1 associates with
the plasma membrane after microinjection of purified protein into MDCK
cells; the protein was visualized by immunofluorescence using a pool of
anti-PLC-
1 monoclonal antibodies. It has been shown also (33) that
deletion of the PH domain (or portions of this domain) resulted in a
loss of membrane association. The data from this type of analysis
cannot be easily quantified and only substantial reduction in membrane
association can be detected as absence of membrane staining.
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DISCUSSION |
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In this article, we describe the structure/function relationship
of PLC-1 PH domain. Results presented here demonstrate that single
amino acid replacements within the PH domain, affecting binding of the
ligands for this domain, can result in reduction of enzyme activity and
loss of PLC-
1 interaction with the plasma membrane.
Our previous studies (1) and studies of others (20, 50) demonstrated
that PLC-1 PH domain, present at the N terminus of the molecule,
binds Ins(1,4,5)P3 and PtdIns(4,5)P2 with high affinity and specificity. Estimated Kd values are
within the range of 3-200 nM for Ins(1,4,5)P3
and 0.1-10 µM for PtdIns(4,5)P2; these
interactions are sufficiently strong to mediate the PH domain binding
to these molecules in vivo at their estimated physiological concentrations (18, 19). Ligand binding studies in vitro
also demonstrated that the binding of Ins(1,4,5)P3 to a
great extent reflects binding of PtdIns(4,5)P2. It has been
shown that the recognition of the head group of this phospholipid is
critical for its binding and interaction with
PtdIns(4,5)P2-containing vesicles, and that this can be
readily inhibited by Ins(1,4,5)P3. The presence of the
lipid moiety seems to restrict rather than facilitate the binding (50).
For comparison of the PH domain ligand binding properties of the
wild-type and mutant PLC-
1 molecules in this study (Figs. 1 and 2),
binding of [3H]Ins(1,4,5)P3 was measured. The
binding of [3H]Ins(1,4,5)P3 to PLC-
1
molecules with mutations K30L, K30E, K32L, K32E, R37L, R37D, R38V,
R38E, R37D/R38E, R40A, R40D, K43E, and K57A was abolished or greatly
reduced, whereas K24A, K43L, K49A, R56A, and R60A resulted only in a
moderate reduction or had no effect.
The enzyme activity of PLC-1 and other phospholipases has been
studied in different assay systems in vitro using substrate presented as detergent mixed micelles, phospholipid vesicles or monolayers (45, 53-55). Studies of the mechanism of
PtdIns(4,5)P2 hydrolysis and binding to
PtdIns(4,5)P2-containing phospholipid vesicles suggested
that the substrate is hydrolyzed in a processive mode of interfacial
catalysis (56, 57). This would involve interaction with the
phospholipid surface via site(s) distinct from the active site followed
by several cycles of PtdIns(4,5)P2 hydrolysis in the active
site. Since the PH domain is required for the processive mode of
catalysis and binding to the vesicles, it has been suggested that this
domain represents a major non-catalytic membrane interaction site (33,
57). Comparison of the full-length and PH domain deletion mutant in the
sodium cholate/PtdIns(4,5)P2 mixed micelle assay, where
processivity cannot be observed, demonstrated similar substrate
hydrolysis (33). Data from analysis of the wild-type and PLC-
1 with
mutations within the PH domain have shown that the specific activity of
all mutants were similar to the wild-type in a mixed micelle assay.
However, when PtdIns(4,5)P2 was presented within
phospholipid vesicles, mutations affecting Ins(1,4,5)P3
binding to the PH domain (Fig. 2) greatly reduced substrate hydrolysis
(Fig. 3). These data are consistent with the proposed function of the
PH domain to provide a non-catalytic PtdIns(4,5)P2 binding
site important for processive catalysis. In a similar study, the impact
of several point mutations within the PH domain on PLC-
1 association
with the lipid vesicles and PtdIns(4,5)P2 hydrolysis was
analyzed (55). It has been found that the mutation K24G resulted in a
moderate reduction of substrate hydrolysis (74% of the wild-type),
whereas mutations K30G, K32G, W36G and R37G had a greater effect
(37-50% of the wild-type). As in our more extensive studies, the
reduction in substrate hydrolysis correlated well with the ability of
PLC
1 mutants to bind the PH domain ligand.
It is becoming clear that, in the cellular environment, the
distribution and availability of PtdIns(4,5)P2 to
phospholipase C is much more complex than in phospholipid vesicles. To
assess an impact of the PH domain mutations on PLC-1 function in
systems closer to the cellular environment, analysis of substrate
hydrolysis in permeabilized cells and the subcellular distribution of
PLC-
1 after microinjection were analyzed (Fig.
5 and Table I). Only mutants selected
among those with greatly reduced in vitro
Ins(1,4,5)P3 binding and PtdIns(4,5)P2
hydrolysis (Figs. 2 and 3) resulted in a loss of the plasma membrane
interaction in MDCK cells (Fig. 4) and also exhibited a greater
reduction of substrate hydrolysis present in permeabilized HL-60 cells
(Table I). These results are consistent with the demonstration that
deletion of the PH domain abolished membrane attachment (33). However,
this previous study did not provide an insight into the nature of these
interactions or identified a ligand present in the membrane. Based on
characterization of binding properties in vitro, it has been
suggested that recognition of the PtdIns(4,5)P2 head group
could be involved. Data presented here, demonstrating that point
mutations that abolish Ins(1,4,5)P3 binding also resulted
in a loss of membrane attachment, further support the proposed role of
PtdIns(4,5)P2 as a membrane component involved in
interaction with the PLC-
1 PH domain. The data are also consistent
with the possibility that Ins(1,4,5)P3, the product of the
enzyme reaction, could interfere with the membrane attachment and
consequently affect PLC-
1 function.
|
All mutations analyzed in this study are present within the N-terminal
portion of PLC-1 PH domain, a region previously identified as
essential for the ligand binding (1, 56). Determination of the crystal
structure of the PLC-
1 PH domain in a complex with
Ins(1,4,5)P3 provided an insight into the overall structure of the domain and identified residues that could interact directly with
the ligand (Fig. 1) (12). Positions of residues selected for
mutagenesis within the PH domain structure and the impact of these
mutations on analyzed functions of PLC-
1 are summarized in Fig. 5.
Residues where replacements have a greater effect on PLC-
1 functions
(with the exception of acidic substitution at position 43) are present
at the surface containing Ins(1,4,5)P3 binding pocket. They
include residues directly involved in interactions with 4- and/or
5-phosphoryl groups: Lys30 and Lys57 (both
position 4 and 5), Lys32 (position 4), and
Arg40 (position 5). Results from another study also
demonstrated inhibition of PLC-
1 by K30G, K32G, and R37G mutations
(55). In addition, it has been shown that the mutation of
Trp36, a residue interacting with phosphoryl group at
position 1, has an inhibitory effect (55). Together, these data suggest
that the recognition of all phosphoryl groups of
Ins(1,4,5)P3 is important for the binding. Residues
exhibiting strong inhibition of PLC-
1 functions also include
residues Arg37 and Arg38 that have not been
directly implicated in Ins(1,4,5)P3 binding but are present
in their vicinity; several mutations of these residues have been
analyzed (R37L, R37D, R37G, R38V, R38E, and R37D/R38E) (this article
and Lomasney et al. (55)). Substitution of two other
residues present at the same part of the molecules, Arg60
and arginine residue involved in Ins(1,4,5)P3 binding
(Arg56), had only moderate effect. However, other mutations
showing moderate inhibition, Lys49, Lys43 and
Lys24 (this article and Lomasney et al. (55)),
are present at the opposite end to the
Ins(1,4,5)P3/PtdIns(4,5)P2 binding pocket and,
as visualized by rotating the model of the PH domain structure (Fig.
5), are roughly in the same vicinity as the binding pocket. Their
influence on Ins(1,4,5)P3/PtdIns(4,5)P2 binding
may imply a secondary low affinity site of interaction. This secondary
site may be involved in interaction with any anionic phospholipids on
the membrane surface and could indirectly influence the binding of
inositol head group to the binding pocket. Involvement of acidic phospholipids in membrane interactions has been suggested by the observation that the presence of acidic phospholipids in phospholipid vesicles increased binding of the isolated PH domain about 10-fold (57)
and that the PLC-
1 activity was stimulated by interaction with
phosphatidic acid (58). It is also intriguing that an acidic-to-basic substitution (E54K) of the PLC-
1 PH domain enhances the enzymatic activity (59).
Studies of PH domains have demonstrated that this conserved structural module can bind a number of different ligands and, potentially, perform a variety of functions (14, 15). It is therefore difficult to predict whether basic residues of other PH domains present at similar positions as those analyzed in this study would affect their function. Nonetheless, the observation that single point mutations within the PH domain can have a dramatic effect on the function of the whole molecule seem to be more general. For example, a point mutation within the PH domain of the serine-threonine kinase PKB/RAC/Akt (R25C) abolished binding of PtdIns(3,4)P2, activation by this phospholipid in vitro, and activation by PI3 kinase in vivo (60). It has also been demonstrated that each of the point mutations identified in the PH domain of Btk and linked to human agammaglobulinemia and murine immunodeficiency, can cause loss of inositol tetrakisphosphate binding in vitro (29).
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ACKNOWLEDGEMENTS |
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We thank Paul Sigler and Kathryn Ferguson for
the coordinates of the PLC-1 PH domain and for Makoto Fujii for
technical help.
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FOOTNOTES |
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* This work was supported by grants from the Hyogo Science and Technology Association (to H. Y.), Naito Foundation (to H. Y.), Ono Medical Research Foundation (to H. Y. and M. H.), and the Ryoichi Naito Foundation for Medical Research (to M. H.); and by grants-in-aid from the Ministry of Education, Science and Culture of Japan (to H. Y. and M. H.), Medical Research Council (to R. L. W.), and Cancer Research Campaign (to M. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 81-7915-8-0204; Fax: 81-7915-8-0197; E-mail: yagisawa{at}sci.himeji-tech.ac.jp.
1
The abbreviations used are: PH, pleckstrin
homology; 204BTN3,
2-O-4-{5-[2-(N-4-azidobenzoyl-N
-biotinyl-L-lysine
amido)ethyl]-2-hydroxyphenylazo}benzoyl-myo-inositol 1,4,5-trisphosphate; GST, glutathione S-transferase;
Ins(1,4,5)P3, myo-inositol 1,4,5-trisphosphate;
MDCK cell, Madin-Darby canine kidney cell; PBS, phosphate-buffered
saline; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3)P, phosphatidylinositol 3-monophosphate; PLC, phosphatidylinositol-specific phospholipase C; PAGE,
polyacrylamide gel electrophoresis; PIPES,
1,4-piperazinediethanesulfonic acid.
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REFERENCES |
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