From the
Pleckstrin is a 40-kDa protein present in platelets and
leukocytes that contains two PH domains separated by a 150-residue
intervening sequence. Pleckstrin is a major substrate for protein
kinase C, but its function is unknown. The present studies examine the
effects of pleckstrin on second messenger generation. When expressed in
cos-1 or HEK-293 cells, pleckstrin inhibited 1) the
G
Proteins that are important in signal transduction often contain
discrete domains that mediate intermolecular interactions.
Well-characterized examples of this include SH2 domains, which interact
with specific tyrosine-phosphorylated sequences, and SH3 domains, which
interact with proline-rich amino acid sequences(1, 2) .
Recently, it has been proposed that the N and C termini of pleckstrin
are the prototypes for a new family of molecular interaction domains
referred to as pleckstrin homology or PH domains
The close conservation of
three-dimensional structure among PH domains helps to establish that
these domains are a valid structural motif, but does not reveal their
function. Although the role of pleckstrin itself is unknown, it has
been proposed that PH domains may mediate either protein-protein or
protein-phospholipid interactions. An interaction with G-protein
G
Collectively,
these studies suggest that in at least some proteins PH domains play a
role in cell signaling, either by targeting G
A cDNA
fragment containing the human thrombin receptor was isolated from a HEL
cell cDNA library and inserted into the expression vector, pRK7. Other
cDNAs used in this study were generous gifts from the following
sources: M
Cos-1 cells,
which normally do not express pleckstrin, were transfected with either
plasmids encoding the human thrombin receptor alone or the thrombin
receptor plus full-length human pleckstrin. Initial studies showed that
thrombin caused a small increase in [
Although the phosphorylation of pleckstrin has long been used
as a marker for protein kinase C activation in platelets, surprisingly
little has been learned about its role. The identification of the
homologous ends of pleckstrin as the prototypes for a structural motif
that is present in a large number of signaling molecules suggests that
pleckstrin may also play a role in cell signaling. In that context, the
present studies demonstrate that when expressed in cos-1 cells,
pleckstrin inhibits 1) the G
The first issue is the mechanism of
inhibition by pleckstrin and the related issue of the identity of its
potential binding partners. Based on experiments with GST fusion
proteins, several recent studies have proposed that PH domains bind to
G
Thus, in contrast to
A second issue is the role of pleckstrin phosphorylation.
The rapid phosphorylation of pleckstrin is one of the hallmarks of
platelet activation and is thought to occur when diacylglycerol and
Ca
Finally, the structure/function
analysis contained in the present studies suggests that the inhibitory
effects of pleckstrin on agonist-induced phosphoinositide hydrolysis
require an intact PH domain at the N terminus. Mutagenesis of a highly
conserved tryptophan residue, located in the
In conclusion, these
observations show that pleckstrin can inhibit agonist-induced
phosphoinositide hydrolysis and suggest that this may be due in part to
a specific interaction between the N-terminal PH domain and
PIP
-mediated activation of phospholipase C
initiated by thrombin, M1-muscarinic acetylcholine, and
angiotensin II receptors, 2) the stimulation of phospholipase
C
by constitutively active G
, 3) the
G
-mediated activation of phospholipase C
caused by
-adrenergic receptors, and 4) the
tyrosine phosphorylation-mediated activation of phospholipase
C
caused by Trk A. However, pleckstrin had no effect on
either the stimulation or inhibition of adenylyl cyclase. The
inhibition of phosphoinositide hydrolysis caused by pleckstrin was
similar in magnitude to that caused by activating protein kinase C with
phorbol 12-myristate 13-acetate (PMA). When combined, pleckstrin and
PMA had an additive effect, inhibiting phosphoinositide hydrolysis by
as much as 90%. Structure-function analysis highlighted the role of
pleckstrin's N-terminal PH domain in these events. Although
deleting the C-terminal PH domain had no effect, deleting the
N-terminal PH domain abolished activity (but not expression) and
mutating a highly conserved tryptophan residue within the N-terminal PH
domain decreased activity by one-third. Notably, however, a pleckstrin
variant in which the N-terminal PH domain was replaced with a second
copy of the C-terminal PH domain was nearly as active as native
pleckstrin. These results show that: 1) pleckstrin can inhibit pathways
leading to both phospholipase C
- and phospholipase
C
-mediated phosphoinositide hydrolysis, 2) this
inhibition affects activation of phospholipase C
mediated by either G
or G
,
but does not affect the regulation of adenylyl cyclase activity by
G
or G
, 3) although pleckstrin is
a substrate for protein kinase C, the effects of pleckstrin and PMA are
at least partially independent, 4) the inhibition caused by pleckstrin
appears to be mediated by the PH domain at the N terminus, rather than
the C terminus of the molecule, and 5) location of the two PH domains
within the molecule clearly contributes to their individual activity.
These results do not appear to be readily attributable to an
interaction between pleckstrin and G
, but they are
consistent with a recent report showing an association between PH
domains and phosphatidylinositol 4,5-bisphosphate in vitro.
(
)(3, 4). Pleckstrin is a 40-kDa protein present in
platelets, lymphocytes, and neutrophils that is phosphorylated by
protein kinase C during platelet activation(5, 6) . When
first cloned in 1988, pleckstrin was shown to consist of 350 amino acid
residues, with an internal homology between the first and last 100
residues(7) . Recent sequence alignments have shown that regions
similar to the two pleckstrin PH domains are present in more than 70
other proteins, many of which are involved in signal transduction,
including Ras-GAP, Ras-GRF, SOS, and
-adrenergic receptor
kinase(3, 4, 8, 9, 10) .
Although in general PH domain sequences vary considerably, a tryptophan
that corresponds to Trp
in the pleckstrin N-terminal PH
domain appears to be absolutely conserved. The three-dimensional
structures of the PH domains from
-spectrin, dynamin, and the N
terminus of pleckstrin have been
determined(11, 12, 13, 14, 15, 16) .
In each case, the PH domains are predicted to assume a barrel framework
comprised of seven
-strands, with three projecting loops comprised
of nonconserved residues. The conserved tryptophan residue is located
in the
-helical cap at the base of the barrel. In the case of
pleckstrin, two of these barrel-like PH domains are located at either
end of the molecule, separated by an intervening 150-residue sequence
whose structure has not been determined.
heterodimers was initially suggested by the
observation that the region in the
-adrenergic receptor kinase
that binds G
partially overlaps with the
-adrenergic receptor kinase's PH domain(8) . In
support of this proposal, it was subsequently shown 1) that chimeric
proteins containing PH domains fused to glutathione S-transferase can capture G
from cell
lysates (17, 18, 19) and 2) that, when expressed
in mammalian cells, PH domain-containing regions of
-adrenergic
receptor kinase and Bruton's tyrosine kinase can inhibit the
ability of G
to regulate phospholipase C
and adenylyl cyclase(18, 20) . More recently, an
alternative hypothesis was suggested by Fesik and co-workers (21) who found that several PH domains, including those from
pleckstrin, can associate with lipid micelles containing small amounts
of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol
4,5-bisphosphate (PIP
). Based upon these observations, they
have proposed that PH domains target proteins to membrane
phospholipids, particularly the phosphoinositides.
or
membrane polyphosphoinositides. However, none of the studies
specifically addresses the function of the prototypical PH domain
protein, pleckstrin. Therefore, in the present studies, we have
attempted, first, to define a role for pleckstrin and, second, to
determine whether either of the two pleckstrin PH domains participate
in that role. To do this, we have examined agonist-induced second
messenger generation in cos-1 and HEK-293 cells co-expressing
pleckstrin with a variety of G-protein-coupled or growth factor
receptors or with constitutively active G
. The results
show that pleckstrin can inhibit phosphoinositide hydrolysis mediated
by both G
and G
, as well as
phosphoinositide hydrolysis stimulated by the neuronal growth factor
receptor, Trk A, but has no effect on G-protein-mediated stimulation or
inhibition of cAMP formation. These results are distinct from those
obtained with
-adrenergic receptor kinase and Bruton's
tyrosine kinase and do not appear to be readily attributable to an
interaction between pleckstrin and G
.
Mammalian Expression Vectors
DNA encoding
full-length human pleckstrin was generated by reverse
transcriptase-polymerase chain reaction from HL60 mRNA with the
following primers: ggcggcaagcttccagctgctgagaggagt and
ggcggcggatccttacttcccagttcggga. The primers incorporated a HindIII and BamHI site that facilitated cloning into
pCMV5. The C-terminal PH deletion variant (pleckstrin
246-350) was generated using the primers:
ggcggcaagcttccagctgctgagaggagt and ggcggcggatccctatctgaattcttctttcag.
The N-terminal PH deletion variant (pleckstrin
6-99) was
generated by the technique of splice overlap extension and the
mutagenesis primers ccaaagcggattaaatgcattgaagga and
gcatttaatccgctttggttccatgct(22) . The W92R variant was generated
using the mutagenesis primer ggaggagagagatgccagggttcgggat and the
technique of Landt et al.(23) . The cDNA sequences of
all clones were fully confirmed and inserted into pCMV5.
-muscarinic acetylcholine receptor, Dr. Ernest
Peralta (Harvard University, Boston, MA); angiotensin II type AT
receptor, Dr. Steven Fluharty (University of Pennsylvania,
Philadelphia, PA);
-adrenergic receptor, Dr. Robert
Lefkowitz (Duke University, Durham, NC); constitutively activated
G
variant with a hemagglutinin epitope tag
(HA-G
Q209L), Dr. J. Silvio Gutkind (National
Institutes of Health, Bethesda, MD); luteinizing hormone (LH/hCG)
receptor, Dr. Dolan Pritchett (University of Pennsylvania,
Philadelphia, PA); high affinity NGF receptor (Trk A), Dr. Luis Parada
(University of Texas, South Western Medical Center, Dallas, TX).
Inositol Phosphate Formation
Two 100-mm tissue
culture plates of cos-1 cells were transfected using DEAE-dextran (24) with either a receptor or GQ209L plus
pCMV5 containing either 1) no insert, 2) wild type pleckstrin, 3)
Trp
-Arg variant pleckstrin, 4) pleckstrin
6-99,
5) pleckstrin
246-350 or 6) a pleckstrin variant containing
two copies of the C-terminal PH domain. Twenty-four hours after
transfection, the cells were trypsinized, and the duplicate plates were
pooled. The cells were divided equally into eight 60-mm tissue culture
plates. [
H]Inositol (4 µCi/ml, ICN) was added
to six of the plates, then all of the cells were incubated at 37° C
for 18 h. The unlabeled cells were used to assess protein expression.
Forty eight hours after transfection, the six
[
H]inositol-labeled plates were divided into
three sets of duplicates which were extracted with perchloric acid
either under resting conditions, after stimulation by thrombin (2
units/ml) for 45 min, or after preincubation with 50-100 nM PMA for 5 min followed by stimulation with thrombin, all in the
presence of 20 mM LiCl. In other experiments, carbachol (100
µM), angiotensin II (1 µM), UK14304 (10
µM), or NGF (100 ng/ml) was used as the agonist. The
neutralized extracts were applied to Dowex 1 columns, which were washed
sequentially with 5 mM inositol and 5 mM sodium
tetraborate, 60 mM ammonium formate. Total
[
H]inositol phosphate was eluted with 0.1 M formic acid plus 1.5 M ammonium formate and quantitated
by scintillation counting.
cAMP Formation
Cyclic AMP production was
determined as described by Shimizu et al.(25) .
Briefly, cos-1 or HEK-293 cells were transfected with
-adrenergic receptors and LH/hCG receptors as
described above, and 24 h later were trypsinized and divided equally
into 24-well plates. After being incubated for an additional 18 h,
duplicate wells were labeled for 2 h with 2 µCi of
[
H]adenine (DuPont NEN). The cells were
stimulated with LH and/or UK14304 for 30 min, either with or without 5
min of prior exposure to 100 nM PMA in the presence of 1
mM isobutylmethylxanthine. The reactions were stopped with 1
ml of ice-cold 5% trichloroacetic acid, and the
[
H]cAMP and [
H]ATP in the
supernatant were separated by Dowex and alumina chromatography. cAMP
production was quantitated by scintillation counting and expressed as %
conversion: ([
H]cAMP
100)/([
H]cAMP +
[
H]ATP).
Other Methods and Materials
Pleckstrin expression
was confirmed by Western blotting of cell lysates after polyacrylamide
gel electrophoresis in SDS using a rabbit polyclonal antiserum (number
354) raised against a recombinant protein corresponding to pleckstrin
residues Glu-Asp
. Trk A and
HA-G
Q209L expression were measured by Western blotting
with antibodies SC11 (Santa Cruz Biotechnology) and 12CA5 (Boehringer
Mannheim), respectively. Thrombin receptor expression was confirmed by
flow cytometry using antibody ATAP2, a peptide-directed monoclonal
antibody that binds to the N terminus of the human thrombin receptor
and recognizes both the cleaved and intact forms of the
receptor(26) . Highly purified human thrombin was a gift from
Dr. John Fenton (New York Public Health Service, Albany, NY). Other
agonists and materials were obtained from the following: hCG and NGF
(Boehringer Mannheim) and UK14304 (Research Biochemical Inc., Natick,
MA). All other reagents were from Sigma.
Pleckstrin Inhibits Thrombin-induced Phosphoinositide
Hydrolysis
When platelets are activated by most agonists,
including thrombin, pleckstrin becomes phosphorylated by protein kinase
C(5, 27) . Since thrombin-induced phosphoinositide
hydrolysis is mediated by a G-protein-coupled receptor (28) and
involves two of the molecules that have been proposed as binding
partners for PH domains, G and PIP
, we
started by asking whether the presence of pleckstrin would affect
signaling initiated by thrombin. Because pleckstrin is a substrate for
protein kinase C, the effects of pleckstrin were compared in the
presence and absence of the phorbol ester, PMA, which has been shown to
acutely inhibit agonist-induced phosphoinositide hydrolysis in a
variety of cells, including
platelets(29, 30, 31) . This effect has been
variously attributed to the phosphorylation of receptors, G
and phospholipase
C(32, 33, 34, 35) .
H]inositol
phosphate formation in nontransfected cos-1 cells and a much larger
increase in cells transfected with the thrombin receptor. This response
was minimally affected by a 16-h incubation with 100 ng/ml pertussis
toxin, suggesting that it is due to phospholipase C
activation mediated by a member of the G
family
rather than G
(data not shown). In the studies shown in Fig. 1, total [
H]inositol phosphate
formation was assayed in cells that were incubated with thrombin either
with or without prior exposure to PMA. Thrombin receptor expression was
determined by flow cytometry using the receptor-directed monoclonal
antibody, ATAP2(26) . Pleckstrin expression was measured using a
polyclonal antibody directed at the entire intervening sequence between
the two PH domains. In the absence of PMA or pleckstrin, thrombin
increased [
H]inositol phosphate levels by 3- to
4-fold (Fig. 1A). Co-expression of pleckstrin decreased
thrombin-induced [
H]inositol phosphate formation
by 33 ± 3% compared with cells transfected with the receptor
alone (Fig. 1B). Preincubating the cells with PMA
inhibited [
H]inositol phosphate formation by 38
± 1% in the absence of pleckstrin and by 81 ± 2% in the
presence of pleckstrin. Similar results were obtained with HEK-293
cells (not shown). These effects of pleckstrin were not due to a
decrease in the level of thrombin receptor expression, which was the
same in the presence or absence of pleckstrin, nor were they due simply
to the presence of a second expressed protein (see below). Therefore,
these results suggest that pleckstrin, like PMA, inhibits
thrombin-induced phosphoinositide hydrolysis. Furthermore, since the
inhibitory effects of pleckstrin and PMA were additive, they are
presumably at least partly independent of each other.
Figure 1:
Effect of pleckstrin on
thrombin-induced inositol phosphate formation. Cos-1 cells were
transfected with the human thrombin receptor (Thr-R), either
alone or in association with wild type pleckstrin (Pleck). A shows total [H]inositol phosphate
formation in cells exposed to thrombin (2 units/ml) for 45 min either
with or without prior incubation with 50 nM PMA for 5 min. B shows the relative thrombin-induced
[
H]inositol phosphate formation in cells with or
without pleckstrin, expressed as a percentage of the response obtained
in the absence of pleckstrin or PMA. Equal levels of thrombin receptor
expression were demonstrated by flow cytometry. The results shown are
the mean ± S.E. from 20 experiments.
Pleckstrin Inhibits Inositol Phosphate Formation
Initiated by Other G-protein-coupled Receptors
In order to
determine whether the inhibitory effects of pleckstrin are limited to
thrombin responses, similar studies were performed with cos-1 cells
expressing M-muscarinic acetylcholine receptors,
angiotensin II type AT
receptors, or
-adrenergic receptors. For the first two receptors,
phosphoinositide hydrolysis was unaffected by pertussis toxin,
suggesting that it is mediated by a member of the G
family.
Phosphoinositide hydrolysis in response to the
-adrenergic receptor agonist, UK14304, on the other
hand, was inhibited approximately 90% by pertussis toxin, suggesting
that it is due to phospholipase C
activation mediated
by G
derived from G
(not shown). In
the studies shown in Fig. 2, each of the receptors was tested in
the presence and absence of pleckstrin. In all cases, pleckstrin
inhibited [
H]inositol phosphate formation, as did
PMA, and the combination was additive. The extent of inhibition varied.
Angiotensin II and UK14304, like thrombin, were inhibited by as much as
90% by the pleckstrin-PMA combination, but the response to carbachol
was inhibited by only 42%. Thus, taken together, the data in Fig. 1and Fig. 2show that pleckstrin inhibits
phosphoinositide hydrolysis initiated by at least four different
G-protein-coupled receptors and affects both G
- and
G
-mediated activation of phospholipase C.
Figure 2:
Effect of pleckstrin on other
G-protein-coupled receptors. Cos-1 cells were transiently transfected
with M-muscarinic acetylcholine receptor (M1-R),
angiotensin II type AT
receptor (AT-R)
or
-adrenergic receptor (
-R) either
with or without pleckstrin. Total [
H]inositol
phosphate formation was measured in response to carbachol (100
µM), angiotensin II (1 µM), or UK14304 (10
µM) for 45 min either with or without prior exposure to
100 nM PMA for 5 min. The data shown are means ± S.E.
from five experiments for M
-muscarinic acetylcholine
receptor, three experiments for the angiotensin II receptor, and four
experiments for the
-adrenergic receptor. In each
case, the results are expressed as a percentage of the response
obtained in the absence of pleckstrin or PMA.
, receptor
alone;
, PMA;
, pleckstrin;
, pleckstrin +
PMA.
Inhibition by Pleckstrin Is
Receptor-independent
To further explore the mechanism by which
pleckstrin inhibits phosphoinositide hydrolysis, additional experiments
were performed in which phospholipase C was stimulated with a
constitutively active form of G, bypassing the
involvement of a receptor. In these studies, cos-1 cells were
transfected with a G
variant in which the substitution
of leucine for Glu
inhibits GTPase
activity(36, 37) . As shown in Fig. 3A,
G
Q209L stimulated phospholipase C activity, raising
total [
H]inositol phosphate levels
15-20-fold above those present in mock-transfected cells. This
increase was only minimally affected by PMA, either because the effects
of PMA are receptor-dependent or because protein kinase C is already
maximally stimulated by the diacylglycerol produced from constitutive
breakdown of the phosphoinositides. On the other hand, pleckstrin
inhibited G
Q209L-induced
[
H]inositol phosphate formation by 40% in the
absence of PMA and by 55% in the presence of PMA. These differences
were not attributable to differences in the level of
G
Q209L expression, which was the same in the presence
or absence of pleckstrin (Fig. 3C). This result suggests
that the inhibition of phosphoinositide hydrolysis by pleckstrin occurs
at or below the level of G-proteins in the signal transduction cascade.
Figure 3:
Pleckstrin inhibits stimulation of
phospholipase C by constitutively active G. Cos-1
cells were transfected with G
Q209L, either alone or
with wild type pleckstrin. A shows total
[
H]inositol phosphate formation in the
transfected cells either before or after a 5-min incubation with 100
nM PMA. The data are from three experiments and are expressed
as a -fold increase over the results obtained in mock-transfected
cells. B shows the data expressed as a percentage of the
[
H]inositol phosphate levels present in cells
transfected with G
Q209L in the absence of pleckstrin
or PMA. C is an immunoblot demonstrating equal expression of
G
Q209L in the presence or absence of
pleckstrin.
Pleckstrin Does Not Affect cAMP Formation
To
investigate whether pleckstrin also affects other signaling events
mediated by G-protein-coupled receptors, cAMP formation was measured in
cos-1 and HEK-293 cells expressing LH receptors and
-adrenergic receptors. LH receptors are coupled to
G
and stimulate adenylyl cyclase(38) , while
-adrenergic receptors inhibit cAMP formation via one
or more members of the G
family(20, 39) . In
cells expressing the LH receptor, hCG caused a marked increase in cAMP
formation (Fig. 4A). This increase was unaffected by
either pleckstrin or PMA. Pleckstrin and PMA also had no effect on the
suppression of cAMP formation seen in cells co-expressing LH and
-adrenergic receptors (Fig. 4B). Taken
together, these results show that pleckstrin affects G-protein-mediated
regulation of phosphoinositide hydrolysis, but not G-protein-mediated
regulation of cAMP formation.
Figure 4:
Pleckstrin does not affect cAMP formation.
In A, cos-1 cells expressing LH receptors with or without
pleckstrin were loaded with [H]adenine,
pretreated with or without 100 nM PMA for 5 min, and then
stimulated with hCG (50 ng/ml) for 30 min.
[
H]cAMP formation (% conversion) was measured as
described under ``Experimental Procedures.'' In B,
HEK-293 cells expressing LH and
-adrenergic receptors
with or without pleckstrin were stimulated with hCG (5 ng/ml) and
UK14304 (10 µM) with or without prior incubation with PMA.
The data shown are the mean ± S.E. of three
experiments.
Pleckstrin Inhibits Phosphoinositide Hydrolysis Activated
by a Growth Factor Receptor
The data described thus far focus on
pathways that activate phospholipase C. To determine
whether pleckstrin also affects phosphoinositide hydrolysis by
phospholipase C
, cos-1 cells were transfected with
plasmids encoding the high affinity NGF receptor, Trk A, either alone
or with pleckstrin. As is shown in Fig. 5A, NGF had no
effect on [
H]inositol phosphate formation in
mock-transfected cells, but caused a 1.7-fold increase in cos-1 cells
expressing Trk A, a response similar to that previously reported in
PC12 cells(40) . Pleckstrin inhibited this increase by 55%,
while having no effect on Trk A expression (Fig. 5, B and C).
Figure 5:
Pleckstrin affects phospholipase
C-mediated inositol phosphate production. Cos-1 cells
were transfected with the high affinity NGF receptor, Trk A, either
alone or with pleckstrin (Pleck) variants. A shows
total [
H]inositol phosphate formation in cells
exposed to 100 ng/ml NGF either with or without a 4-h prior incubation
with 200 ng/ml pertussis toxin (PTX). B shows the
relative NGF-induced [
H]inositol phosphate
formation in cells with or without pleckstrin and pertussis toxin. C demonstrates equal levels of protein expression by anti-Trk
A and anti-pleckstrin immunoblots from a typical experiment. The data
shown are the mean ± S.E. of 3-7
experiments.
This suggests that pleckstrin can inhibit
phospholipase C, as well as phospholipase
C
. However, earlier studies have shown that some growth
factor receptors can stimulate phosphoinositide hydrolysis through a
pertussis toxin-sensitive mechanism, implying that in addition to
activating phospholipase C
by direct phosphorylation
some growth factors activate phosphoinositide hydrolysis via
G
(41, 42). To test whether NGF can do the same,
cos-1 cells expressing Trk A were incubated with pertussis toxin. Under
conditions in which pertussis toxin inhibited UK14304-induced
phosphoinositide hydrolysis by 90% (data not shown), the response to
NGF was inhibited by 51%. The combination of pleckstrin and pertussis
toxin had an even greater effect (91% inhibition), showing that
pleckstrin can inhibit the portion of the NGF stimulation of
phospholipase C that is not mediated by a pertussis toxin-sensitive
G-protein.
The Role of the Pleckstrin PH Domains
To begin the
process of understanding the structural basis for the inhibition of
phosphoinositide hydrolysis by pleckstrin, two variants of pleckstrin
were prepared in which either the complete N-terminal PH domain
(6-99) or the complete C-terminal PH domain
(
246-350) was deleted. As seen in Fig. 6, pleckstrin
6-99 was unable to inhibit thrombin-induced phosphoinositide
hydrolysis, while pleckstrin
246-350 was as active as the
intact molecule. Pleckstrin
6-99 was also unable to inhibit
phosphoinositide hydrolysis in response to NGF (Fig. 5). However,
based on immunoblotting, all three forms of pleckstrin were expressed
to the same extent (Fig. 6D).
Figure 6:
The effect of pleckstrin variants on
thrombin-induced inositol phosphate formation. Cos-1 cells were
transfected with the human thrombin receptor, either alone or in
association with wild type pleckstrin (WT), or with pleckstrin
variants lacking the complete N-terminal (6-99) or
C-terminal (
246-350) PH domains. A shows a
schematic of the pleckstrin deletion variants. B shows total
[
H]inositol phosphate formation. C shows
the relative effect of thrombin-induced
[
H]inositol phosphate formation caused by the
addition of pleckstrin variants compared to cells with no pleckstrin.
Where indicated, 50 nM PMA was added 5 min before thrombin (2
units/ml). D demonstrates comparable levels of wild type and
variant pleckstrin expression as detected by an anti-pleckstrin
immunoblot. Equal levels of thrombin receptor expression were
demonstrated by flow cytometry. The data shown are the mean ±
S.E. of four experiments.
To further explore the
role of the N-terminal PH domain, residue Trp was mutated
to arginine. This conserved tryptophan is the one invariant residue
found in every PH domain identified to date and is postulated to play a
role in the structural framework of the PH domain(8) . As is
shown in Fig. 7, the Trp
-Arg (W92R) pleckstrin
variant inhibited thrombin-induced inositol phosphate formation only
two-thirds as well as wild type pleckstrin (22 ± 5% versus 34 ± 4%, p < 0.04). Taken together, these
results suggest that a region necessary for pleckstrin's ability
to regulate phosphoinositide hydrolysis includes the N-terminal PH
domain, and that the C-terminal PH domain is not required. However,
since the two pleckstrin PH domains are 30% identical and 50% similar,
and since both domains have been shown to bind to phospholipid
vesicles(21) , we next asked whether we could restore activity
to the N-terminal PH deletion variant by adding back a second copy of
the C-terminal PH domain. This ``double C-PH'' variant
inhibited thrombin-induced inositol phosphate formation better than the
6-99 N-terminal deletion variant, but not quite as well as
did wild type pleckstrin: 29 ± 2% (double C-PH) versus 34 ± 4% (wild type) in the absence of PMA and 63 ±
2% versus 77 ± 1% in the presence of PMA (n = 3) (Fig. 8). This suggests that location of the PH
domains within the molecule, as well as their sequence, contributes to
activity.
Figure 7:
Effect of mutating a conserved tryptophan
residue in the N-terminal PH domain. Cos-1 cells were co-transfected
with the human thrombin receptor and either wild type pleckstrin (WT) or pleckstrin variant containing a Trp-Arg
mutation (W92R). Effects on thrombin-induced
[
H]inositol phosphate formation expressed in A shows total [
H]inositol phosphate
formation. B shows the relative effect of thrombin-induced
[
H]inositol phosphate formation caused by the
addition of pleckstrin W92R compared to cells transfected with wild
type pleckstrin. C shows comparable levels of pleckstrin or
variant expression by anti-pleckstrin immunoblot on a typical
experiment. Equal levels of thrombin receptor expression were
demonstrated by flow cytometry. The mean and S.E. are derived from
three experiments.
Figure 8:
Effect of replacing the N-terminal PH
domain with a second copy of the C-terminal PH domain. Cos-1 cells were
co-transfected with the human thrombin receptor and either wild type
pleckstrin (WT) or pleckstrin variant containing two copies of
the C-terminal PH domain (Double C-PH). A schematic showing
the double C-PH pleckstrin variant is shown in A. Effects on
thrombin-induced [H]inositol phosphate formation
expressed in B shows total [
H]inositol
phosphate formation. C shows the relative effect of
thrombin-induced [
H]inositol phosphate formation
caused by the addition of the double C-PH variant compared to cells
transfected with wild type pleckstrin. D demonstrates
comparable levels of wild type and variant pleckstrin expression as
detected by an anti-pleckstrin immunoblot. Equal levels of thrombin
receptor expression were demonstrated by flow cytometry. The data shown
are the mean ± S.E. of three
experiments.
-mediated activation of
phospholipase C
initiated by thrombin,
M
-muscarinic acetylcholine, and angiotensin II receptors,
2) the activation of phospholipase C
by
G
Q209L, 3) the G
-mediated
activation of phospholipase C
caused by
-adrenergic receptors, and 4) the activation of
phospholipase C
caused by Trk A. However, under the
same conditions, pleckstrin has no effect on the regulation of adenylyl
cyclase by LH and
-adrenergic receptors mediated by
either G
or G
. These observations raise a
number of questions, including the mechanism by which pleckstrin
inhibits signaling, the contribution of pleckstrin's two PH
domains in this process, and the impact of the phosphorylation of
pleckstrin by protein kinase C.
and possibly to other proteins containing the
WD-40 motif as well(17, 19) . Among the proteins that
have been studied in this manner are
-adrenergic receptor kinase
and Bruton's tyrosine kinase. Evidence obtained prior to the
recognition of PH domains had suggested that
-adrenergic receptor
kinase interacts with and is regulated by G
(43).
Subsequent studies showed that expression of all or part of
-adrenergic receptor kinase or Bruton's tyrosine kinase in
mammalian cells will inhibit G
-mediated activation
of phospholipase C(18, 20) . Inhibition of
G
-mediated phospholipase C
activation
was either not found (20) or not sought(18) , and effects
on phospholipase C
were not examined. Of note, however,
the
-adrenergic receptor kinase fragment that was studied included
only part of the
-adrenergic receptor kinase PH domain, and
results similar to those found with
-adrenergic receptor kinase
have been reported recently with phosducin, a
G
-binding protein that does not contain a PH
domain(44) .
-adrenergic receptor
kinase and phosducin, the inhibitory effects of pleckstrin are not
limited to signaling events mediated by G
. In
fact, given 1) the lack of an effect of pleckstrin on adenylyl cyclase
regulation, 2) the results obtained with G
Q209L, and
3) the inhibition of phosphoinositide hydrolysis stimulated by Trk A,
our data are more consistent with an interaction between pleckstrin and
either phospholipase C or PIP
than with an interaction
between pleckstrin and G
. Notably, a recent study
by Fesik and co-workers (21) has shown that PH domains,
including the two pleckstrin PH domains, can associate with micelles
containing PIP
, suggesting that PH domains are phospholipid
binding motifs(21) . If so, then one possible mechanism for the
inhibition of phosphoinositide hydrolysis by pleckstrin would be
competition between pleckstrin and the phospholipases for access to
PIP
, particularly if the interaction with PIP
was mediated in part by PH domains present in these proteins. PH
domains have been described in the
,
, and
forms of
phospholipase C (45) and, in at least one recent study, a
proteolytic fragment of phospholipase C
lacking the
first 60 residues of its PH domain was found to be profoundly impaired
in its ability to bind to PIP
(46) . This would also
suggest that PH domains bind to PIP
in vivo.
However, whether or not pleckstrin ultimately proves to interact with
PIP
, its ability to inhibit phosphoinositide hydrolysis
provides a potential regulatory mechanism that can inhibit signaling
through both G-protein-coupled receptors and growth factor receptors
that targets a specific class of effectors, particularly since its
activity is enhanced by phosphorylation (see below). Notably, other
proteins that can bind to PIP
, such as the actin filament
regulatory protein, profillin, have also been shown to inhibit
phosphoinositide hydrolysis by phospholipase C(47) . However,
unlike pleckstrin, the inhibition by profillin seemed to be limited to
hydrolysis mediated by phospholipase C
. This difference
in specificity may be due to the mechanism by which profillin interacts
with PIP
, since profillin associates with PIP
via a peptide sequence that does not contain a recognized PH
domain.
activate protein kinase C(5, 27) .
Phosphoamino acid analysis shows that pleckstrin is phosphorylated
exclusively on serine and threonine residues and occurs at multiple
sites(6) . One plausible hypothesis is that the phosphorylation
of pleckstrin promotes its activity. In the transfected cos-1 and
HEK-293 cells, phosphorylation could occur either by stimulating
protein kinase C with PMA or by activating protein kinase C as a
consequence of phosphoinositide hydrolysis. If the phosphorylation of
pleckstrin were critical for its activity, then the latter would
account for the inhibitory effects of pleckstrin in the absence of PMA.
In preliminary studies, we have found the phosphorylation sites in
pleckstrin are outside the PH domains, and that mutagenesis of these
sites results in a 50% decrease in the ability of pleckstrin to inhibit
phosphoinositide hydrolysis.
(
)This suggests that
phosphorylation is one of the factors affecting pleckstrin function,
but not the only factor. Since PMA induces inhibition of
phosphoinositide hydrolysis in the absence of pleckstrin, protein
kinase C must exert this effect by phosphorylating other participants
in the signaling cascade, or, more intriguingly, by phosphorylating
unidentified pleckstrin homolog(s).
-helical cap of the
PH barrel framework, partially blocked the inhibitory effects of
pleckstrin, while deletion of the N-terminal PH domain completely
suppressed inhibition. Interestingly, while deletion of the C-terminal
PH domain had no effect, suggesting that it is not directly involved in
pleckstrin function, replacing the N-terminal PH domain with a second
copy of the C-terminal PH domain produced a variant molecule nearly as
active as native pleckstrin. This is reminiscent of the modular nature
of SH2 or SH3 domains, and suggests that the location of the PH domain
at the N terminus of pleckstrin is important for its activity. It is
also consistent with the observation that isolated N- and C-terminal
pleckstrin PH domains bind PIP
in lipid micelles with
comparable affinity(21) . This leaves the role of the second PH
domain in pleckstrin an open question.
. The results also suggest that this interaction may in
some way be enhanced by the phosphorylation of pleckstrin's
intervening sequence by protein kinase C. How this process occurs and
whether or not the hypothesized interaction between pleckstrin and
PIP
has additional purposes remains to be determined.
, phosphatidylinositol 4,5-bisphosphate;
NGF, nerve growth factor.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.