(Received for publication, June 8, 1995; and in revised form, July 31, 1995)
From the
Pleckstrin is a substrate for protein kinase C in activated
platelets that contains at its N and C termini two of the pleckstrin
homology (PH) domains that have been proposed to mediate
protein-protein and protein-lipid interactions. We have recently shown
that pleckstrin can inhibit agonist-induced phosphoinositide hydrolysis
and that this inhibition requires an intact N-terminal PH domain
(residues 6 to 99). In the present studies, we have identified the
sites of phosphorylation in pleckstrin and examined their contribution
to pleckstrin function. In human platelets activated with thrombin or
phorbol esters, and in COS-1 cells expressing pleckstrin, a combination
of phosphopeptide analysis and site-directed mutagenesis shows that
three residues in the intervening sequence between the two pleckstrin
PH domains become phosphorylated: Ser, Thr
,
and Ser
. Replacing all three of these sites with glycine
decreased phosphorylation by >90% and reduced pleckstrin's
ability to inhibit phosphoinositide hydrolysis by as much as 80%.
Replacing the phosphorylation sites with alanine residues had a similar
effect, while substitution with aspartate, glutamate, or lysine
residues produced pleckstrin variants that were fully active even in
the absence of phosphorylation. These results suggest that
phosphorylation enhances pleckstrin's activity by introducing a
cluster of charges into a region adjacent to, but not within, the
N-terminal PH domain. This may have an allosteric effect on the
N-terminal PH domain, regulating its interaction with other molecules
necessary for the inhibition of phosphoinositide hydrolysis.
Proteins that are important in signal transduction often contain
discrete domains that mediate protein-protein interactions. Examples of
this include Src homology domains 2 and 3 (SH2 and SH3) which interact
with specific tyrosine-phosphorylated and proline-rich amino acid
sequences, respectively(1, 2) . It has been proposed
that the N and C termini of the hematopoietic protein, pleckstrin, are
the prototypes for a new family of intermolecular interaction domains,
referred to as pleckstrin homology or PH ()domains(3, 4) . Within the past year,
similar three-dimensional structures have been reported for the PH
domains from
-spectrin, dynamin, and the N terminus of pleckstrin,
supporting their status as a bona fide structural motif,
despite large differences in their primary
sequences(5, 6, 7, 8, 9, 10) .
Although it is generally believed that PH domains will turn out to
mediate intermolecular interactions and may be involved in membrane
targeting, there is as yet no consensus on the specifics of this
interaction. Both G(11) and
phosphatidylinositol 4,5-bisphosphate (PIP
) (12) have been proposed as potential partners for PH domains,
and several recent reports have suggested that the PH domain of the
-adrenergic receptor kinase may interact individually (13) or simultaneously with both(14, 15) .
Pleckstrin or p47 is a 40-kDa protein present in platelets and
leukocytes that becomes phosphorylated when platelets are activated by
agonists that directly or indirectly activate protein kinase
C(16) . Phosphoamino acid analysis and two-dimensional
electrophoresis suggest that pleckstrin is phosphorylated
heterogeneously on one or more serine and threonine residues, but not
tyrosine residues(17) . The function of pleckstrin has not been
fully characterized, but we have shown previously that when transfected
into COS-1 or HEK-293 cells pleckstrin can inhibit agonist-induced
phosphoinositide hydrolysis initiated by G-protein-coupled receptors
and growth factor receptors(18) . In that model system,
pleckstrin also inhibited the increase in inositol phosphate formation
caused by the expression of a constitutively active variant of
G
, but had no effect on G
- or
G
-mediated regulation of adenylyl cyclase. The
inhibition of phosphoinositide hydrolysis required an intact N-terminal
PH domain and was additive with that observed when mock-transfected
cells were preincubated briefly with PMA, suggesting that it is
independent of the phosphorylation of receptors, G-proteins, and
phospholipase C known to occur under the same
conditions(19, 20, 21, 22) .
Given the ability of pleckstrin's PH domains to bind to lipid
micelles containing PIP(12) , one possible
explanation for these results is that pleckstrin inhibits
phosphoinositide hydrolysis by binding to PIP
and that
phosphorylation of pleckstrin by protein kinase C promotes this
interaction. In this context, pleckstrin could play a role in the
feedback regulation of phosphoinositide hydrolysis following activation
of protein kinase C, limiting the duration of inositol
1,4,5-trisphosphate formation. Conceivably, pleckstrin might also
interact with G
, such an interaction has been
demonstrated for GST fusion proteins containing pleckstrin's PH
domains(23) , (
)but this alone would not readily
account for the observed ability of pleckstrin to inhibit
phosphoinositide hydrolysis initiated by TrkA receptors and
constitutively active G
.
In the present studies, we have identified the sites of phosphorylation within pleckstrin and examined their relationship to pleckstrin's ability to inhibit agonist-induced phosphoinositide hydrolysis. The results show that: 1) pleckstrin is variably phosphorylated on a cluster of residues located near, but not within, the N-terminal PH domain, 2) elimination of any one of these sites does not alter the overall phosphorylation of the molecule, presumably because of compensatory increases in phosphorylation at the other sites, 3) phosphorylation of pleckstrin is required for maximal inhibition of phosphoinositide hydrolysis, and 4) the effects of phosphorylation may be due largely to the introduction of charged residues into the region between the two PH domains.
CNBr mapping
was performed on immunoprecipitated pleckstrin which was gel-purified
by SDS-polyacrylamide gel electrophoresis. A gel slice containing the
phosphopleckstrin was mixed with 50 mM ammonium carbonate (pH
8.5), 0.1% SDS, 1% -mercaptoethanol. Pleckstrin contained in the
eluate was passed through glass wool, trichloroacetic
acid-precipitated, washed twice with a 50:50 mixture of cold
ethanol:ether, and then vacuum-dried. The sample was resuspended in 30
µl of 50 mg/ml CNBr in 70% formic acid and incubated at room
temperature for 1 h. At this point, the digest was lyophilized with 1
ml of distilled water and again vacuum-dried. When noted, the CNBr
fragments were further digested by incubating them overnight in the
dark at room temperature in 30 µl of 10 mg/ml iodosobenzoate (IBZO)
in 80% acetic acid with 4 M guanidine HCl(26) . All
samples were then lyophilized with 1 ml of distilled water,
vacuum-dried an additional three times, and fractionated on a Tricine
gel(27) .
Figure 1:
CNBr and IBZO digest of
[P]pleckstrin from platelets. A,
pleckstrin that had been immunoprecipitated from
P-labeled
platelets with an anti-pleckstrin antibody was digested with CNBr alone
or with CNBr followed by iodosobenzoate (IBzo). The digest was
electrophoresed on a Tricine polyacrylamide gel and analyzed by
autoradiography. B, a map of pleckstrin highlighting the CNBr
and IBZO cleavage sites, as well as the locations of the PH domains and
potential sites of phosphorylation.
Of the six
potential sites for phosphorylation, four are located in the 8-kDa
Phe-Met
CNBr fragment: Thr
,
Ser
, Thr
, and Ser
(Fig. 1). To determine which of these four sites becomes
phosphorylated, the
P-labeled CNBr fragments were
incubated with IBZO, which cleaves after tryptophan
residues(26) . IBZO is predicted to liberate two fragments of 3
kDa (Phe
-Trp
) and 5 kDa
(Val
-Met
) from the 8-kDa CNBr fragment. As
is shown in the right lane of Fig. 1A, only
the 5-kDa fragment contained
P, effectively limiting the
potential phosphorylation site(s) to residues Ser
,
Thr
, and Ser
. Although not formally
excluded by this part of the analysis, the only other serine and
threonine residues within the 5-kDa CNBr/IBZO fragment are Thr
and Ser
, neither of which are predicted sites for
phosphorylation by protein kinase C.
Figure 2:
Phosphorylation mutants in vivo. COS-1
cells were transfected with wild type pleckstrin or pleckstrin variants
in which the 3 potential sites were individually (S113G, T114G, and
S117G) or collectively (3 Gly) mutated to glycine. A shows the
relative phosphorylation of the pleckstrin variants when the cells were
labeled with [P]PO
and stimulated
with 50 nM PMA. The expressed pleckstrin was
immunoprecipitated, fractionated by SDS-polyacrylamide gel
electrophoresis, and quantitated by a PhosphorImager. B shows
a rabbit anti-pleckstrin immunoblot demonstrating equivalent
immunoprecipitation of the wild type and variant pleckstrin. C shows that the phosphorylated CNBr fragments produced from wild
type and variant pleckstrin are similar in size to each other and to
those produced from platelet pleckstrin. Note that the amount of
protein applied to lane 2 of C is 4 times that
applied to the other lanes. Despite this, there was no phosphorylation
on the major 8-kDa CNBr fragment in the triple glycine
mutant.
Previous
studies have shown that phosphorylated pleckstrin is comprised of
multiple isoforms that can be resolved by two-dimensional
electrophoresis(17) . Together with the present results, this
suggests that pleckstrin normally becomes phosphorylated on residues
Ser, Thr
, and Ser
near, but
not within, the N-terminal PH domain and that no one site is
predominant. Since elimination of any one of these sites has no effect
on overall phosphorylation, there is apparently a compensatory increase
in phosphorylation at the other sites, which is also consistent with
the observation that the stoichiometry of phosphorylation of
recombinant pleckstrin is 2:1 rather than 3:1. An alternative
interpretation of the data, which is that phosphorylation actually
occurs at unidentified sites elsewhere in the molecule and is indirectly affected by mutagenesis of Ser
,
Thr
, and/or Ser
is less likely when the
results of the mutagenesis studies are combined with those from the
CNBr/IBZO double digest of
P-labeled platelet pleckstrin.
The minor CNBr fragment that at times becomes phosphorylated,
Ser
-Ala
, accounts for <10% of total
phosphorylation and does not show increased phosphorylation when
Ser
, Thr
, and Ser
are
individually mutated.
To accomplish this, COS-1 cells were
transfected with thrombin receptors and either wild type pleckstrin or
the pleckstrin variant described above in which all three sites of
phosphorylation were mutated to glycine. In the absence of PMA, wild
type pleckstrin inhibited thrombin-induced
[H]inositol phosphate formation by 46% (Fig. 3). As we have shown previously, PMA in the absence of
pleckstrin, or pleckstrin in the absence of PMA, each inhibited
thrombin-induced phosphoinositide hydrolysis by 40-50%. The
combination of pleckstrin plus PMA was additive, inhibiting
thrombin-induced [
H]inositol phosphate formation
by 91%. Keeping in mind that thrombin, like PMA, causes the
phosphorylation of pleckstrin, we found that replacing residues
Ser
, Thr
, and Ser
with
glycine (3 Gly) reduced pleckstrin's ability to inhibit
phosphoinositide hydrolysis by at least two-thirds in either the
presence or absence of PMA (Fig. 3B). This was not due
to a decrease in the level of pleckstrin expression, which was the same
for the glycine variant as it was for wild type pleckstrin (Fig. 3C). Replacing the sites of phosphorylation with
another neutral amino acid, alanine, had a similar effect (data not
shown). These results suggest that phosphorylation is required for
maximal inhibition of thrombin-induced phosphoinositide hydrolysis by
pleckstrin, but also show that some inhibition can occur even in the
absence of phosphorylation.
Figure 3:
Effect on phosphoinositide hydrolysis of
substitution of the phosphorylation sites in pleckstrin with glycine
residues. COS-1 cells were transfected with the human thrombin
receptor, either alone or in association with either wild type
pleckstrin or variants in which Ser, Thr
,
and Ser
were replaced with glycine. A shows
total [
H]inositol phosphate formation in cells
exposed to 50 nM thrombin either with or without prior
incubation with 50 nM PMA. B shows the thrombin
response of the cells expressed as a fraction of thrombin response in
the absence of pleckstrin. C shows a typical anti-pleckstrin
immunoblot of total cell lysates from cells transfected with pleckstrin
or phosphorylation-deficient variant. Equal levels of thrombin receptor
expression were demonstrated by flow cytometry. The mean and S.E. are
derived from six to seven experiments.
Finally, to assess the importance of any one of the phosphorylation sites, COS-1 cells were transfected with the pleckstrin variants in which the phosphorylation sites were mutated to glycine individually or in pairs. The results that were obtained mirrored the effects on phosphorylation: the single-site mutants behaved like wild type pleckstrin and the double-site mutants had an intermediate effect (data not shown). These results suggest that the phosphorylation of at least two of the sites is required for maximal pleckstrin activity and that this phosphorylation can occur on any two of the three sites.
Figure 4: Effect of substitution of charged residues into pleckstrin's regulatory region. Studies identical with those in Fig. 3were performed after transfecting COS-1 cells with thrombin receptors plus either wild type pleckstrin or pleckstrin variants in which the sites of phosphorylation were replaced with glutamate (3 Glu) or lysine (3 Lys). In A, the results are expressed as a percentage of the thrombin response in cells transfected with the receptor in the absence of pleckstrin. B shows a typical anti-pleckstrin immunoblot of total cell lysates from transfected cells. The mean and S.E. are derived from three to seven experiments.
These results raise the
question of how phosphorylation regulates pleckstrin's activity.
It is conceivable that the phosphorylation of pleckstrin affects its
cellular location, in the process affecting its proposed interaction
with PIP. However, preliminary studies in platelets and
pleckstrin-transfected COS-1 cells indicate that this is not the case. (
)We have previously shown that the N-terminal PH domain of
pleckstrin is critical for its ability to inhibit phosphoinositide
hydrolysis. Since the N-terminal PH domain and the 3 sites of
phosphorylation are close to each other in the linear sequence, one
alternative explanation is that the first portion of the intervening
sequence between the PH domains obstructs the access of ligands to
binding sites within the N-terminal PH domain. Under this model,
phosphorylation relieves this obstruction, allowing the N-terminal PH
domain to interact with other molecules necessary for the inhibition of
phosphoinositide hydrolysis. Alternatively, the first part of the
intervening sequence may undergo a conformational change upon
phosphorylation that affects necessary spatial relations between the
N-terminal and C-terminal PH domains or an as-yet-unidentified
accessory protein. The PH domain from the N-terminal PH domain of
pleckstrin, like that from dynamin and
-spectrin, possesses a
highly polarized electrostatic potential. It is possible that this
asymmetry of charges contributes to an interaction between the
N-terminal PH domain and the charged residues surrounding the protein
kinase C phosphorylation site. However, regardless of which model
ultimately proves to be correct, phosphorylation appears to affect
pleckstrin activity by altering the charge distribution in a region
proximal to the N-terminal PH domain. Somewhat surprisingly, the
effects of phosphorylation could be mimicked by replacing the
phosphorylation sites with either a negatively charged glutamate
residue or a positively charged lysine residue. This suggests that it
is the presence of charged residues in this region of pleckstrin that
is important and not just the negative charge carried by the phosphate
groups.
Finally, what is the role of pleckstrin in cells that
normally contain it, such as platelets and other blood cells? The rapid
phosphorylation of pleckstrin is one of the hallmarks of platelet
activation and is thought to occur when diacylglycerol and
Ca activate protein kinase C. This suggests that
phosphorylated pleckstrin could play a regulatory role, helping to
limit the duration of agonist-induced phospholipase C activity and
perhaps preventing premature platelet activation. Whether it does so in
blood cells remains to be demonstrated.