* Department of Medicine, Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania 19104
Pleckstrin homology (PH) domains are sequences of ~100 amino acids that form "modules" that
have been proposed to facilitate protein/protein or protein/lipid interactions. Pleckstrin, first described as a
substrate for protein kinase C in platelets and leukocytes, is composed of two PH domains, one at each end
of the molecule, flanking an intervening sequence of
147 residues. Evidence is accumulating to support the
hypothesis that PH domains are structural motifs that
target molecules to membranes, perhaps through interactions with G or phosphatidylinositol 4,5-bisphosphate (PIP2), two putative PH domain ligands. In the
present studies, we show that pleckstrin associates with
membranes in human platelets. We further demonstrate that, in transfected Cos-1 cells, pleckstrin associates with peripheral membrane ruffles and dorsal membrane projections. This association depends on
phosphorylation of pleckstrin and requires the presence of its NH2-terminal, but not its COOH-terminal, PH domain. Moreover, PH domains from other molecules cannot effectively substitute for pleckstrin's NH2terminal PH domain in directing membrane localization. Lastly, we show that wild-type pleckstrin actually
promotes the formation of membrane projections from
the dorsal surface of transfected cells, and that this
morphologic change is similarly PH domain dependent.
Since we have shown previously that pleckstrin-mediated inhibition of PIP2 metabolism by phospholipase C
or phosphatidylinositol 3-kinase also requires pleckstrin phosphorylation and an intact NH2-terminal PH
domain, these results suggest that: (a) pleckstrin's NH2terminal PH domain may regulate pleckstrin's activity
by targeting it to specific areas within the cell membrane; and (b) pleckstrin may affect membrane structure, perhaps via interactions with PIP2 and/or other
membrane-bound ligands.
Signal transduction pathways frequently use proteins
that contain discrete domains that can facilitate intermolecular interactions. Well-characterized examples of such protein "modules" include the Src homology 2 and phosphotyrosine binding domains, which target phosphorylated tyrosine residues (21, 40), and Src homology 3 domains, which bind polyproline sequences, (21, 36). Pleckstrin homology (PH)1 domains, first described as 100-
amino acid sequences found at the amino and carboxyl
termini of the hematopoietic protein, pleckstrin, have
been proposed as a new class of functional domains. The
structures of PH domains from dynamin, spectrin, phospholipase C Pleckstrin is a 40-kD protein found solely in cells of hematopoietic origin and was first described as a major substrate for protein kinase C in human platelets (15). When
expressed in Cos-1 cells, pleckstrin can inhibit phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis initiated by
both G protein-coupled and growth factor receptors (1).
This effect is dependent on the presence of an intact PH
domain at the amino terminus of pleckstrin and can be
regulated by phosphorylation of the molecule (2). Recombinant phospho-pleckstrin can also inhibit G More than 100 proteins have been reported to contain
PH domains. Among these are signaling molecules such as
Ras-GAP, Ras-GRF, Sos, Expanding the hypothesis that PH domains direct membrane targeting, we show in this report that pleckstrin
interacts with membranes in human platelets. We also
demonstrate that pleckstrin associates with peripheral membrane ruffles and membrane projections at the dorsal surface of transfected Cos-1 cells. The localization appears to
be regulated by pleckstrin's sites of phosphorylation and
to require the presence of the native NH2-terminal PH domain, the same structural features that we have previously
shown to be necessary for pleckstrin's inhibition of PIP2
metabolism. Moreover, pleckstrin also appears to promote
the formation of membranous projections from the dorsal
surface of transfected cells, thus suggesting a new role for
pleckstrin in mediating morphologic changes at the cell
membrane.
Platelet Fractionation
Human platelets from normal volunteers were isolated by differential centrifugation of whole blood anticoagulated with acidified citrate dextrose
(85 mM sodium citrate, 11 mM dextrose, 71 mM citric acid). Platelets were
washed in Hepes/Tyrode buffer (129 mM NaCl, 8.9 mM NaHCO3, 2.8 mM
KCl, 0.8 mM KH2PO4, 56 mM dextrose, 10 mM Hepes, pH 7.4, 0.8 mM MgCl2)
with 1 µM prostaglandin E1, resuspended in ice-cold 30 mM KCl, 5 mM
Pipes, pH 6.5, 5 mM MgCl2, 2 mM DTT, 2 mM PMSF, 2 mM vanadate,
0.2% aprotinin, and 1 µg/ml leupeptin, and then lysed by nitrogen cavitation. The lysate was spun first at 500 g for 15 min to remove intact cells.
The supernatant from this low speed spin was then spun at 100,000 g for
60 min, and the high speed membrane pellet was separated from the supernatant. The high speed pellet was then extracted with 1% Triton X-100 in 30 mM KCl, 5 mM Pipes, pH 6.5, 5 mM MgCl2, 2 mM DTT, 2 mM
PMSF, 2 mM vanadate, 0.2% aprotinin, and 1 µg/ml leupeptin, and then spun again at 100,000 g for 30 min. The Triton-soluble supernatant was
separated from the Triton-insoluble pellet. Protein concentrations in each
sample were determined using a bicinchoninic microassay kit (Pierce
Chemical Co., Rockford, IL), and 50 µg of protein from each sample was
run on SDS-PAGE gels and transferred to nitrocellulose membranes,
which were then probed with either rabbit anti-pleckstrin antisera or an
antibody to the well-described membrane-bound protein, Gq Lactate dehydrogenase activity assays, used to demonstrate that cytoplasmic contents were not trapped in the membrane fraction, were performed as follows: 100 µg of protein, 0.17 mg of NADH, and 80 µg of
pyruvate (Sigma Chemical Co., St. Louis, MO) were simultaneously
added to a cuvette containing 1 ml of 20 mM Hepes, pH 7.0, and the absorbance at 342 Å was measured every 15 s.
Construction of Pleckstrin Expression Vectors
The generation of expression plasmids encoding full-length human pleckstrin, the NH2-terminal PH deletion variant ( Cell Culture and Transient Transfections
Cos-1 cells (American Type Cell Culture, Rockville, MD) were grown in
DME with 10% FBS (GIBCO BRL, Gaithersburg, MD) and 1% penicillin-streptomycin (GIBCO BRL) under 5.5% CO2. Plasmid DNA coprecipitated with calcium phosphate was layered on Cos-1 cells for 24 h before
cells were shocked with 10% glycerol for 2 min. Cells were washed three
times with DME, trypsinized (GIBCO BRL), and then replated onto two-well
chamber slides (Nunc, Naperville, IL) in preparation for immunostaining.
Antibody Preparation
Rabbit polyclonal antiserum (No. 354) was raised against a recombinant
protein corresponding to pleckstrin residues Glu104-Asp233. Murine ascites
from hybridoma cells expressing the mAb 12CA5 against the hemagglutinin antigen were purified on a protein A column (Affi-Gel; Bio Rad Laboratories, Hercules, CA), concentrated to 1.57 mg/ml, and stored at 4°C.
The anti-Gq Indirect Immunofluorescence
48 h after transient transfection, slides were washed with PBS, and then
fixed with freshly prepared 4% paraformaldehyde, 100 mM Pipes, pH 6.8, 2 mM EGTA, 2 mM MgCl2 for 30 min. Three washes in 150 mM Tris HCl,
pH 7.4, were performed to quench free aldehyde groups, followed by 10min permeabilization with 0.2% Triton X-100 in PBS. Cells were blocked
with 10% normal goat serum (Biosource Intl., Camarillo, CA) in PBS for
30 min at room temperature. 12CA5 antibody, prepared as above, was diluted 1:100 in 10% normal goat serum in PBS and applied to cells for 1 h
at 37°C. Cells were washed in PBS, and then incubated at 37°C for 1 h in
affinity-purified, FITC-labeled goat F(Ab)2 anti-mouse antisera (Biosource Intl.) diluted 1:200 in 10% normal goat serum in PBS. Slides were
washed in PBS, and then mounted in Citifluor antifadant mounting material (University of Kent, Canterbury, United Kingdom).
For fluorescent double labeling of the cell surface and intracellular
pleckstrin, living transfected cells on chamber slides were washed with
PBS at room temperature, cooled on ice for 3 min, and then externally labeled with TRITC-conjugated WGA (Sigma Chemical Co.). After washing in PBS, cells were fixed, permeabilized, and stained as before.
Two methods were used for image collection and analysis. Conventional fluorescence microscopy was performed using a Microphot-SA microscope and camera (Nikon Inc., Garden City, NJ). We also used the
resources of the University of Pennsylvania Cancer Center Confocal Microscopy Core Facility. Confocal images were acquired from a 4D Upright
microscope (TCS-Medical Products Co., Huntington Valley, PA) and processed on an IBM OS9 workstation (IBM Instruments, Inc., Danbury,
CT), using Scanware software. Three-dimensional reconstruction was performed using a 3D software package provided to the Confocal Core on
loan from Leica Inc. (Deerfield, IL). All light microscopic figures were
shot with a ×40 objective.
In Vivo Phosphorylation Studies
48 h after transient transfection, cells were incubated for 4 h in phosphatefree DME, containing 0.25 mCi/ml of 32Pi. Cells were lysed in Frackleton
buffer (10 mM Tris, pH 7.6, 50 mM NaCl, 30 mM sodium pyrophosphate,
50 mM NaF) with 1% Triton X-100, 1 mM PMSF, 0.1% aprotinin, and 25 µg/ml leupeptin. After cell lysis, pleckstrin and pleckstrin variants were
immunoprecipitated with 12CA5 and run on a polyacrylamide gel. Counts
on the dried gel were quantified using a phosphor imager (Molecular Diagnostics, Sunnyvale, CA) to determine the degree of phosphorylation of
the different pleckstrin variants.
Pleckstrin Associates with Membrane
in Human Platelets
Pleckstrin is a major constituent of human platelets, comprising 0.65% of all platelet protein (26). To determine the
subcellular localization of pleckstrin within human platelets, platelet lysates were spun at 100,000 g and separated
into particulate and supernatant fractions. Equivalent
amounts of protein were run on SDS-PAGE gels and immunoblotted with the anti-pleckstrin antisera. Though pleckstrin has previously been reported as a cytosolic
platelet protein (19, 26), the anti-pleckstrin immunoblot
shown in Fig. 1 A shows that pleckstrin can be detected in
both the membrane pellet and the cytosolic supernatant of
human platelets. When this pellet was extracted with 1%
Triton X-100, the pleckstrin was found predominantly in
the Triton-soluble fraction, consistent with membrane association (Fig. 1 B).
To demonstrate that the supernatant fraction did not
contain membrane components, we performed immunoblotting with antisera to the membrane-associated protein,
Gq Pleckstrin Associates with Peripheral
Membrane Ruffles and Dorsal Membrane Projections
in Transfected Cos-1 Cells
Having determined that pleckstrin associates with platelet
membranes, we next sought to identify the regions of
pleckstrin required for this interaction. To do this, we used
indirect immunofluorescence to study the intracellular location of hemagglutinin (HA) epitope-tagged wild-type
and variant pleckstrins expressed in transfected Cos-1 cells. Fig. 2 shows schematic representations of the different pleckstrin variants used in this series of experiments.
The attachment of the HA tag has been shown to have no
effect on the ability of the pleckstrin variants to inhibit
PLC activity (Abrams, C., unpublished observation).
Expressed wild-type pleckstrin was found diffusely
throughout the cytoplasm but concentrated most intensely
around the plasma membrane, where it associated with areas of membrane folding or ruffles along the periphery of
the cell (Fig. 3 a). In general, cells expressing wild-type
pleckstrin were flatter and broader than mock-transfected
cells or untransfected cells. Notably, cells transfected with
wild-type pleckstrin also demonstrated membranous projections from their dorsal surface, and pleckstrin was also concentrated within these structures (Fig. 3 a). Confocal
microscopy with three-dimensional reconstruction of images confirmed that these regions of intense pleckstrin
staining were projections from the surface of the cell,
rather than intracytoplasmic inclusions (Fig. 4 a).
The NH2-terminal PH Domain Is Required for
Membrane Localization
To determine whether pleckstrin was associating with
membranes via its PH domains, Cos-1 cells were transfected with a variant of pleckstrin lacking both PH domains
( To examine the relative contribution from each of pleckstrin's two PH domains to its membrane localization, pleckstrin mutants individually deleted of either the NH2-terminal PH domain ( Pleckstrin Must Be Phosphorylated to Associate
with Membranes
Pretreating Cos-1 cells with 50 nM PMA affected neither
the morphology of the cell nor the localization of expressed wild-type pleckstrin (data not shown). However,
we have previously determined that, when overexpressed
in Cos-1 cells, wild-type pleckstrin predominately exists in
the phosphorylated state (2). Thus, it is still possible that
phosphorylation regulates the ability of pleckstrin to associate with peripheral membranes and induce morphologic
changes. To test this hypothesis, we analyzed two pleckstrin variants in which Ser113, Thr114, and Ser117, the residues normally phosphorylated by protein kinase C (2), were collectively mutated to either glutamates (3-phos
glu) or to glycines (3-phos gly). We have previously shown
that replacing the sites of phosphorylation with glutamate
residues mimics the state of phosphorylation by introducing a cluster of negative charges in a region adjacent to the
NH2-terminal PH domain. This variant was previously
shown to be constitutively active in inhibiting PLC and
PI3-K activity (2, 3). When this constitutively active pleckstrin variant was tested, it produced a phenotype and pattern of staining identical to that of the wild-type molecule (Figs. 3 a and 5 a).
The pleckstrin variant containing glycine substitutions
at the sites of phosphorylation (3-phos gly) is relatively inactive biochemically when compared with the wild-type
protein. For example, this mutant is much less efficient
than wild-type pleckstrin at inhibiting PLC and PI3-K activity (2, 3). Cells expressing the 3-phos gly variant stained
diffusely and were of the same size and shape as mocktransfected cells (Fig. 5 b). Immunolocalization of this
variant in transfected Cos-1 cells produces a diffuse cytoplasmic pattern, similar to the variant truncated of its
NH2-terminal PH domain (pleckstrin
The requirement for pleckstrin to be phosphorylated to
associate with Cos-1 cell membranes could be explained
by two possible hypotheses: (a) the NH2-terminal PH domain, which is structurally regulated by pleckstrin phosphorylation, is critical for membrane association; or (b)
pleckstrin phosphorylation, which is regulated by the NH2terminal PH domain, is required for membrane localization. To address this issue, we analyzed the state of phosphorylation of each of the expressed pleckstrin variants.
Transfected Cos-1 cells were labeled with 32Pi, and the pleckstrin variants were immunoprecipitated and fractionated by
SDS-PAGE. The relative radioactivity of each variant was
compared by analysis on a phosphor imager. The wild-type
pleckstrin and the pleckstrin variants missing either the
NH2-terminal PH domain (
Thus, it is the NH2-terminal PH domain of pleckstrin
that is critical for its association with the plasma membrane. The phosphorylation of pleckstrin appears to regulate this PH domain but does not, by itself, appear to be
sufficient for membrane association.
Other PH Domains Cannot Substitute for Pleckstrin's
NH2-terminal PH Domain
Since the structures of PH domains from a number of different proteins are all remarkably similar, we determined
if different PH domains could mediate membrane localization when substituted in place of pleckstrin's NH2-terminal
PH domain. Plasmids directing the expression of chimeric
pleckstrin variants were constructed in which pleckstrin's
NH2-terminal PH domain was replaced by the PH domain
from either
PIP2-binding Residues in the NH2-terminal PH Domain
Are Necessary for Pleckstrin's Membrane Association
Fesik and co-workers mapped the sites of PIP2 interaction
within the NH2-terminal PH domain of pleckstrin, based
on 31P chemical shifts seen in PIP2 upon addition of the
N2-terminal PH domain (14). A cluster of three lysine residues (K13, K14, and K22) was shown to be critical in mediating the association of pleckstrin's NH2-terminal PH
domain with PIP2. When any one of these residues was
mutated to an asparagine, the affinity of the resultant molecule for PIP2 decreased 10-fold (14). Therefore, we generated a plasmid that directed the expression of a pleckstrin variant in which the first two of these lysines were
mutated to asparagines (N13, N14) and tested this variant
for its ability to associate with membranes. This pleckstrin
variant also migrated at its predicted molecular weight on
SDS polyacrylamide gels, and in vivo labeling studies demonstrated that its phosphorylation was approximately
equal to that of the wild-type protein when overexpressed
in Cos-1 cells (data not shown). Cells expressing this variant stained diffusely, had no dorsal projections, and were
not broadened. This finding argues that interactions between pleckstrin's NH2-terminal PH domain and PIP2 may
be critical for its association with membranes (Fig. 8).
Pleckstrin Promotes the Formation
of Membrane Projections from the Dorsal Surface
of Transfected Cells
To determine if pleckstrin was inducing the formation of
the dorsal villous projections or was merely being recruited to these membrane structures, we used fluorescently labeled WGA, which binds to cell surface glycoproteins and thus labels the surfaces of all cells, including
cells not expressing pleckstrin. By fluorescently labeling
the cell surface and then staining for expressed pleckstrin, we could study whether cells expressing pleckstrin differed
in their surface morphology from those cells not expressing pleckstrin. Under these conditions, only cells expressing wild-type pleckstrin demonstrated surface projections
(Fig. 9, a and b). This is in contrast to cells transfected with
the pleckstrin variant missing its PH domains and nontransfected cells, which did not have dorsal projections
(Fig. 9, c and d). This observation is consistent with pleckstrin actually inducing the formation of these membrane structures and changing cell surface architecture, in a fashion dependent on the presence of its PH domains.
Although the phosphorylation of pleckstrin has long been
used as a marker for platelet activation, we are only now
beginning to understand its intracellular role. Previous
works have suggested that pleckstrin may: (a) inhibit the
hydrolysis of PIP2 by multiple isoforms of PLC (1); (b) inhibit the phosphorylation of PIP2 by a G Since PH domains were first reported as components of
signaling molecules other than pleckstrin itself, they have
been proposed as structural motifs important in recruiting
molecules to the plasma membrane. A preponderance of
evidence now exists supporting this hypothesis (22, 25, 31-
33, 41, 45). However, even though the three-dimensional
structures from different PH domains are strikingly similar
in their backbone, there exist regions of marked primary
and tertiary structural diversity (6, 11, 27, 37, 46). It
stands to reason that these signaling modules may not be
interchangeable between molecules. Consistent with this
proposal, PH domains from different proteins have different binding affinities for inositol phosphates (13, 24), G The identity of the specific ligands targeted by PH domains remains unresolved. Several classes of molecules
have now been proposed as potential PH domain ligands,
including PIP2, G The appearance of expressed pleckstrin within dorsal
projections of transfected cells is intriguing, especially
since pleckstrin variants lacking the NH2-terminal PH domain are not seen in these structures. Moreover, we have
shown that pleckstrin, in a fashion dependent on its PH
domains, is in fact inducing the formation of these projections. Interestingly, the appearance of these projections is
reminiscent of the findings seen in CV-1 cells expressing
the actin-binding protein, villin (9, 10). Since villin also binds PIP2 (20), it is tempting to speculate that pleckstrin may be causing the appearance of these villous projections
in a PIP2-dependent manner. Determining whether pleckstrin is inducing the formation of these structures, and the
other factors that may regulate this process, are areas of
ongoing investigation in our laboratory.
It is striking that the structural regions within pleckstrin
that regulate its ability to inhibit the accumulation of second messengers are the same regions that affect its intracellular localization. Both pleckstrin-mediated biochemical effects and the recruitment of pleckstrin to peripheral
membranes require pleckstrin phosphorylation and an intact NH2-terminal PH domain. Control of the intracellular
location of signaling molecules is a common method of
regulating their function. An attractive hypothesis is that
pleckstrin is recruited to the peripheral membrane upon cell activation by potential ligands such as PIP2 or G1 (PLC
1), and the NH2 terminus of pleckstrin have been determined and are virtually superimposable, giving credence to their inclusion as a genuine family
of structural motifs, despite significant divergence in their
primary sequences (6, 11, 27, 37, 46). The molecular targets with which PH domains interact have not yet been
clearly elucidated. PH domains have been suggested to
bind to inositol phosphates (13), and the structures of inositol 1,4,5-trisphosphate complexed to the PH domains
from spectrin and PLC
1 have been recently reported (8,
18). The
subunits from heterotrimeric G proteins (G
)
have also been proposed as binding partners for PH domains (28, 39, 42), and we, along with others, have shown
that PH domains from a number of different proteins can
interact with G
(4, 22, 33, 34, 38).
-activated
phosphatidylinositol 3-kinase (PI3-K) activity in human
platelets (3). These data suggest a role for pleckstrin in the
inhibition of cellular pathways involving PIP2 and/or G
.
-adrenergic receptor kinase
(
ARK), phospholipase C, insulin receptor substrates
(IRS-1 and 2), and structural molecules, including spectrin
and dynamin (12, 16, 29, 30, 35). Common to many of
these proteins is a functional requirement for membrane localization, but there is a lack of traditional membraneanchoring groups such as lipophilic helices or sites for
posttranslational addition of lipid moieties. Several lines
of evidence support the hypothesis that PH domains serve
as membrane-targeting motifs. First,
ARK must be located near the cell membrane in order for it to become activated and phosphorylate its receptor substrate. Deletion
of a region overlapping the PH domain of
ARK diminishes its kinase activity, and replacing this sequence with
an isoprenylation site restores function to the molecule
(22, 33). Second, the transforming ability of the Lfc protein requires an intact PH domain, and an isoprenylation
site can effectively substitute for the PH domain in this activity (45). Third, IRS-1 mutants truncated of their PH domains are less effective substrates of the transmembrane
insulin receptor than is the wild-type IRS-1 molecule (31).
Fourth, a point mutation found within the PH domain of
Bruton's tyrosine kinase activates the enzyme and also causes increased membrane association (25). Fifth, the PH
domain at the carboxy terminus of
G spectrin has been
shown to interact with brain membranes and to target
plasma membranes in vivo (41, 43), and, lastly, PLC
1 was
demonstrated to require a PH domain for association with
the plasma membrane (32).
Materials and Methods
(44).
6-99), and pleckstrin variants containing mutations at Ser113, Thr114, and Ser117 has been described
previously (1, 2). A vector encoding a COOH-terminal PH domain deletion variant (
246-345) with the hemagglutinin antigen epitope tag fused
to the carboxy terminus of the molecule was generated by PCR overlap
extension according to the technique of Ho et al. (17). The hemagglutinin
antigen epitope tag was fused to the carboxy terminus of wild-type pleckstrin and the other pleckstrin variants using PCR mutagenesis. A variant deleted of both PH domains was constructed using a unique Pst site found
within the region between the two PH domains. Chimeric variants of
pleckstrin with the NH2-terminal PH domain replaced by the PH domain
from either
ARK or dynamin were generated by PCR overlap extension
(17). A variant of pleckstrin in which two lysine residues (K13 and K14)
were mutated to asparagines was constructed by PCR mutagenesis, using
the technique of Landt et al. (23). The DNA sequences of all clones were
confirmed and inserted into pCMV5.
antiserum was kindly supplied by Dr. David Manning (Department of Pharmacology, University of Pennsylvania, Philadelphia).
Results
Fig. 1.
Localization of pleckstrin to membranes. (a) Antipleckstrin immunoblot of membrane pellet (lane 1) and
cytosolic supernatant (lane 2)
fractions from nitrogen-cavitated human platelets showing that a fraction of cellular pleckstrin is found in the membrane pellet. (b) As a control, an identical immunoblot probed with antisera against the subunit if
G protein, Gq, showing that Gq-
is found only in the membrane
pellet (lane 1) and not in the supernatant fraction (lane 2).
These blots are from an experiment representative of seven
performed to date. (c) Antipleckstrin immunoblots of the
Triton-soluble (lane 1) and -insoluble (lane 2) fractions of the high speed pellet from A are shown.
Pleckstrin is found predominantly in the Triton-soluble fraction,
consistent with membrane localization.
[View Larger Version of this Image (45K GIF file)]
(44), and showed that this protein was not found in
the supernatant fraction (Fig. 1 A). Similarly, the absence
of lactate dehydrogenase activity in the pellet fraction argues that it was not contaminated with cytosolic components (data not shown).
Fig. 2.
Pleckstrin variants. Wild-type pleckstrin
and pleckstrin variants were
tagged with the hemagglutinin epitope (HA) recognized by the mAb 12CA5 (Boehringer Mannheim Biochemicals, Indianapolis, IN).
[View Larger Version of this Image (22K GIF file)]
Fig. 3.
Effect of PH domain deletions on localization of pleckstrin. Indirect
immunofluorescence was performed on Cos-1 cells transiently transfected with HAtagged pleckstrin variants, using the 12CA5 mAb against
the HA epitope. Wild-type
pleckstrin (a) and COOHterminal PH-deleted pleckstrin (246-345) (c) localize
to peripheral ruffles and projections from dorsal surface
of the cell. Double PH-deleted pleckstrin (
6-99,
246-345)
(b) and NH2-terminal PHdeleted pleckstrin (
6-99)
(d) are diffusely localized.
This suggests that the NH2terminal PH domain but not
the COOH-terminal PH domain is critical for pleckstrin's localization to membrane structures. Bar, 50 µm.
[View Larger Version of this Image (97K GIF file)]
Fig. 4.
Confocal microscopy with three-dimensional image reconstruction was performed to better demonstrate membrane structures
projecting from the dorsal surface of the cell. (a) Cells expressing wild-type pleckstrin exhibit bright staining within dorsal projections
and peripheral ruffles. (b) Cells expressing double PH-deleted pleckstrin (6-99,
246-345) exhibit diffuse staining. These images show that wild-type pleckstrin staining is not within intracytoplasmic structures, but rather within projections from the cell surface.
[View Larger Version of this Image (66K GIF file)]
6-99,
246-345). Cells expressing this variant demonstrated a diffuse pattern of staining, consistent with loss of
membrane interaction (Fig. 3 b). Unlike cells transfected
with wild-type pleckstrin, cells expressing this variant were
not broad and flattened, and intracytoplasmic vacuoles
could be clearly seen. Moreover, no dorsal projections could be demonstrated by either light or confocal microscopy (Figs. 3 b and 4 b). To ascertain if these pleckstrininduced changes were related to the level of protein expression, we varied the concentration of plasmid used for
transfections, and then analyzed cells expressing different
levels of protein. The relative concentration of expressed
protein was compared with anti-HA immunoblots of transfected cell lysates. Both the wild-type protein and the PH domain-truncation variant (
6-99,
246-345) migrated on
an SDS polyacrylamide gel at their predicted molecular
masses of 40 and 18 kD, respectively (data not shown).
Though lower levels of expression of wild-type pleckstrin
produced less peripheral staining and fewer dorsal projections, these structural features were still present, even
when the expression of the wild-type protein was lower
than that of the pleckstrin
6-99,
246-345 variant. Thus, the complete absence of peripheral staining and dorsal
projections in cells expressing the pleckstrin variant missing its PH domains cannot be explained on the basis of levels of expression.
6-99) or the COOH-terminal PH domain
(
246-345) were studied. Cells expressing the COOHterminal PH domain-deleted variant (
246-345) stained in
a pattern indistinguishable from that of the wild-type molecule, (Fig. 3 c). As predicted by their amino acid sequences, these expressed proteins both migrated at 29 kD
on SDS-PAGE as determined by immunoblotting with the
anti-HA tag antibody, 12CA5. The cells were flat and
showed peripheral staining and dorsal projections. In contrast, the NH2-terminal PH domain-deleted mutant (
699) was diffusely localized and had neither peripheral
staining nor dorsal projections (Fig. 3 d). This suggests
that the NH2-terminal PH domain, and not the COOHterminal PH domain, is critical in targeting pleckstrin to
membranes and mediating morphologic changes.
6-99). This suggests
that pleckstrin must be phosphorylated or contain charged
residues in place of the sites of phosphorylation in order
for it to bind to its target within the plasma membrane.
This result may contrast with the finding that a fraction of
cellular pleckstrin was associated with the cell membrane
in unstimulated platelets. However, at this point we cannot exclude the possibility that these platelets were partially activated during our preparative process, thus resulting in pleckstrin phosphorylation.
Fig. 5.
Effect of pleckstrin phosphorylation on intracellular localization. Cos-1 cells were transfected with HA-tagged variants
of pleckstrin that had the residues normally phosphorylated by
protein kinase C (Ser113, Thr114, Ser117) mutated to either glutamates (3-phos glu) or to glycines (3-phos gly). (a) The 3-phos glu
variant produced the same pattern of staining as did the wild-type
molecule, showing staining at the periphery and within dorsal
projections. This variant has previously been shown to be constitutively active in mediating inhibition of PIP2 metabolism. (b)
The 3-phos gly mutant, which cannot be phosphorylated, is diffusely localized and does not associate with membrane structures.
This suggests that pleckstrin phosphorylation is critical for its effective recruitment to membranes.
[View Larger Version of this Image (70K GIF file)]
6-99) or the COOH-terminal
PH domain (
246-345) were radiolabeled in vivo approximately equally (Fig. 6 A). Anti-pleckstrin immunoblots were performed in parallel to verify that each variant was
expressed and immunoprecipitated equivalently (Fig. 6 B).
Fig. 6.
Effect of PH domain deletions on pleckstrin variant
phosphorylation. Cos-1 cells were transfected with either wildtype pleckstrin or pleckstrin variants missing either the NH2-terminal PH domain (6-99) (lane 2) or the COOH-terminal PH domain (
246-345) (lane 3). 48 h after transient transfection, cells
were labeled with 32Pi, immunoprecipitated, and run on SDSPAGE gels. A shows that wild-type pleckstrin (lane 1) and the
pleckstrin variants missing either the NH2-terminal PH domain
(lane 2) or the COOH-terminal PH domain (lane 3) were phosphorylated equally. B shows an antipleckstrin immunoblot performed in parallel, demonstrating that these variants all expressed and immunoprecipitated equally well.
[View Larger Version of this Image (54K GIF file)]
ARK (NPH-
ARK) or dynamin (NPH-dynamin). These two chimeras were expressed at their predicted molecular weight as judged by anti-HA immunoblots.
Neither chimera behaved like wild-type pleckstrin. Cells
expressing either of these molecules stained diffusely, had
no dorsal projections, and were of normal size and shape
(Fig. 7, a and b). This suggests that pleckstrin's native
NH2-terminal PH domain makes specific interactions with ligands in the plasma membrane, and that, despite having
similar structures, other PH domains cannot effectively
substitute in these associations.
Fig. 7.
Localization of pleckstrin NH2-terminal PH domain
chimeras. Cos-1 cells were transfected with HA-tagged variants
of pleckstrin in which the NH2-terminal PH domain had been replaced with either the PH domain of ARK (a) or dynamin (b).
Neither chimera behaved like wild-type pleckstrin, suggesting
that pleckstrin's native NH2-terminal PH domain is making specific interactions with membrane-bound ligands in which other
PH domains, despite having striking structural similarities, cannot effectively substitute.
[View Larger Version of this Image (78K GIF file)]
Fig. 8.
Effect of mutating PIP2-binding residues in NH2-terminal PH domain. Cos-1 cells were transiently transfected with a
vector encoding the HA-tagged pleckstrin variant K13N, K14N,
and then stained with 12CA5. This variant produced a diffuse
pattern of staining, thus demonstrating that mutating two residues in pleckstrin's NH2-terminal PH domain can disrupt the
membrane association of the entire molecule. This suggests a critical role for interactions between PIP2 and pleckstrin's NH2-terminal PH domain in mediating recruitment of the molecule to the
plasma membrane.
[View Larger Version of this Image (97K GIF file)]
Fig. 9.
Effect of pleckstrin variants on cell surface morphology. Cells expressing either HA-tagged pleckstrin or the HA-tagged double PH-deleted pleckstrin (6-99,
246-345) were surface labeled with TRITC-conjugated WGA, and then fixed and stained with
12CA5. Cells expressing HA-tagged wild-type pleckstrin are shown in a and b, and cells expressing the HA-tagged double PH-deleted
pleckstrin variant are shown in c and d. a and c show 12CA5 staining for the HA epitope, and b and d show TRITC-conjugated WGA
staining of the cell surface. Cells expressing wild-type pleckstrin exhibit projections from the dorsal surface of the cell (b), and pleckstrin is found within these projections (a). In contrast, cells expressing the double PH domain-deleted variant of pleckstrin exhibit diffuse pleckstrin staining (c) and fail to form dorsal projections (d). This suggests a new role for pleckstrin in mediating morphologic changes
at the cell surface.
[View Larger Version of this Image (114K GIF file)]
Discussion
-activated isoform of PI3-K (3); and (c) activate an inositol 1,4,5-trisphosphate-specific 5
inositol phosphatase (5). These
activities are all regulated by phosphorylation of pleckstrin near its NH2-terminal PH domain (2, 3, 5). This
present study builds on these findings and shows that
pleckstrin associates with the plasma membrane and peripheral ruffles by a mechanism dependent on both pleckstrin phosphorylation and an intact NH2-terminal PH domain. In addition, we show that pleckstrin induces the
formation and associates with dorsal membrane projections in transfected Cos-1 cells. These observations raise a
number of questions, including the specificity of the PH
domains in membrane targeting, the potential membranebound ligands for pleckstrin, and the significance of both
the dorsal projections and pleckstrin's intracellular localization.
(28), and brain membranes (41). Our results support the
hypothesis that PH domains have different specificities for
ligands within the membrane by demonstrating that pleckstrin can be recruited to membrane structures by its own
NH2-terminal PH domain, but not by other PH domains.
, and phosphotyrosine residues. The
data showing that pleckstrin is able to inhibit PLC
as well
as PLC
suggest a direct interaction between pleckstrin
and PIP2. However, the observation that pleckstrin is able
to inhibit only the G
-activated isoform of PI3-K and not
the isoforms of PI3-K activated by growth factor receptors suggests that G
may as well be able to modulate pleckstrin's interaction with other ligands. The data presented
here showing that targeted mutations of PIP2-binding residues within the NH2-terminal PH domain disrupt pleckstrin's membrane association support a role for PIP2 as a
physiologic ligand for pleckstrin and its NH2-terminal PH
domain. Additionally, our preliminary studies in Cos-1 cells reveal that overexpression of either wild-type or nonisoprenylated G
heterodimers has no effect on pleckstrin's
membrane localization.
. We
are actively investigating whether pleckstrin's activity is
regulated by localization to the proximity of these biochemical targets.
Received for publication 31 July 1996 and in revised form 14 November 1996.
Address all correspondence to Dr. Charles S. Abrams, Hematology- Oncology Division, University of Pennsylvania School of Medicine, 422 Curie Blvd., Stellar-Chance Labs #1005, Philadelphia, PA 19104. Tel.: (215) 898-1058. Fax: (215) 662-7617. e-mail: abramsc{at}mail.med.upenn.eduThese studies were supported in part by funds from the National Institutes of Health (HL07439-17, HL53545, and HL54500) and the American Heart Association (AHA) (95014910). C.S. Abrams is a recipient of an AHA-Sanofi Winthrop Award.
ARK,
-adrenergic receptor kinase;
G
,
gamma;
subunit of heterotrimeric GTP-binding proteins; HA, hemagglutinin;
PH, pleckstrin homology;
PI3-K, phosphatidylinositol 3-kinase;
PIP2, phosphatidylinositol 4,5-bisphosphate;
PLC, phospholipase C.