Correspondence to: Charles S. Abrams, Hematology-Oncology Division, University of Pennsylvania, 421 Curie Blvd., Basic Research Bldg. II/III, Rm. 912, Philadelphia, PA 19104. Tel:(215) 898-1058 Fax:(215) 573-7400
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Abstract |
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Pleckstrin is a 40-kD phosphoprotein containing NH2- and COOH-terminal pleckstrin homology (PH) domains separated by a disheveled-egl 10-pleckstrin (DEP) domain. After platelet activation, pleckstrin is rapidly phosphorylated by protein kinase C. We reported previously that expressed phosphorylated pleckstrin induces cytoskeletal reorganization and localizes in microvilli along with glycoproteins, such as integrins. Given the role of integrins in cytoskeletal organization and cell spreading, we investigated whether signaling from pleckstrin cooperated with signaling pathways involving the platelet integrin, IIbß3. Pleckstrin induced cell spreading in both transformed (COS-1 & CHO) and nontransformed (REF52) cell lines, and this spreading was regulated by pleckstrin phosphorylation. In REF52 cells, pleckstrin-induced spreading was matrix dependent, as evidenced by spreading of these cells on fibrinogen but not on fibronectin. Coexpression with
IIbß3 did not enhance pleckstrin-mediated cell spreading in either REF52 or CHO cells. However, coexpression of the inactive variant
IIbß3 Ser753Pro, or ß3 Ser753Pro alone, completely blocked pleckstrin-induced spreading. This implies that
IIbß3 Ser753Pro functions as a competitive inhibitor by blocking the effects of an endogenous receptor that is used in the signaling pathway involved in pleckstrin-induced cell spreading. Expression of a chimeric protein composed of the extracellular and transmembrane portion of Tac fused to the cytoplasmic tail of ß3 completely blocked pleckstrin-mediated spreading, whereas chimeras containing the cytoplasmic tail of ß3 Ser753Pro or
IIb had no effect. This suggests that the association of an unknown signaling protein with the cytoplasmic tail of an endogenous integrin ß-chain is also required for pleckstrin-induced spreading. Thus, expressed phosphorylated pleckstrin promotes cell spreading that is both matrix and integrin dependent. To our knowledge, this is the first example of a mutated integrin functioning as a dominant negative inhibitor.
Key Words: pleckstrin, integrins, platelets, cell spreading, PH domain
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Introduction |
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Phosphorylation of pleckstrin, a 4047-kD protein present in platelets and leukocytes, is one of the earliest detectable events after platelet stimulation (130 other proteins, and likely constitute phosphoinositide-binding motifs (
When overexpressed in tissue culture cells, pleckstrin induces the formation of, and localizes within, lamellipodia, ruffles, and microvilli. Coincident with the formation of these structures is dissolution of central actin fibers and the formation of cortical actin cables (
Integrins are a ubiquitous family of adhesion receptors that mediate cellcell and cellmatrix interactions in processes as diverse as embryogenesis, metastasis, host defense, hemostasis, and wound repair. In platelets, the integrin IIbß3 is required for platelet aggregation (
IIbß3 that is associated with an increased affinity for the ligands fibrinogen and von Willebrand factor (
IIbß3 initiates "outside-in" signaling, characterized by activation of protein and lipid kinases and, ultimately, remodeling of the platelet's actin cytoskeleton.
Although pleckstrin and IIbß3 each play a role in platelet cytoskeletal reorganization, a direct connection between the cytoskeletal effects of each protein has not been identified. In this report, we demonstrate that pleckstrin overexpression induces the spreading of a number of transformed and nontransformed cell lines. Furthermore, we demonstrate that this effect of pleckstrin is matrix specific and can be regulated by pleckstrin phosphorylation and by the ß subunit of
IIbß3.
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Materials and Methods |
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Tissue Culture and Reagents
REF52 and COS-1 cells were maintained in DME supplemented with L-glutamine, penicillin/streptomycin, and 10% FBS. CHO cells were maintained in F-12 Nutrient Mixture (HAM) supplemented with L-glutamine, penicillin/streptomycin, and 10% FBS. In selected experiments, fibrinogen (Calbiochem-Novabiochem">Calbiochem-Novabiochem) or fibronectin (Sigma-Aldrich) were applied to chamber slides according to the manufacturer's instruction. The cDNA encoding IIb, ß3, or ß3 Ser753Pro were inserted into pcDNA3.1+. Plasmids directing the expression of hemagglutinin antigen (HA) epitopetagged wild-type pleckstrin, as well as pseudo-phosphorylated (3 Glu) and nonphosphorylatable (3 Gly) variants of pleckstrin, were generated by subcloning HindIIIBamHI fragments of previously described plasmids into pcDNA3.1+ (
IIb, Tac-ß3, and Tac-ß3 S752P were a gift from Dr. Timothy O'Toole (Scripps Research Institute, La Jolla, CA) and have been described previously (
Heterologous Expression of Pleckstrin, Pleckstrin Mutants, and IIbß3
Pleckstrin, pleckstrin mutants, and IIbß3 were expressed transiently in COS-1 or CHO cells. In brief, cells were cultured on 100-mm polystyrene tissue culture dishes (Falcon) and transfected with plasmid DNA by calcium phosphate coprecipitation or Lipofectamine (Life Technologies) 24 h after transfection, cells were shocked with 10% glycerol, treated with trypsin, and replated onto 2-well chamber slides (Falcon). After an additional 24 h incubation, the cells were fixed using 3% neutral buffered formalin and stained with fluorescent antibodies.
REF52 cells were plated onto gridded fibronectin or fibrinogen coated coverslips (Bellco Glass) at 60% confluency. To prepare quiescent cells for microinjection, the cells were incubated in medium containing 0.1% FBS for 36 h. The cells were then microinjected with plasmid DNA in microinjection buffer (100 mM Hepes, pH 7.2; 200 mM KCl; 10 mM NaPO4, pH 7.2) according to the following protocol: (i) IIb (12.5 ng/µl) -ß3 (12.5 ng/µl) and pcDNA3.1+ (vector; 25 ng/µl); (ii) pseudo-phosphorylated pleckstrin (25 ng/µl) and
IIb (12.5 ng/µl) -ß3 (12.5 ng/µl); (iii) pseudo-phosphorylated pleckstrin (25 ng/µl) and pcDNA3.1+ (vector; 25 ng/µl). In selected experiments, ß3 Ser753Pro was substituted for the wild-type ß3. Microinjection was performed using Eppendorf Transjector 5246 (P2 = 80, P3 = 30, injection time = 0.3 s). Unless otherwise indicated, cells were fixed 4 h after injection in 3% neutral buffered formalin.
Immunofluorescence and Quantitation of Cell Spreading
Cells were stained for proteins of interest using the appropriate primary antibody, and counterstained with a fluorescent secondary antibody as previously described (
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Results |
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Overexpression of Phosphorylated Pleckstrin Produces Cell Spreading
Having previously determined that pleckstrin overexpression alters cytoskeletal organization in various tissue culture cells (65% larger than control cells. The difference in cell size was reproducible and statistically significant (P < 0.0001). Similar effects were also found when pleckstrin was expressed in CHO cells (data not shown). Moreover, the pleckstrin-induced increase in footprint size was larger than that induced by constitutively active Rac L61, a potent activator of cell spreading (P < 0.0001;
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Substitution of phosphorylated Ser113, Thr114, and Ser117 by glycine (nonphosphorylatable pleckstrin) inhibits pleckstrin function (
Effect of IIbß3 and Fibrinogen on Pleckstrin-induced Cell Spreading
Pleckstrin and integrins colocalize in the lamellipodia of pleckstrin-transfected COS-1 cells (IIbß3 is the most abundant platelet integrin and pleckstrin is present in platelets, we asked whether pleckstrin-induced cell spreading was affected by the presence of
IIbß3 or its ligand, fibrinogen. To address the effect of
IIbß3 and fibrinogen, we examined the consequences of microinjecting cDNA that direct the expression of
IIbß3, pleckstrin variants, or both into adherent REF52 cells. REF52 cells are a nontransformed cell line that can be serum-starved into quiescence.
We first examined the effect of pleckstrin and IIbß3 on the spreading of REF52 cells adherent to surfaces coated with fibrinogen. As shown in Fig 2 and Fig 3, microinjecting wild-type
IIbß3 did not induce cell spreading when compared with control cells microinjected with a plasmid that directed the expression of GFP (P = 0.38). Similarly, microinjecting the nonphosphorylatable pleckstrin mutant with and without
IIbß3 had no effect on cell footprint size (P = 0.79 and P = 0.66, respectively). As indicated by the quantitative analysis shown in Fig 3, microinjection of pseudo-phosphorylated pleckstrin significantly increased the footprint of adherent REF52 cells compared with cells microinjected with either
IIbß3 or GFP (P < 0.002 and P < 0.0002, respectively). On the other hand, the effect of microinjecting cells with pseudo-phosphorylated pleckstrin and
IIbß3 was no different than microinjecting cells with pseudo-phosphorylated pleckstrin alone (P = 0.32). Time course experiments indicate that some pleckstrin expression and cell spreading is seen as soon as 30 min after microinjection with plasmids. Pleckstrin expression and cell spreading reached a maximum 4-6 h after microinjection. This is consistent with the time course of expression of most proteins after microinjection of plasmids. PMA stimulation of these cells without pleckstrin-expression induces ruffling, but not spreading (data not shown). Together these results imply that pleckstrin-mediated cell spreading requires PKC; however, in these cells PKC activation alone is not sufficient to induce cell spreading.
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We next compared the effect of pseudo-phosphorylated pleckstrin and IIbß3 on the spreading of REF52 cells on tissue culture slides coated with fibrinogen or fibronectin. Fibrinogen is the predominant ligand for
IIbß3, and fibronectin is the predominant ligand for integrins such as
5ß1. As shown in Fig 4 (and graphically in Fig 3), we found that pseudo-phosphorylated pleckstrin induced REF52 cell spreading on fibrinogen-coated surfaces, but failed to do so when the surfaces were coated with fibronectin. Moreover, there was no significance difference in footprint size of cells plated on fibronectin regardless of whether they were expressing any combination of GFP, pseudo-phosphorylation pleckstrin, or
IIbß3 (ANOVA, P = 0.11). Thus, these results indicate that not only does pleckstrin induce cell spreading but its effect is dependent on the substrate for cell adhesion. The data also suggest that the pleckstrin-induced spreading of REF52 cells requires the presence of an adhesion molecule that is capable of interacting with fibrinogen.
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Although expressing IIbß3 in REF52 cells does not appear to effect pleckstrin-mediated cell spreading, it is possible that
IIbß3 had no effect because endogenous integrins in these cells were sufficient for this function. Accordingly, we reasoned that the participation of these endogenous integrins might become apparent if their function was specifically impaired. The ß3 mutation Ser753Pro abrogates agonist-induced
IIbß3 function in platelets and, when expressed in CHO cells, reduces ß3-mediated cell spreading (
IIbß3 or
IIbß3 Ser753Pro. As shown by the quantitation in Fig 3 and photomicrographs in Fig 5, microinjecting
IIbß3 Ser753Pro into REF52 cells abrogated cell spreading induced by pleckstrin, whereas wild-type
IIbß3 had no effect (P < 0.0001 and P = 0.32, respectively). Moreover, there was no difference in footprint size between REF52 cells expressing pseudo-phosphorylated pleckstrin along with either wild-type
IIbß3 or
IIbß3 Ser753Pro when plated on fibronectin-coated surfaces (P = 0.30). Thus, these experiments suggest that integrins modulate the effect of pleckstrin in REF52 cells and that ß3 Ser753Pro functions as a dominant negative inhibitor in this regard.
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We found that, like our results using REF52 cells plated on fibrinogen, expression of wild-type IIbß3 in CHO cells does not influence pleckstrin-mediated cell spreading, whereas spreading was abrogated by
IIbß3 S752P. Moreover, ß3 S752P alone abrogated the pleckstrin effect (Fig 6A and Fig B). Because ß3 is not expressed on the cell surface unless it is coupled to an
-subunit, it is likely that ß3 S752P is associated with the widely expressed
v integrin subunit (
vß3).
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We next tested whether "outside-in" integrin signaling was critical for pleckstrin-mediated cell spreading by measuring the effect of chimeric proteins composed of the extracellular and transmembrane portions of Tac fused to the cytoplasmic tail of or ß integrin subunits on pleckstrin-mediated cell spreading. Tacintegrin fusion proteins have previously been shown to disrupt integrin function, presumably by competing for intracellular factors that associate with integrin cytoplasmic tails (
IIb and Tacß3 S752P had no effect. Thus, these experiments demonstrate that overexpression of the wild-type ß3 tail alone specifically inhibits the ability of pleckstrin to induce cell spreading, suggesting that it specifically impairs the ability of endogenous ß3 integrins to support pleckstrin function. ß3 S752P, on the other hand, had no effect because S752P is an inactivating mutation.
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Discussion |
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We reported previously that expressed pleckstrin will bind to plasma membranes and induce lamellipodia and actin reorganization (
The observation that pleckstrin-induced cell spreading is inhibited by the dominant-negative IIbß3 Ser753Pro mutant implies that pleckstrin cooperates with integrins to mediate cell signaling. Most integrins respond to agonist-stimulated signals ("inside-out" signaling) that regulate their ability to bind to extracellular matrix proteins, where binding to the matrix itself initiates signals ("outside-in" signaling) that increases the cytosolic concentration of calcium, causes the phosphorylation of a number of signaling proteins, and induces the reorganization of the cytoskeleton (
IIbß3, where it induces the integrin to bind its ligand, or downstream of
IIbß3, where it enhances integrin-initiated cell spreading. Given current information, either of these possibilities is equally likely to contribute to this phenomenon.
It is not currently known how an adapter protein such as pleckstrin, with no apparent enzymatic activity, could induce cell spreading. One possibility is that pleckstrin directly interacts with and alters the cytoplasmic tail of IIbß3. However, we have not been able to detect pleckstrin in immunoprecipitates of
IIbß3 or vice versa (Abrams, C.S., and L. Brass, unpublished observation). An alternative explanation is that pleckstrin binds and sequesters a critical phospholipid cofactor required for integrin signaling. For example,
Lß2 when it is overexpressed in Jurkat cells. Finally, it is possible that the effect of pleckstrin on the cytoskeleton is downstream of integrins, potentially via a small GTP-binding protein of the Rho family or an actin capping protein (
IIbß3.
Many integrins interact with a variety of extracellular matrix proteins. We found that pleckstrin promotes spreading on fibrinogen, but not on fibronectin. This observation, coupled with the finding that the ß3 mutant Ser753Pro inhibits pleckstrin-induced spreading, suggests that the pleckstrin effect may be specific for ß3 integrins and their ligands. This would also include a possible pleckstrin effect on the endogenously expressed vß3 integrins found in fibroblasts.
Our data also indicates an important role for integrin ß subunits signaling effectors. IIb or Tac-ß3 S752P, inhibited pleckstrin-mediated spreading. This suggests that direct binding of integrin cytoplasmic tails to associated proteins is critical for this phenomenon.
The identity of the cytoplasmic protein that associates with ß-integrin subunit to cooperate with pleckstrin in mediating cell spreading is unclear. Numerous candidate proteins that directly or indirectly associate with ß-chain cytoplasmic tails include: paxcillin, talin, vinculin, Src, FAK, ß3-endonexin, -actinin, ILK, ICAP-1, filamin, cytohesin-1, p27BBP, and rack 1 (
Our data suggest that overexpressed pleckstrin contributes to the process of integrin-mediated cytoskeletal change. An important issue that has not yet been determined is whether pleckstrin plays a similar role in cells in which it is normally expressed. Approximately 1% of total cellular protein in platelets and leukocytes is pleckstrin and its rapid phosphorylation is a hallmark of platelet activation. Accordingly, the agonist-stimulated behavior of platelets and leukocytes from pleckstrin-deficient mice may provide the most facile, and only readily available, way to verify pleckstrin function in blood cells.
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Footnotes |
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1 Abbreviations used in this paper: DEP, disheveled-egl 10-pleckstrin; GFP, green fluorescent protein; HA, hemagglutinin antigen; PH domain, pleckstrin homology domain; PKC, protein kinase C.
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Acknowledgements |
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These studies were supported in part by funds from the National Institutes of Health (grant Nos. P50 HL54500 and P01 HL40387).
Submitted: 27 March 2000
Revised: 14 June 2000
Accepted: 4 August 2000
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References |
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