©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Pleckstrin Inhibits Phosphoinositide Hydrolysis Initiated by G-protein-coupled and Growth Factor Receptors
A ROLE FOR PLECKSTRIN'S PH DOMAINS (*)

Charles S. Abrams (1) (3)(§), Hung Wu (1), Wei Zhao (1), Elizabeth Belmonte (1), David White (1), Lawrence F. Brass (1) (2)

From the (1)Departments of Medicine and (2)Pathology, University of Pennsylvania and the (3)Philadelphia Veterans Administration Medical Center, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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-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.


INTRODUCTION

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()(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.

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 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.

Collectively, these studies suggest that in at least some proteins PH domains play a role in cell signaling, either by targeting G 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.


EXPERIMENTAL PROCEDURES

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.

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-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-GQ209L), 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-GQ209L 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.


RESULTS

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) .

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 [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, GQ209L 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 GQ209L-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 GQ209L 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 GQ209L, 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 GQ209L in the absence of pleckstrin or PMA. C is an immunoblot demonstrating equal expression of GQ209L 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.




DISCUSSION

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-mediated activation of phospholipase C initiated by thrombin, M-muscarinic acetylcholine, and angiotensin II receptors, 2) the activation of phospholipase C by GQ209L, 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.

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 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) .

Thus, in contrast to -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 GQ209L, 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 PIPin 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.

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 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).

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 -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.

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. 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.


FOOTNOTES

*
These studies were supported in part by National Institutes of Health Grants 5K11 HL02464 and P01 HL40387, the American Heart Association Southeastern Pennsylvania Affiliate, the Diabetes Center of the University of Pennsylvania, and the University of Pennsylvania Penn HomeIT fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Hematology-Oncology Division, University of Pennsylvania, 415 Curie Blvd., CRB 678, Philadelphia, PA 19104. Tel.: 215-898-1058; Fax: 215-573-2189; E-mail: abrams@mail.med.upenn.edu.

The abbreviations used are: PH domain, pleckstrin homology domain; PMA, phorbol 12-myristate 13-acetate; hCG, human chorionic gonadotropin; LH receptor, luteinizing hormone receptor; PIP, phosphatidylinositol 4-phosphate; PIP, phosphatidylinositol 4,5-bisphosphate; NGF, nerve growth factor.

C. S. Abrams, W. Zhao, E. Belmonte, and L. F. Brass, unpublished observations.


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