©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sequestration of a G-protein Subunit or ADP-ribosylation of Rho Can Inhibit Thrombin-induced Activation of Platelet Phosphoinositide 3-Kinases (*)

(Received for publication, October 4, 1994; and in revised form, January 3, 1995)

Jin Zhang Jun Zhang Jeffrey L. Benovic Motoyuki Sugai (1) Reinhard Wetzker (2) Ivan Gout (3) Susan E. Rittenhouse (§)

From the  (1)Department of Pharmacology/Jefferson Cancer Institute and Cardeza Foundation for Hematologic Research, Thomas Jefferson Medical College, Philadelphia, Pennsylvania 19107, the Department of Microbiology, Hiroshima University School of Dentistry, Kasumi 1-2-3, Minami-ku, Hiroshima City, Japan, the (2)Max Planck Research Group ``Growth Factor Signal Transduction,'' University of Jena, Jena 07747, Federal Republic of Germany, and the (3)Ludwig Institute for Cancer Research, University College, London W1P 8BT, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Stimulation of platelets by thrombin leads to an increased association of activated phosphoinositide 3-kinase (PI 3-K) with a membrane cytoskeletal fraction (CSK). Activation of PI 3-K is dependent upon GTP-binding protein(s), since PI 3-K in permeabilized platelets is stimulated by GTPS (guanosine 5`-3-O-(thio)triphosphate), and stimulation of platelet cytosolic PI 3-K by GTPS requires a functional small G-protein, Rho. Recent reports indicate that cytosolic PI 3-Ks can also be activated by the beta subunits of heterotrimeric G-proteins (Gbeta). We now report that the activated PI 3-K that is associated with CSK can be inhibited by a recombinant protein containing the Gbeta-binding pleckstrin homology domain of beta-adrenergic receptor kinase 1 (betaARK-PH). Inhibition is blocked by Gbeta. PI 3-K in nonactivated platelet CSK is activated by GTPS but unaffected by betaARK-PH or Gbeta. Western blots indicate that activated platelet CSK contains a novel 110-kDa PI 3-K() that has been shown to be stimulated by Gbeta and to lack binding sites for the 85-kDa subunit of conventional PI 3-K. PI 3-K in immunoprecipitates obtained via p85 subunit-directed antibodies can be activated by GTPS but not by Gbeta. PI 3-K that is stimulatable by Gbeta remains soluble, as does PI 3-K(), and is unaffected by Rho. In contrast, ADP-ribosylation of Rho present in p85 immunoprecipitates is inhibitory. Further, activation of PI 3-K in permeabilized platelets exposed to thrombin or GTPS is inhibited by betaARK-PH and/or Rho-specific ADP-ribosylating enzymes. We conclude that Rho and Gbeta each, respectively, contributes to the activation of different PI 3-Ks (p85-containing heterodimer and PI 3-K ()) in thrombin-stimulated platelets.


INTRODUCTION

GTP-binding proteins (G-proteins), (^1)both heterotrimeric and small, are known to be involved in platelet activation by receptor-directed agonists, controlling the inhibition of adenylyl cyclase, the stimulation of phosphatidylinositide-specific phospholipase C, and platelet aggregation(1, 2, 3, 4, 5) . The significance of phospholipase C and adenylyl cyclase in cell signaling is by now well documented. More recently, attention has focused on an enzyme which, in other cells, can regulate events such as the mitogenic response to growth factors (6) and the respiratory burst in activated neutrophils (7) , phosphoinositide 3-kinase (PI 3-K). PI 3-K has been proposed to play a role in cytoskeletal/integrin reorganization, and activated PI 3-K becomes associated with the alphabeta(3) integrin-containing membrane cytoskeleton of thrombin-activated platelets(8) . In platelets exposed to thrombin, PI 3-K rapidly phosphorylates PtdIns(4,5)P(2) at the 3-OH position to form PtdIns(3,4,5)P(3)(9) . We have reported that PI 3-K in permeabilized platelets (10) and in platelet cytosolic fractions (11) can be activated by GTPS and that such activation in cytosol is dependent upon the small G-protein Rho, since ADP-ribosylation of endogenous Rho by C3 transferase is inhibitory(11) . Rho has also been implicated in the control of platelet aggregation(5) , a response to platelet agonists that is dependent upon activated alphabeta(3)(12) . PI 3-K may participate in regulating alphabeta(3) activation.

Studies with cytosolic fractions from myeloid cells (13) and platelets (14) indicate that PI 3-K activity also can be stimulated by a product of heterotrimeric G-protein dissociation, Gbeta, although direct evidence for the role of either pathway in regulating the activation of PI 3-K in receptor-stimulated cells has not yet been provided. We have addressed this issue, and the nature of the PI 3-Ks involved, in the present work. In doing so, we have taken advantage of the observations that the carboxyl-terminal portion of betaARK1 (which contains a PH domain) binds Gbeta (15, 16) and that EDIN or C3 transferase can ADP-ribosylate Rho selectively in permeabilized platelets, whose PI 3-K activity is ordinarily responsive to thrombin or GTPS(10) .


EXPERIMENTAL PROCEDURES

Materials

Purified bovine brain G-protein beta subunits were generously provided by P. Casey (Duke University). A glutathione S-transferase-betaARK fusion protein construct, containing a PH domain (betaARK-PH), was generated by ligating a polymerase chain reaction-amplified carboxyl-terminal domain of betaARK (encoding residues 466-689) into the vector pGEX-2T (Pharmacia Biotech Inc.). The glutathione S-transferase-betaARK-(466-689) fusion protein was then expressed in BL21 cells and purified using a glutathione-agarose column (Sigma), essentially as described(17) . Lipid substrates and [-P]ATP were prepared as described(8, 11, 18) . [P]P(i) and [alpha-P]betaNAD were purchased from DuPont NEN. Recombinant Clostridium botulinum C3 transferase was a gift from S. Dillon and L. Feig (Tufts University). alpha-Thrombin was purchased from Hemetech (Burlington, VT). The thrombin receptor tethered ligand analogue, SFLLRN, was synthesized by the Jefferson Cancer Institute peptide facility. The thrombin-binding inhibitor, hirudin, was purchased from Sigma. EDIN was isolated from Staphylococcus aureus E-1 culture media according to Sugai et al.(19) . Antibodies to the p85 subunit of PI 3-K (rabbit polyclonal) and to RhoA were purchased from Upstate Biotechnology, Inc. and Santa Cruz Biotechnology, Inc., respectively, and were also contributed by I. Gout (mouse monoclonals to alpha, beta, and forms of p85, i.e. N1, T4, and N2) and A. Hall (University College, London), respectively. Rho-glutathione S-transferase was prepared as before(11) . Antibodies to beta subunits of Gbeta were kindly provided by M. Woolkalis (Thomas Jefferson University, Philadelphia, PA). A rabbit antibody was raised against a nine-amino acid peptide corresponding to a domain of PI 3-K present uniquely in p110 and was affinity-purified via protein A and the peptide antigen. (^2)

Studies with Permeabilized Platelets

Fresh human platelets were prepared as described (10, 18) and suspended to 10 cells/ml. Platelets (100 µl) were added to 37 °C permeabilization buffer (400 µl) containing saponin (20 µg/ml) with or without betaNAD (200 µM) and/or [alpha-P]NAD (20 µCi/ml), ATP (500 µM) with or without [-]P]ATP (40 µCi/ml), and 0-10 µM betaARK-PH or equivalent concentrations of glutathione S-transferase, as described (10) . After 90 s, EDIN (0-22 µg/ml) or C3 transferase (0-10 µg/ml) was added (when C3 transferase was added, 20 units/ml hirudin were present to bind any thrombin contaminating the recombinant protein), followed after 6 min by buffer, thrombin (2 units/ml), SFLLRN (10 µM; added when C3 transferase/hirudin was used), or GTPS (10 µM). Incubations continued up to 5 min further and were terminated with either CHCl(3)/MeOH(10, 18) (for [P]ATP incubations) or Laemmli buffer (11) (for P-NAD incubations), followed by resolution and quantitation of P-labeled phosphoinositides(10, 18) /P-labeled PtdOH (20) or P-labeled Rho, respectively. The identification of Rho was confirmed by Western blotting/ECL(11) .

Studies with Platelet Cytoskeletal Fractions or Cytosol

Human platelets were prepared freshly as described(8) . Platelets (10/ml), at 37 °C, were incubated with thrombin (5 units/ml) for 0, 15, 45, and 180 s. Incubations were terminated with Triton lysis buffer (8) and CSK (15,000 times g), and Triton-soluble fractions were prepared(8) . Triton X-100 was removed from supernatants with ExtractiGel(8) . CSK or supernatant (0.2-1 mg/ml, final concentration) fractions were incubated in the PI 3-K assay system (11) (containing [-P]ATP and using PtdIns(4,5)P(2) lipid substrate) for 5 min, following exposure of CSK or supernatant to 0-10 µM betaARK-PH for 5 min at 37 °C. In some studies, incubations contained 1 µM Gbeta or Gbeta buffer (where Gbeta was added to the lipid substrate mixture prior to addition of CSK) with or without 1 µM betaARK-PH or equivalent concentrations of glutathione S-transferase. Incubations were terminated and lipids resolved as described(11, 18) . The relative content of p85/PI 3-K in CSK and supernatant was determined by Western blotting(8) /ECL detection (11) and densitometry in a linear response range. In other studies, cytosol from sonicated, nonstimulated platelets (11) or CSK was incubated for 5 min at 37 °C with 200 µM betaNAD with or without 80 µg/ml C3 transferase prior to addition to PI 3-K assay mixtures with or without betaARK-PH (5 µM), Gbeta (1 µM), Rho-glutathione S-transferase (1 µM), and/or GTPS (5 µM). Incubations were terminated and lipids resolved and quantitated as above. Additional experiments were performed with platelet cytosol using immunoprecipitation. In some experiments, cytosol (11) was incubated with buffer, a rabbit polyclonal antibody to the 85-kDa subunit of heterodimeric PI 3-K, or a mixture of mouse monoclonal antibodies to the alpha, beta, and forms of the p85 subunit, and PI 3-K activity was assayed with or without GTPS (5 µM), Gbeta (1 µM), and/or EDIN (20 µg/ml), or protein A with or without antimouse IgG was added, and precipitation was undertaken prior to assay. Precipitates were suspended to the same volume as the original cytosol, and PI 3-K activity was assayed in the post-precipitation supernatant as well as the resuspended precipitate, as above. In some cases, Rho-glutathione S-transferase was added to assay mixtures. Efficiency of immunoprecipitation was determined by Western blotting/ECL, using rabbit polyclonal antibody to p85 or antibodies to alpha, beta, or forms of p85, followed by laser-scanning densitometry. CSK from control and thrombin-activated platelets and cytosol (before and after immunoprecipitations with anti-p85 antibodies) were also examined for the presence of PI 3-K() and quantitated after Western blotting/ECL with anti-p110 antibody. Unfortunately, under the conditions used, the PI 3-K() antibody was not efficient for immunoprecipitations.


RESULTS

As demonstrated in Fig. 1, enzymes known to ADP-ribosylate Rho (either EDIN or C3 transferase) inhibit the activation of PI 3-K in permeabilized platelets in a dose- and NAD-dependent manner. This inhibitory effect is both selective, i.e. not observed with respect to phospholipase C activation (gauged indirectly by PtdOH, Fig. 2, A and B) or PtdIns(4,5)P(2) metabolism (not shown), and transient (Fig. 2, C and D). The transience is not attributable to a reversal of ADP-ribosylation, since levels of [P]ADP-ribosylated Rho remain essentially constant during the period of incubation with agonist (not shown). It is possible that a portion of Rho is protected from ADP-ribosylation by binding to RhoGDI (21) and is later freed of RhoGDI after exposure to platelet agonists but further protected by interaction with effector(s). It also seemed possible, however, that inhibition might be overcome by increasing accumulations of Gbeta (13, 14) as a function of the stimulated dissociation of heterotrimeric G-protein(s).


Figure 1: Effect of varied ADP-ribosylation of Rho on GTPS-stimulated PI 3-K in permeabilized platelets. Permeabilized platelets were incubated with [P]ATP and the indicated concentrations of EDIN (bullet) or C3 transferase (), in the presence or absence (*) of NAD, 5 min prior to incubation with buffer or GTPS for 2 min. Labeled lipids were extracted and quantitated as described. Basal quantities of labeled lipids were unaffected by ADP-ribosylation conditions. Results are presented as the average percentage of inhibition of stimulated accumulations of PtdIns(3,4)P(2) ± the range for a representative experiment performed in duplicate. Error bars are included within symbols. Similar effects were observed for PtdIns(3,4,5)P(3). No significant GTPS-stimulated changes were observed in PtdIns3P during this period, as noted(10) . Changes in PtdIns(4,5)P(2) and PtdOH were unaffected by ADP-ribosylation. 3PPI, 3-phosphorylated phosphoinositide.




Figure 2: Effects of EDIN on GTPS- or thrombin-stimulated accumulations of PtdIns(3,4,5)P(3), PtdIns(3,4)P(2), and PtdOH in permeabilized platelets with time. Platelets were incubated, as in Fig. 1, with (opensymbols) or without (closedsymbols) 22 µg/ml EDIN prior to addition of buffer, GTPS (A and C), or thrombin (B and D) for 2 min. Lipids were resolved and quantitated as described. No significant changes were observed in the absence of GTPS or thrombin. Data are shown as the averages ± ranges (included within symbols) for a representative experiment performed in duplicate. C and D, PtdIns(3,4,5)P(3) (circle, bullet) and PtdIns(3,4)P(2) (up triangle, ) are shown. A and B, PtdOH (box, ) is shown. Similar results were observed when C3 transferase and GTPS/SFLLRN were employed in place of EDIN and thrombin.



That Gbeta becomes available to activate PI 3-K in thrombin-stimulated platelets is indicated by the data in Fig. 3. The CSK fraction from platelets exposed to thrombin for 45 s exhibits an elevated specific activity of PI 3-K in comparison with the Triton-soluble fraction (supernatant) from similarly incubated cells (Fig. 3A), and this elevated PI 3-K activity gradually decreases as increasing concentrations of Gbeta-binding betaARK-PH are added to CSK. Equivalent amounts of glutathione S-transferase are without effect (not shown). The inhibitory effects of betaARK-PH are maximal at 5 µM and bring the specific activity of CSK PI 3-K down to that of supernatant, which is unaffected by betaARK-PH. That PI 3-K in supernatant is unaffected by betaARK-PH argues against the PH domain of betaARK acting as an inhibitor by binding to PtdIns(4,5)P(2) substrate(22) . Furthermore, Gbeta additionally activates the PI 3-K activity of CSK from stimulated platelets and competes with betaARK-PH, consistent with a specific effect of betaARK-PH on Gbeta. The effect of thrombin in promoting an increased specific activity of PI 3-K in CSK is rapid, achieving near maximal levels within 15 s of thrombin addition (Fig. 3B). Most of this increase is inhibited by 5 µM betaARK-PH, causing the specific activity to approach that of PI 3-K in unstimulated platelet CSK.


Figure 3: Effects of betaARK-PH and Gbeta on PI 3-K activity in platelet CSK and supernatant. CSK and supernatant derived from platelets exposed to thrombin for 45 s (A) were incubated with varied concentrations of betaARK-PH in the absence (bullet) or presence (circle) of 1 µM Gbeta and assayed for PI 3-K activity. Supernatant was also assayed with varied concentrations of betaARK-PH (box). Results are presented as the mean ± S.D. for three incubations in duplicate. CSK fractions were also obtained from platelets incubated for 0-3 min with thrombin (B) and assayed for PI 3-K activity in the presence (bullet) or absence (circle) of betaARK-PH (5 µM).



betaARK-PH also inhibits the accumulation of labeled PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) but not PtdOH (or metabolism of PtdIns(4,5)P(2); not shown) in permeabilized platelets stimulated with GTPS (Fig. 4), implying an impairment of PI 3-K but not of phospholipase C activities. The combination of EDIN and betaARK-PH is more inhibitory than is either agent alone; however, neither totally blocks the activation of PI 3-K after 5 min, leaving it likely that an additional factor, relatively insensitive to EDIN and betaARK-PH, contributes to the stimulated activity observed.


Figure 4: Effects of varied concentrations of betaARK-PH with or without EDIN on GTPS-stimulated accumulations of PtdIns(3,4,5)P(3), PtdIns(3,4)P(2), and PtdOH in permeabilized platelets. Platelets were incubated as in Fig. 1with up to 10 µM betaARK-PH in the presence (*) or absence of 22 µg/ml EDIN, prior to addition of buffer or GTPS (10 µM). Incubations were terminated after 5 min, and lipids were extracted and resolved as described. Results shown are the averages ± ranges (in some cases ranges are included within symbols) of two experiments performed in duplicate and are presented as the -fold increase over GTPS-free controls. Basal values with or without betaARK-PH or EDIN were not significantly different. No effects of glutathione S-transferase in betaARK-PH buffer were observed. bullet, PtdIns(3,4,5)P(3); circle, PtdIns(3,4)P(2); up triangle, PtdOH. Note that values for PtdOH with EDIN are not shown, since they were not different from values without EDIN (see also Fig. 2).



Interestingly, the lower PI 3-K activity associated with the small amount of CSK present in unstimulated platelets (8) is unaffected by betaARK-PH (Fig. 3B). Not only is betaARK-PH without inhibitory effect (which might have been attributed to a lack of Gbeta), but, as illustrated in Fig. 5, Gbeta is not stimulatory. Yet, PI 3-K in this fraction (which contains Rho) can be stimulated by GTPS. The stimulation is impaired by ADP-ribosylation of Rho and restored by exogenous Rho, implying that Rho/GTPS-stimulatable PI 3-K is different from Gbeta-stimulatable PI 3-K. Indeed, the Rho-containing p85-directed immunoprecipitates derived from resting platelet cytosol (using polyclonal antibody to the 85-kDa subunit of PI 3-K (Fig. 6) or a combination of monoclonals to p85 isoforms (Fig. 7)) show similar characteristics: no activation of PI 3-K by Gbeta but activation by GTPS ( Fig. 6and Fig. 7), in a manner inhibited by ADP-ribosylation (Fig. 7). The antibody has no effect on total PI 3-K activity with or without Gbeta (Fig. 6). As gauged by Western blotting, the efficiency of p85 immunoprecipitation achievable with the polyclonal antibody ranges from 38 to 42% and is equally efficacious for alpha, beta, and isoforms, whereas the combination of monoclonal antibodies is more efficient (up to 96%). GTPS-stimulated activity in the post-immunoprecipitation supernatant decreases in proportion to decreases in p85, whereas Gbeta-stimulatable PI 3-K remains in the supernatant at levels relatively unaffected by immunoprecipitation. Notably, platelet Rho also sediments with p85-containing PI 3-K, in proportion to the efficiency of immunoprecipitation. Under conditions in which 38-42% of p85 is immunoprecipitated, approximately the same amount of Rho is sedimentable, and when more than 90% of p85 is immunoprecipitated, most of the Rho is immunoprecipitated as well. One might predict, therefore, that, in contrast to the case for the assayed immunoprecipitate, ADP-ribosylation of the Rho-depleted post-immunoprecipitation supernatant would have no effect on PI 3-K activity. Indeed, GTPS is ineffective in stimulating, and ADP-ribosylation cannot inhibit this activity (Fig. 7). Even when exogenous Rho is provided to the p85 and Rho-depleted supernatant, however, there is no enhancement of PI 3-K activity (Fig. 8). Thus, in contrast to the case for p85-containing PI 3-K, Gbeta-stimulatable PI 3-K is not modulated by Rho. The platelet cytosol presumably contains relatively low but variable amounts of heterotrimeric G-protein(s), since we have found GTPS-stimulated PI 3-K to be inhibited variably by betaARK-PH. This supposition is confirmed by the variable detectability of Gbeta in Western blots of platelet cytosol (data not shown).


Figure 5: Effects of Gbeta, ADP-ribosylation (ADP-R), Rho, and GTPS on PI 3-K activity in CSK from unstimulated platelets. Triton-insoluble (CSK) fractions from unstimulated platelets were incubated under various conditions as described under ``Experimental Procedures,'' and PI 3-K activity was assayed. *, with Rho (5 µM), added after ADP-ribosylation of endogenous Rho. Results are the means ± S.D. of two experiments in duplicate. Openbars, without GTPS; hatchedbars, with GTPS.




Figure 6: Effects of polyclonal antibody to p85 on Gbeta- or GTPS-stimulatable PI 3-K activity. Platelet cytosol (Cytosol) was incubated with buffer (- p85 Ab for control and later ``mock'' immunoprecipitation) or polyclonal rabbit antibody to the p85 subunit of PI 3-K (+ p85 Ab) at 4 °C. A portion of these incubation mixtures was combined as well with protein A-Sepharose, followed by sedimentation. The supernatant (Cytosol, Post-spin) was removed and saved, and the pellet was resuspended in the same volume of original buffer as the cytosol from which it came. Efficiency of immunoprecipitation was monitored by Western blotting with polyclonal antibody to p85, and the percentage of the original p85 present was calculated (values in parentheses). PI 3-K activities in cytosol, post-spin cytosol, and resuspended pellets (from true and mock immunoprecipitations) were assayed as described under ``Experimental Procedures'' in the presence (hatchedbars) or absence (openbars) of GTPS (5 µM) with or without Gbeta (1 µM). Data are results of an experiment performed in duplicate and are representative of two experiments.




Figure 7: Effects of immunoprecipitation with combined monoclonal antibodies to p85 on activation of PI 3-K by GTPS or Gbeta and inhibition by EDIN. Incubations proceeded as in Fig. 6except that antibodies were added to all cytosols and assayed, or immunoprecipitated and then assayed, i.e. no mock immunoprecipitations were performed. Combined monoclonal antibodies to the alpha, beta, and forms of p85 were added to cytosol, and immunoprecipitation with anti-mouse IgG/protein A-Sepharose was performed. Pellets were suspended to the same original volume. Western blotting was with combined monoclonal antibodies to p85. The percentage of original p85 is shown in parentheses. PI 3-K was assayed following pretreatment for 5 min with beta-NAD (200 µM) with or without EDIN (20 µg/ml). Assays were in the presence (hatchedbars) or absence (openbars) of GTPS (5 µM) with or without Gbeta (1 µM) and are the results of an experiment in duplicate. ADP-R, ADP-ribosylation; I. P., immunoprecipitate.




Figure 8: Effects of exogenous Rho on Gbeta-stimulated PI 3-K activity in cytosol depleted of p85 and Rho. Platelet cytosol was incubated with a mixture of monoclonal antibodies to p85 alpha, beta, and isoforms as in Fig. 7and then centrifuged with anti-mouse IgG and protein A-Sepharose. PI 3-K activity in the resulting supernatant fractions was assayed in the presence or absence of recombinant Rho-glutathione S-transferase (1 µM) with or without Gbeta (1 µM) and with or without GTPS (5 µM). Hatchedbars, with GTPS. The antibody mixture was >90% efficient in immunoprecipitating both p85 and endogenous Rho. Results are the average ± the range of an experiment performed in duplicate.



Using Western blotting, we have detected a new form of PI 3-K in platelet cytosol and in activated CSK (Fig. 9). This PI 3-K() has been cloned and expressed recently and has been detected in U937 and K562 cells.^2 A cDNA sequence encoding this novel kinase is also present in a leukemic cell line derived from a platelet progenitor cell, the megakaryoblast. (^3)PI 3-K() is not present in CSK of quiescent platelets but appears in the CSK after platelet activation (Fig. 9), as does Gbeta-stimulatable (Fig. 9,) and betaARK-PH-inhibitable (Fig. 9, *) PI 3-K activity, and Gbeta (not shown). Further, PI 3-K() is not immunoprecipitated by our p85-directed antibodies, even when >90% of p85 is immunoprecipitated (Fig. 9).


Figure 9: Presence of PI 3-K() in cytoskeleton and cytosol of human platelets. Platelet cytosol before (lane1) and after (lane2) immunoprecipitation with a mixture of monoclonal antibodies to p85 isoforms (as in Fig. 8, >90% of p85 immunoprecipitated) was monitored by Western blotting with an affinity-purified antibody to PI 3-K(), after resolution on SDS-polyacrylamide gel electrophoresis and transfer to nitrocellulose. Similarly, Triton-insoluble cytoskeletal fractions were prepared from quiescent (lane3) and thrombin-activated (45 s) platelets (lane4), as described in Fig. 3. The same amount of protein from resting and activated platelets was applied per lane for SDS-polyacrylamide gel electrophoresis and Western blotting. The arrow indicates PI 3-K(). For cytoskeletal fractions (lanes3 and 4) assayed for PI 3-K activity (see also Fig. 3and Fig. 5), inhibition by betaARK-PH is shown (*), and stimulation by Gbeta (**) is shown.




DISCUSSION

Our studies indicate that thrombin-induced stimulation of platelet PI 3-K activity is dependent upon both functional Rho and Gbeta. This conclusion is based upon the inhibitory effects of ADP-ribosylating C3 transferase/EDIN and Gbeta-binding betaARK-PH, respectively. Further, the Rho-responsive versus Gbeta-responsive forms of PI 3-K differ. Our findings suggest that Gbeta-stimulatable PI 3-K is a distinct PI 3-K, probably not a conventional p85-containing PI 3-K. Rho-responsive PI 3-K activity precipitates with antibodies directed to the 85-kDa subunits of the most thoroughly characterized (to date) form of PI 3-K, i.e. the heterodimer. Consistent with this, the immunoprecipitates also contain the 110-kDa catalytic subunit of PI 3-K known as p110(alpha), detected by Western blotting (not shown), as well as Rho and CDC42Hs, (^4)another member of the Rho family of small G-proteins(23) . These immunoprecipitates, however, do not contain a newly identified PI 3-K, called PI 3-K().^2 In these respects, the cytoskeleton of ``resting'' platelets and p85-directed immunoprecipitates resemble each other. PI 3-K() is a 110-kDa protein that shares the kinase domain of the other mammalian p110 subunits (alpha and beta) known to associate with p85 alpha and beta subunits but lacks homology in the N-terminal region responsible for binding to p85, which explains why recombinant p110(), in contrast to recombinant p110(alpha), does not bind p85. Significantly, recombinant PI 3-K() is activated directly in vitro by Gbeta.^2 Platelet PI 3-K() remains in the supernatant following immunoprecipitation of p85 PI 3-K, as does Gbeta-stimulatable PI 3-K activity, and both PI 3-K() and Gbeta-stimulatable PI 3-K activity appear in the cytoskeletal fraction of thrombin-activated platelets. It therefore seems likely that the Gbeta-activated PI 3-K present in our platelet subfractions is PI 3-K(). These observations are consistent with the finding of chromatographically separable PI 3-Ks in the cytosol of myeloid cells and the lack of p85 isoforms alpha, beta, or in the Gbeta-activable PI 3-K from the cytosolic subfractions of such cells(13) . Our results, however, differ from those of Thomason et al.(14) who, using a monoclonal antibody to p85 different from those that we employed, found that Gbeta-stimulatable PI 3-K co-precipitated with p85. Conceivably, an epitope is shared between p85 and PI 3-K() or another component to which PI 3-K() binds. Another possibility is that the platelets from which the cytosolic fraction was derived were activated inadvertently and that, in fact, a cytoskeletal complex containing both p85/PI 3-K and PI 3-K() was isolated with the immunoprecipitates.

The observation that two forms of PI 3-K, regulated by Gbeta and Rho, respectively, are activated in thrombin-stimulated platelets is noteworthy. By definition, both routes of activation are controlled by GTP, the former via the receptor and GTP-dependent dissociation of Galpha from Gbeta and the latter via the binding of GTP to the small G-proteins of the Rho family (the connection between the receptor and Rho being as yet unknown). Why two such mechanisms for regulating PI 3-K are available in a cell exposed to one initial agonist is an intriguing question. Further, although betaARK-PH was used as a Gbeta-sequestering tool in our experiments, platelets contain betaARK1 and betaARK2. (^5)It is thus possible that platelet betaARK or other Gbeta-binding entities in addition to Galpha play a physiological role in regulating platelet PI 3-K.

That activated cytoskeletal PI 3-K is inhibited almost completely by betaARK-PH does not necessarily preclude Rho-dependent PI 3-K being associated with activated CSK. Increased amounts of both Rho and PI 3-K (gauged by the 85-kDa subunit of PI 3-K) have been found in the CSK of activated platelets(8, 11) . It may be, however, that Rho-dependent activation cannot be maintained easily during CSK isolation, whereas Gbeta is isolated with PI 3-K in CSK and is activating as such. Some questions in this regard are whether PI 3-K() associates with CSK directly or via Gbeta and how p85/Rho PI 3-K and PI 3-K() each binds to CSK. An additional complication is posed by the observation that Rho does not appear to bind to PI 3-K directly (23) although it is present in p85-directed immunoprecipitates from platelet cytosol, yet CDC42Hs, also present in platelet cytosol,^3 has been shown in reconstitution experiments to bind and activate p85-containing PI 3-K(23) . It thus seems likely that the presence of Rho in PI 3-K immunoprecipitates from platelet cytosol is dependent upon additional factor(s) and that Rho may regulate the interaction between endogenous platelet CDC42Hs and p85-containing PI 3-K. These are subjects currently under investigation in our laboratory.


FOOTNOTES

*
This work was supported in part by Grant HL 38622 (to S. E. R.) from the NHLBI and Grant GH 44944 (to J. L. B.) of the National Institutes of Health. 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 should be addressed. Fax: 215-923-7145.

(^1)
The abbreviations used are: G-protein, GTP-binding protein; PI 3-K, phosphoinositide 3-kinase; PtdIns, phosphatidylinositol (locants of other phosphates on inositol ring shown in parentheses); PtdOH, phosphatidic acid; betaARK, beta-adrenergic receptor kinase; PH, pleckstrin homology; Gbeta, beta subunits of heterotrimeric G-proteins; GTPS, guanosine 5`-3-O-(thio)triphosphate; EDIN, epidermal cell differentiation inhibitor; CSK, membrane cytoskeleton; SFLLRN, Ser-Phe-Leu-Leu-Arg-Asn peptide; ECL, enhanced chemiluminescence; RhoGDI, Rho GDP-dissociation inhibitor.

(^2)
B. Stoyanov, S. Volinia, T. Hanck, I. Rubio, M. Lubchenkov, S. Stoyanova, B. Vanhaesenbroek, R. Dhand, M. Zvelebil, D. Maleek, P. Gierschik, K. Seedorf, J. J. Hsuan, M. D. Waterfield, and R. Wetzker, submitted for publication.

(^3)
S. E. Rittenhouse, S. Volinia, and M. D. Waterfield, unpublished results.

(^4)
J. Zhang and S. E. Rittenhouse, unpublished results.

(^5)
J. L. Benovic, unpublished results.


ACKNOWLEDGEMENTS

We thank the blood drawing services of the Blood Center, Cardeza Foundation for Hematologic Research.


REFERENCES

  1. Aktories, K., and Jakobs, K. H. (1984) Eur. J. Biochem. 145, 333-338 [Abstract]
  2. Shenker, A., Goldsmith, P., Unson, C. G., and Spiegel, A. M. (1991) J. Biol. Chem. 266, 9309-9313 [Abstract/Free Full Text]
  3. Brass, L. F., Laposata, M., Banga, H. S., and Rittenhouse, S. E. (1986) J. Biol. Chem. 261, 16838-16847 [Abstract/Free Full Text]
  4. Banga, H. S., Walker, R. K., Winberry, L. K., and Rittenhouse, S. E. (1988) Biochem. J. 252, 297-300 [Medline] [Order article via Infotrieve]
  5. Morii, N., Teru-uchi, T., Tominaga, T., Kumagai, N., Kozaki, S., Ushikubi, F., and Narumiya, S. (1992) J. Biol. Chem. 267, 20921-20926 [Abstract/Free Full Text]
  6. Valius, M., and Kazlauskas, A. (1993) Cell 73, 321-334 [Medline] [Order article via Infotrieve]
  7. Okada, T., Sakuma, L., Fukui, Y., Hazeki, O., and Ui, M. (1994) J. Biol. Chem. 269, 3563-3567 [Abstract/Free Full Text]
  8. Zhang, J., Fry, M. J., Waterfield, M. D., Jaken, S., Liao, L., Fox, J. E. B., and Rittenhouse, S. E. (1992) J. Biol. Chem. 267, 4686-4692 [Abstract/Free Full Text]
  9. Carter, A. N., Huang, R., Sorisky, A., Downes, C. P., and Rittenhouse, S. E. (1994) Biochem. J. 301, 415-420 [Medline] [Order article via Infotrieve]
  10. Kucera, G. L., and Rittenhouse, S. E. (1990) J. Biol. Chem. 265, 5345-5348 [Abstract/Free Full Text]
  11. Zhang, J., King, W. G., Dillon, S., Hall, A., Feig, L., and Rittenhouse, S. E. (1993) J. Biol. Chem. 268, 22251-22254 [Abstract/Free Full Text]
  12. Plow, E. F., D'Souza, S. E., and Ginsberg, M. H. (1992) Semin. Thromb. Hemostasis 18, 324-331 [Medline] [Order article via Infotrieve]
  13. Stephens, L., Smrcka, A., Cooke, F. T., Jackson, T. R., Sternweis, P. C., and Hawkins, P. T. (1994) Cell 77, 83-93 [Medline] [Order article via Infotrieve]
  14. Thomason, P. A., James, S. R., Casey, P. J., and Downes, C. P. (1994) J. Biol. Chem. 269, 16525-16528 [Abstract/Free Full Text]
  15. Pitcher, J. A., Inglese, J., Higgins, J. B., Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264-1267 [Medline] [Order article via Infotrieve]
  16. Koch, W. J., Inglese, J., Stone, W. C., and Lefkowitz, R. J. (1993) J. Biol. Chem. 268, 8256-8260 [Abstract/Free Full Text]
  17. Smith, D. B., and Johnson, K. S. (1988) Gene (Amst.) 67, 31-40 [CrossRef][Medline] [Order article via Infotrieve]
  18. Rittenhouse, S. E. (1995) in Methods in Enzymology: Small GTPases and Their Regulators, Part B, Rho Family (Hall, A., ed), Academic Press, Orlando, Florida, in press
  19. Sugai, M., Enomoto, T., Hashimoto, K., Matsumoto, K., Matsuo, Y., Ohgai, H., Hong, Y.-M., Inoue, S., Yoshikawa, K., and Suginaka, H. (1990) Biochem. Biophys. Res. Commun. 173, 92-98 [Medline] [Order article via Infotrieve]
  20. Huang, R., Kucera, G. L., and Rittenhouse, S. E. (1991) J. Biol. Chem. 266, 1652-1655 [Abstract/Free Full Text]
  21. Kikuchi, A., Kuroda, S., Sasaki, K., Kotani, K., Hirata, K., Katayama, M., and Takai, Y. (1992) J. Biol. Chem. 267, 14611-14615 [Abstract/Free Full Text]
  22. Harlan, J. E., Hajduk, P. J., Yoon, H. S., and Fesik, S. W. (1994) Nature 371, 168-170 [CrossRef][Medline] [Order article via Infotrieve]
  23. Zheng, Y., Bagrodia, S., and Cerione, R. A. (1994) J. Biol. Chem. 269, 18727-18730 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.