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Physical and Functional Interaction between Protein Kinase C (cid:1) and Fyn Tyrosine Kinase in Human Platelets*

An increasing number of tyrosine kinases have been shown to associate with isoforms of the protein kinase C (PKC) family. Here, we show evidence for physical and functional interaction between PKC (cid:1) and the Src family kinase Fyn in human platelets activated by alboaggre-gin-A, a snake venom capable of activating both GPIb-V-IX and GPVI adhesion receptors. This interaction in-volves phosphorylation of PKC (cid:1) on tyrosine and is specific in that other isoforms of PKC, PKC (cid:2) and (cid:3) , which also become tyrosine-phosphorylated, do not interact with Fyn. In addition, PKC (cid:1) does not interact with other platelet-expressed tyrosine kinases Syk, Src, or Btk. Stimulation also leads to activation of both Fyn and PKC (cid:1) and to serine phosphorylation of Fyn within a PKC consensus sequence. Alboaggregin-A-dependent activation of Fyn is blocked by bisindolylmaleimide I, suggesting a role for PKC isoforms in regulating Fyn activity. Platelet activation with alboaggregin-A induces translocation of the two kinases from cytoplasm to the plasma membrane of platelets, as observed by confocal immunofluorescence micros-copy. Translocation of Fyn and PKC (cid:1) are blocked by PP1 and bisindolylmaleimide I, showing a dependence upon Src and PKC kinase

The protein kinase C (PKC) 1 family comprises 10 isozymes grouped into three classes: conventional (␣, ␥, ␤I, and ␤II), novel (␦, ⑀, /L, and ), and atypical ( and /). In addition, PKC and are considered as a fourth class, although some authors classify them as a separate family termed protein kinase D (1). Protein kinases C are critical for the regulation of several functional events in cells, including exocytosis and adhesion. In platelets, activation of PKC by diacylglycerol or phorbol esters can induce platelet degranulation (2), and inhibitors of PKC effectively inhibit receptor-mediated platelet secretion (3)(4)(5)(6), although synergism between Ca 2ϩ -and PKCmediated processes is required for a full secretory response of platelets to external stimuli (4,(7)(8)(9). Stimulation of PKC also leads to activation of the integrin ␣ IIb ␤ 3 (10), although there is conflicting evidence concerning the sensitivity of agonist-induced aggregation to pharmacological inhibition of PKC (4,(11)(12)(13). It is now becoming clear, however, that different isoforms of PKC may play differential roles in the regulation of these events. Leitges et al. (14) have recently shown that PKC␦ is a negative regulator of degranulation in mast cells, demonstrating a more complex role for the PKC family in control of this functional event.
There are now known to be a variety of different mechanisms by which PKC activity and localization may be regulated, important among which is phosphorylation of serine, threonine, and more recently tyrosine residues (15). We have recently shown that the novel isoform, PKC, may be phosphorylated on tyrosine through a physical and functional association with the non-receptor tyrosine kinase Btk (16). The closely related kinase PKC␦ has also been shown to be phosphorylated on tyrosine residues in a variety of cell types and in response to a variety of stimuli, including phorbol ester, growth factors, and hormones (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). We had shown that phosphorylation of PKC on tyrosine is associated with an inhibition of this isoform, but there is presently controversy concerning the functional role of tyrosine phosphorylation of PKC␦. In some reports activity of PKC␦ is reduced by tyrosine phosphorylation (20,21,32), whereas in others the modification enhances its activity (15,17,22,23,31). Differential effects upon activity may be accounted for by different sites of tyrosine phosphorylation within the protein by different upstream kinases. It has been shown that PKC␦ may be phosphorylated by Fyn (18,20,21), Lyn (24,33), Src (18,20,26,(33)(34)(35), Abl (36,37), and growth factor receptor kinases (20,23).
In addition to tyrosine phosphorylation of PKC isoforms, tyrosine kinases may in turn become reciprocally phosphorylated on serine and threonine residues. There is substantial evidence of this for members of the Src family kinases. Src itself is phosphorylated by PKC isoforms (38,39), and one isoform, PKC⑀, has been shown to lie upstream of activation of Src and Lyn in cardiac myocytes (40). On the other hand, PKC␦ has been shown to associate with Src and Lyn in mast cells, leading to reciprocal phosphorylation and a decrease in functional activity of Src and Lyn (33). Fyn has also been shown to be phosphorylated on serine residues (41,42). Cabodi et al. (43) have recently shown that one isoform of PKC, PKC, associates with Fyn and is necessary and sufficient for activation of Fyn in keratinocytes. There is therefore evidence that, in other cell types, Fyn may associate physically and functionally with PKC isoforms, leading to modulation of its activity.
In the present report we were interested in investigating the mutual regulation of PKC␦ and Fyn in human platelets. We were able to show a physical interaction between the two kinases and phosphorylation of each kinase on both serine/threonine residues and tyrosines. Functionally, Fyn is shown to lie upstream of PKC␦, and PKC isoforms other than PKC␦ lie upstream of Fyn positively regulating its activity and translocation from cytosol to plasma membrane.

Materials
Trimeresurus albolabris venom was a kind gift from Professor R. G. D. Theakston (Liverpool, UK). Alboaggregin-A was prepared from venom by ion-exchange chromatography as previously described (44). Anti-phosphotyrosine monoclonal antibody 4G10 was from Upstate Biotechnology Inc. (TCS Biologicals Ltd., Bucks, UK). All anti-PKC antibodies were from Transduction Laboratories (BD Biosciences, Oxford, UK). Anti-Btk, Fyn, Src, and Syk antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-specific anti-Src (Tyr-416) and phospho-specific anti-PKC substrate antibodies were from Cell Signaling Technology (New England BioLabs, UK

Preparation and Stimulation of Human Platelets
Human blood was drawn from healthy, drug-free volunteers on the day of the experiment. Acid citrate dextrose (ACD: 120 mM sodium citrate, 110 mM glucose, 80 mM citric acid, used at 1:7 v/v) was used as anticoagulant. Platelet-rich plasma (PRP) was prepared by centrifugation at 200 ϫ g, for 20 min, and platelets were then isolated by centrifugation for 10 min at 1000 ϫ g, in the presence of prostaglandin E 1 (40 ng/ml). The pellet was resuspended to a density of 4 ϫ 10 8 platelets/ml in a modified Tyrode's solution-HEPES buffer (145 mM NaCl, 2.9 mM KCl, 10 mM HEPES, 1 mM MgCl 2 , 5 mM glucose, pH 7.3). To this platelet suspension, 10 M indomethacin was added, and a 30-min resting period was allowed before stimulation. Stimulation of platelets was performed in an aggregometer at 37°C, with continuous stirring at 800 rpm, unless stated. Unless otherwise specified, all platelet stimulation occurred in the presence of EGTA (1 mM). Alboaggregin A was used at 3.5 g/ml, unless otherwise stated, a concentration previously determined to be the EC 50 value for induction of 5-HT release (44).

Immunoprecipitation with Immobilized Antibody
For experiments involving assessment of phosphorylation of Fyn, to avoid contamination of immunoprecipitation samples with IgG heavy chain, it was necessary to covalently couple immunoprecipitating antibody to beads. This was achieved using a Seize X protein G immunoprecipitation kit (Perbio Science UK, Cheshire, UK), following the described protocol. Briefly, 100 g of antibody was incubated with protein G-agarose beads for 1 h at room temperature to allow binding. Crosslinking reagent disuccinimidyl suberate was added for 1 h, and beads were then washed extensively to remove all free antibody. Antibodylinked beads were then added to samples and were incubated overnight at 4°C. Beads were then washed, and trapped protein was eluted by addition of elution buffer (pH 2.8). To the eluate, 5ϫ Laemmli sample solvent was added, and samples were boiled for 5 min.

Electrophoresis of Proteins and Western Blotting
Proteins were resolved by electrophoresis in 10 -15% gradient SDS-PAGE gels. Samples were then transferred to polyvinylidene difluoride membranes, using a Bio-Rad Trans-Blot SD semi-dry transfer cell, blocked with 10% bovine serum albumin, and incubated for 1 h at room temperature with appropriate primary antibody (1 g/ml). Membranes were then washed before incubation with appropriate secondary antibody followed by thorough washing. Bound peroxidase activity was detected using enhanced chemiluminescence (ECL, Amersham Biosciences).

In Vitro Kinase Assays
Autophosphorylation-PKC␦ was immunoprecipitated from platelets lysed into Nonidet P-40 buffer and re-suspended in 20 l of kinase assay (KA) buffer (5 mM MgCl 2 , 5 mM MnCl 2 , 100 mM NaCl, 10 M ATP, 2 mM Na 3 VO 4 , 20 mM HEPES, pH 7.2), and the reaction was started by adding [␥-32 P]ATP (250 Ci/ml). After incubation for 10 min at room temperature, the reaction was terminated by adding 0.5 ml of ice-cold 100 mM EDTA. Immunoprecipitated proteins were then washed in RIPA buffer before separation by SDS-PAGE and detection of phosphorylated proteins by autoradiography.
Raytide Phosphorylation-Fyn activity was assayed using Raytide peptide as an exogenous substrate. Immunoprecipitated kinase was resuspended in 20 l of KA buffer and 10 g of Raytide peptide added to each sample. The reaction was started by addition of 10 l of ATP buffer (0.15 mM ATP, 30 mM MgCl 2 , and 200 Ci/ml [␥-32 P]ATP in KA buffer). After incubation at 30°C for 30 min, the reaction was terminated by addition of 10% phosphoric acid. Samples were applied to 2 ϫ 2 cm squares of P81 ion exchange chromatography paper, extensively washed in 0.5% phosphoric acid followed by a wash in acetone. Papers were then dried, and labeled Raytide was quantified by liquid scintillation counting.

Measurement of Cytosolic Calcium
Measurement of cytosolic calcium was performed as previously described (45). Briefly, 3 M Fura 2-AM was added to platelet-rich plasma, and the mixture was incubated at 30°C for 45 min in the presence of 10 M indomethacin. Platelets were washed and re-suspended in modified Tyrode's solution and stimulated at room temperature in the absence of EGTA. Fluorescence excitation was made at 340 and 380 nm, and emission at 510 nm was measured using a PerkinElmer Life Sciences LS5 spectrofluorometer. Data are presented as the excitation fluorescence ratio (340:380 nm).

Measurement of Released 5-HT
Platelets were loaded by incubation of PRP with 0.2 Ci/ml [ 3 H]5-HT for 1 h at 37°C. Platelets were preincubated with 1 mM EGTA before stimulation, to prevent aggregation. Reactions were terminated by addition of an equal volume of 6% glutaraldehyde in Tyrode's solution, followed by brief microcentrifugation, and [ 3 H]5-HT released into the supernatant was determined by liquid scintillation counting and expressed as a percentage of the total tissue content, as described previously (46).

Immunofluorescence Confocal Imaging of Platelets
Platelets were stimulated with alboaggregin-A, and reactions were terminated by addition of 4% paraformaldehyde in phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 ). Samples were left under agitation for 30 min at room temperature. Platelets were then pelleted by centrifugation at 4000 rpm for 2 min in a microcentrifuge and washed twice in PBS. Platelets were immobilized on poly-lysine-coated coverslips overnight, permeabilized by incubation of coverslips with 0.05% Triton X-100/PBS at room temperature for 10 min, and then incubated for 30 min at room temperature with PBS/1% bovine serum albumin (BSA) to block nonspecific antibody binding. Samples were then incubated overnight with primary antibody at a concentration of 1 g/ml in PBS, 1% BSA at 4°C. Coverslips were then washed three times in PBS, 0.05% Triton X-100. Fluorescein isothiocyanate (FITC)-labeled secondary antibody was then added at a concentration of 2 ng/ml in PBS/1% BSA, for 1 h at room temperature. Subsequent to this, coverslips were washed three times in PBS/0.05% Triton X-100 and then mounted onto slides using a 13.5% Mowiol solution containing 2.5% 1,4-diazobicyclo[2.2.2]octane to prevent bleaching of fluorescence. Platelets were imaged using a Leica TCS-NT confocal laser scanning microscope equipped with Kr/Ar laser (488-, 568-, and 647-nm lines) attached to a Leica DM IRBE inverted epifluorescence microscope with phase-contrast. (15,32,47), and we have recently reported tyrosine phosphorylation of PKC induced by alboaggregin A in platelets (16). Alboaggregin A is a purified lectin-type snake venom capable of binding to and activating both GP Ib and GP VI adhesion receptors on platelets for von Willebrand factor and collagen, respectively. Here we show that several other PKC isoforms are also phosphorylated on tyrosine upon alboaggregin-A stimulation of platelets (Fig. 1). These include the classic isoform ␤, the novel isoforms ⑀ and ␦ and PKC, all of which were not phosphorylated under basal conditions, but became so upon platelet activation. PKC was also investigated, but showed no tyrosine phosphorylation under these conditions.

Several PKC Isoforms Become Tyrosine-phosphorylated upon Platelet Activation by Alboaggregin-A-Tyrosine phosphorylation of various isoforms of PKC has been demonstrated in a number of cell types
Association of PKC Isoforms with Platelet Non-receptor Tyrosine Kinases-We had previously shown that PKC physically and functionally associates with the non-receptor tyrosine kinase Btk upon platelet activation (16). It was important therefore to investigate whether other isoforms of PKC that become tyrosine-phosphorylated also associate with non-receptor tyrosine kinases. Syk, Src, Fyn, and Btk are four major platelet tyrosine kinases known to be involved in the alboaggregin-A-activated signaling pathway through activation of both GPIb-V-IX receptor complex and GPVI (16,44,48,49). Each of these kinases was immunoprecipitated from alboaggregin-A-activated platelets, and Fig. 2A shows that there is specific association between PKC␦ and Fyn, whereas there is no co-association between PKCs ⑀ or with any of the tyrosine kinases investigated. The association between PKC␦ and Fyn was dependent upon activation (Fig. 2B) and was inhibited by PP1, a selective inhibitor of Src family kinases. Bisindolylmaleimide I (BIM), the broad-spectrum PKC inhibitor, had no effect upon the association between PKC␦ and Fyn (Fig. 2C).
Tyrosine Phosphorylation and Activity of PKC␦ Is Dependent upon Src Kinase Activity and Is Up-regulated by Pre-treatment of Platelets with PKC Inhibitors- Fig. 3A shows that, under basal conditions, PKC␦ is not tyrosine-phosphorylated. By 30 s of alboaggregin A stimulation, PKC␦ had become tyrosinephosphorylated, reaching a maximal by 60 s, and then subsequently decreasing by 120 s, although not back to basal levels. As PKC␦ and Fyn associate, it was decided to investigate whether inhibition of either of these kinase activities would affect the level of tyrosine phosphorylation of PKC␦. Fig. 3B shows that preincubation of platelets with the Src family kinase inhibitor PP1 ablates the alboaggregin-A-induced tyrosine phosphorylation of PKC␦. However, pretreatment of platelets with non-isozyme-specific inhibitor BIM (20 M), or specific inhibition of PKC␦ using rottlerin (also called mallotoxin, 10 M), caused a marked increase in the tyrosine phosphorylation of PKC␦. Immunoprecipitated PKC␦ was subjected to in vitro kinase assay where activity is measured by autophosphorylation, and incorporation of 32 P-labeled phosphate is visualized by autoradiography. Fig. 3C shows that PKC␦ from resting platelets had a low level of activity but that, subsequent to alboaggregin A stimulation, its activity increased. On addition of BIM or rottlerin to the kinase assay buffer, activity of PKC␦ was ablated. When PKC␦ was immunoprecipitated from platelets preincubated with either BIM or rottlerin prior to stimulation, however, the in vitro activity of PKC␦ was enhanced. PKC␦ activity in contrast was fully inhibited when platelets were preincubated with the Src family kinase inhibitor PP1.
PKC␦ Translocates to the Plasma Membrane upon Platelet Activation in a Manner Dependent upon Src Kinase Activity-Translocation of PKC isoforms from cytosol to plasma mem- brane is characteristic of PKC activation. It was therefore decided to examine the subcellular localization of PKC␦ upon activation of platelets by alboaggregin-A, and to investigate the effect of inhibition of Src and PKC upon this translocation. Fig.  4A shows that, upon stimulation of platelets with alboaggregin-A, PKC␦, along with the other tyrosine-phosphorylated isoforms PKC and PKC⑀, translocates from the cytosol to the plasma membrane. This translocation is prevented by pretreatment of platelets with PP1, the inhibitor of Src family kinases, as shown in Fig. 4B. This effect is specific to inhibition of Src kinases, because inhibition of Btk with LFM-A13 had no effect on translocation of PKC␦. Fig. 4B shows that the activity of PKC is essential for translocation of PKC␦ to take place, because it is blocked by BIM, although not the activity of PKC␦ itself, because inhibition of PKC␦ with rottlerin did not affect its redistribution to the cell surface.
Fyn Is Phosphorylated on Tyr-419 upon Activation in a Manner Dependent upon PKC and Src Kinase Activity-For full activity of Src kinases, a tyrosine residue within the activation loop of the catalytic domain (Tyr-419 for Fyn) must undergo autophosphorylation (reviewed in Refs. 50 and 51). Using a phosphorylation site-specific antibody, it is possible to assess the phosphorylation state of this residue. Fig. 5A shows that under basal conditions there is some minimal phosphorylation of this residue (Tyr-419) but that, upon stimulation of platelets with alboaggregin-A, tyrosine phosphorylation is enhanced. Addition of the Src family kinase inhibitor PP1 to the platelets (20 M) ablated the observed phosphorylation. Addition of the non-selective PKC inhibitor BIM (20 M) to the platelets resulted in a decrease in the stimulated level of phosphorylation back to, but not below, the basal level of phosphorylation, whereas addition of rottlerin (10 M) had no effect.
Fyn Is Phosphorylated on Serine within a PKC Consensus Sequence-It has long been known that, in various cell types, Fyn may be phosphorylated on serine residues (41,42). Fig. 5B shows that, upon platelet activation by alboaggregin-A, Fyn becomes phosphorylated on serine residues that are located within a PKC consensus sequence. This was shown by Western blotting using an antibody that recognizes phosphoserine within a PKC phosphorylation consensus sequence, specifically phosphoserine with arginine or lysine residues at the Ϫ2 and ϩ2 positions with a hydrophobic residue at the ϩ1 position. Furthermore, this stimulation-dependent phosphorylation was shown to be dependent on the activity of PKC, because inhibition with BIM (20 M) ablated the phosphorylation.
Fyn Is Activated by Alboaggregin and Is Positively Regulated by PKC Activity-It has previously been shown that Fyn is activated in platelets downstream of collagen and thrombin receptor activation (52,53). In Fig. 5C, the activity of Fyn immunoprecipitated from platelets is assayed in vitro by examining its ability to phosphorylate the exogenous substrate peptide Raytide TM . Inhibitors were added either to the platelets, prior to stimulation with alboaggregin A, or to the kinase assay buffer (KAB) during the in vitro assay stage, to observe the role of PKC and Src family kinases in regulating the activity of Fyn. There is considerable Fyn activity even in basal platelets, because addition of the Src family kinase inhibitor PP1 (20 M) to the KAB ablated this measured activity. Upon stimulation of platelets with alboaggregin A, however, the activity increases. Preincubation of platelets with PP1 prior to stimulation caused ablation of Fyn activity. Preincubation of platelets with the non-selective PKC inhibitor BIM (20 M) prevented the stimulation-dependent increase in Fyn activity but did not decrease activity below basal, unlike PP1. Preincubation of platelets with rottlerin (10 M) had no effect on Fyn activity.
Fyn Translocates to the Plasma Membrane in an Src Kinaseand PKC Kinase-dependent Manner-There are various contrasting reports concerning the subcellular localization of Fyn in basal and activated platelets. Although some suggest that Fyn translocates to the cytoskeleton in an aggregation-dependent manner (53), others have reported a constitutive association of Fyn with the GPVI-Fc receptor ␥ chain complex (52,54), indicating that some Fyn would be located at the plasma membrane regardless of stimulation. Fig. 6 shows that in resting platelets most Fyn is diffusely distributed throughout the platelet, as indicated by the uniform distribution of staining, whereas, upon stimulation of platelets with alboaggregin A, Fyn becomes localized to the plasma membrane, thus demonstrating a stimulation-dependent translocation. Preincubation of platelets with the non-selective PKC inhibitor BIM (20 M) prevented the alboaggregin-A-induced translocation, however, when platelets were preincubated with rottlerin (10 M), Fyn still translocated as normal upon alboaggregin-A stimulation. Preincubation of platelets with the Src family kinase inhibitor PP1 (20 mM), however, prevented translocation of Fyn to the plasma membrane.
Rottlerin Potentiates Alboaggregin-A-induced Platelet Aggregation, Dense Granule Secretion, and Calcium Response- Fig.  7A shows that rottlerin, at both 5 and 20 mM, markedly potentiated alboaggregin-A-induced platelet aggregation, when the agonist was used at concentrations of 1.75 or 0.875 g/ml, which are submaximal for induction of aggregation. In addition, when alboaggregin-A-induced dense granule secretion was examined, by means of measurement of release of [ 3 H]5-HT from platelets loaded with this compound, it was observed that inhibition of PKC␦ caused a marked increase in [ 3 H]5-HT released (Fig. 7B). This potentiatory effect was also seen when the effects of rottlerin on calcium mobilization were examined. Fig. 7C shows that preincubation of platelets with 5 and 20 M rottlerin caused concentration-dependent increases in the maximum intracellular calcium concentration achieved by stimulation of platelets with alboaggregin A. DISCUSSION It is well established that the PKC and Src kinase families play key roles in signaling events in most cell types. In platelets it has been known for many years that PKC is essential for full activation of secretion and aggregation responses, although not for the shape change response, induced by a wide variety of platelet agonists from adhesion receptors such as GP VI to G-protein-coupled receptors such as the thrombin and thromboxane receptors. Activation of PKC is not an immediate early event, but is generally subsequent to activation of phospholipase C and release of diacylglycerol. The Src family of kinases has been shown to play a vital early signaling role in GP VI signaling, being responsible for phosphorylation of FcR ␥-chain (52, 55). We and others have also shown them to be involved in a similar early event in signaling downstream of GP Ib-V-IX (44,48), and in this report we use the snake venom alboaggre- gin A to activate both these adhesion receptors to induce early phosphorylation of the ␥-chain by Src kinases.
It has become clear, however, that there is a greater level of complexity involved in these signaling events. In particular, the multiple of isoforms of PKC and multiple members of the Src kinase family are likely each to play different signaling and functional roles. We have recently shown for example that one novel PKC isoform, PKC, physically and functionally associates with the tyrosine kinase Btk. This is a selective association, because other members of the PKC family do not associate with Btk. In this report we have chosen to extend this study of cross-talk to investigate further selective interactions between PKC family members and tyrosine kinases. In so doing we have uncovered a selective physical and functional association between PKC␦ and Fyn, which is summarized in the diagram shown in Fig. 8.
PKC␦: Tyrosine Phosphorylation, Activation, and Translocation to the Plasma Membrane-Because we had already shown that PKC becomes phosphorylated on tyrosine upon activation of platelets with alboaggregin A (16), it was important to assess whether other members of the PKC family would also become tyrosine-phosphorylated upon activation. The classic isoform ␤, the novel isoforms ⑀ and ␦, and the atypical isoform were all tyrosine-phosphorylated in a stimulation-dependent manner. The novel isoform PKC was not phosphorylated on tyrosine. Several of these isoforms have previously been shown to be phosphorylated on tyrosine in other cell types in response to a variety of stimuli (15,27,47,56), but their phosphorylation is clearly a cell-and stimulus-specific event. Many different agonists have been shown to cause tyrosine phosphorylation of the novel PKC isoform PKC␦, including growth factors (19,20,25,47), H 2 O 2 (57), and G-protein-coupled receptors (25, 58 -60).
Indeed one of these reports shows phosphorylation of PKC␦ in platelets in response to thrombin (60), showing the phosphorylation to be biphasic, possibly corresponding to the phases of activation of Src kinases in platelets in response to this agonist (61). In the present study, in response to alboaggregin A, the time course peaked at 60 s and then dropped to a sustained plateau over the 5-min time course studied. This pattern is similar to, although more rapid to peak than, that seen in other cell types such as pancreatic acinar cells in response to CCK (27) and skeletal muscle cells in response to insulin (47).
It was then important to address the mechanism and role of tyrosine phosphorylation of PKC␦, which has previously been shown to be a calcium-independent, diacylglycerol-dependent kinase (1,19). First, PP1 was used as a selective Src family kinase inhibitor and was shown to abolish tyrosine phosphorylation of PKC␦ in response to alboaggregin A. Src family kinases are therefore likely to lie upstream of this event, as expected, because we and others (44,52,55) have shown Src kinases to lie very proximal in the GP VI and GP Ib-V-IX signaling pathways. Therefore, blockade of these kinases would lead to inhibition of most signaling and functional events. Src kinases may also be directly responsible for the phosphoryla-tion of PKC␦, and this is in agreement with other reports (18,20,24,26,(33)(34)(35) in other cell types showing that Src kinases are capable of phosphorylating this PKC isoform. Src kinases clearly lie functionally upstream in this signaling pathway, because PP1 also led to inhibition of PKC␦ activity (Fig. 3C). Addition of the non-selective inhibitor bisindolylmaleimide (BIM) and the selective inhibitor rottlerin to the kinase assay buffer caused full inhibition of PKC␦ activity (Fig. 3C). This is in contrast to Davies et al. (62) who showed in a purified enzyme in vitro kinase assay system that rottlerin did not inhibit PKC␦ but did inhibit p38-regulated/activated kinase and mitogen-activated protein kinase-activated protein kinase-2. In addition, others have shown poor activity and specificity of rottlerin in vivo (63) and in vitro (64). Our assay system differed from Davies et al. (62) and involved the use of native PKC␦ isolated from activated platelets by immunoprecipitation, which may explain the difference between our data and theirs, although it is clear that the specificity of rottlerin for PKC␦ may be poor. This limits us from drawing definitive conclusions regarding the role of PKC␦ in signaling pathways, but because we were able to show clear and complete inhibition of PKC␦ by rottlerin in platelets, this allows us to rule out a role for PKC␦ if rottlerin is shown to have no effect upon specific cellular responses. When inhibitors were added to the platelet suspension, both BIM and rottlerin were shown to enhance tyrosine phosphorylation of PKC␦ (Fig. 3B), and in the case of rottlerin, this was associated with an increase in the activity of PKC␦. This may indicate a negative feedback inhibition of PKC␦ by itself or other kinases inhibited by rottlerin, leading to enhanced apparent activity when isolated from platelets pretreated with the PKC inhibitor. These data are in agreement with a recent report showing enhanced tyrosine phosphorylation of PKC␦ upon activation of pancreatic acinar cells by phorbol ester in the presence of BIM-1 at concentrations less than 30 M (27).
It has long been recognized that translocation of PKC isoforms from cytosol to plasma membrane is characteristically associated with activation of the kinase (1). Here we have used a confocal immunofluorescence approach to examine movement of PKC isoforms from cytosol to membrane upon cell activation. PKCs ␦, ⑀, and , all of which become phosphorylated on tyrosine, translocate from cytosol to a peripheral position in the platelet very rapidly upon activation by alboaggregin A. It was important to ascertain the role of PKC and Src kinase activities upon this translocation event for PKC␦. In correlation with the phosphorylation and activation of PKC␦, addition of PP1 blocked translocation to the membrane. Addition of BIM, however, also blocked translocation to the membrane, although rottlerin did not do so. This suggests that the activity of PKC isoforms other than PKC␦ are important in controlling the localization of PKC␦. One intriguing possible explanation for this may be that Fyn, which clearly associates with PKC␦, may act as a scaffold, or novel RACK protein (receptor for activated protein kinase C) for PKC␦, mediating its transport to the FIG. 6. Translocation of Fyn to the platelet plasma membrane is blocked by PP1 and BIM. Fixed platelets were stained with antibodies to Fyn, followed by FITC-conjugated anti-rabbit secondary antibody as described under "Experimental Procedures." Samples were either from basal platelets or platelets stimulated with alboaggregin-A (3.5 g/ml) for 60 s without stirring as indicated. Some samples were pretreated with either PP1 (20 M), BIM (20 M), or rottlerin (10 M) as indicated before stimulation. Images shown are from a single experiment but are representative of at least three repetitions. plasma membrane, because BIM-1 was also shown to inhibit Fyn activity, whereas rottlerin did not (Fig. 5C). Interestingly, however, BIM did not inhibit the interaction between PKC␦ and Fyn (Fig. 2C). Therefore PKC activity is not required for interaction between the two kinases, but is required for translocation to the membrane, as has been shown previously for novel isoforms of PKC, which require phosphorylation on serines within their C2 domain for full translocation to the plasma membrane (65).
Fyn: Activation and Phosphorylation by PKC and Translocation to the Plasma Membrane-Interestingly, Fyn is phosphorylated by a PKC isoform in a BIM-sensitive manner. Phosphorylation of Fyn by PKC correlates with activity of Fyn as assessed either by phosphorylation of tyrosine 419 (Fig. 5A), the autophosphorylation site within the catalytic domain of the kinase required for full activity of Fyn, or phosphorylation of an exogenous peptide Raytide (Fig. 5C). Alboaggregin A induces an activation of Fyn from a high resting basal level, and inhibition of PKC activity by BIM leads to inhibition of Fyn activity to the resting level. The high basal activity of Fyn is in contrast to the low basal activity of Src 2 and is in agreement with data showing a constitutive association of active Fyn and Lyn with GP VI (54). Interestingly, PP1, the Src kinase inhibitor, reduces the activity and phosphorylation of Fyn on Tyr-419 apparently to zero, to a level well below that of the basal resting state. From this we infer that there are therefore two phases of Fyn activity: a basal activity that does not require PKC activity and a stimulated activity that is dependent upon PKC activity. This stimulated activity may, however, not be dependent upon PKC␦ activity, because rottlerin is not able to inhibit either the PKC-dependent phosphorylation of Fyn or its activity. We interpret this as meaning that Fyn activity is positively regulated  8. Model depicting the physical and functional interaction between PKC␦ and Fyn. Upon activation of platelets through the adhesion receptors GP Ib-V-IX and GP VI, Fyn and PKC␦ physically associate, translocate to the plasma membrane, and become activated and phosphorylated as shown. The physical interaction between the two kinases is independent of PKC activity, however, the translocation of both kinases to the plasma membrane is dependent upon the activity of a PKC isoform other than PKC␦ itself. Fyn becomes phosphorylated on a serine by a PKC isoform other than PKC␦ and is activated in a PKC-dependent manner. PKC␦ activity subsequently negatively modulates platelet activation processes as shown.
scintillation counting. Data shown are mean Ϯ S.E. (n ϭ 3). C, platelets were loaded by preincubation for 45 min with the calcium indicator Fura 2-AM (3 M). Results show the rise in cytosolic calcium in response to alboaggregin A (3.5 g/ml) in the presence or absence of rottlerin as indicated. Data shown are the results of one experiment but are representative of at least three repetitions. by a PKC kinase activity, which does not include PKC␦.
The activity of Src kinases correlates with their translocation from cytosol to plasma membrane in platelets and other cells (66). Fig. 6 showed that Fyn translocates upon alboaggregin A stimulation and that this translocation depends upon a PKC activity, because it is inhibited by BIM. This PKC activity does not, however, include PKC␦, because rottlerin was not able to ablate this response, and therefore we conclude that, as for Fyn activity, Fyn translocation requires a PKC isoform other that PKC␦. In parallel with its activity, Fyn translocation also depends upon its own activity, because PP1 fully inhibits it.
We had already shown that BIM and PP1 markedly inhibit the secretion of 5-HT induced by alboaggregin A (16,44), demonstrating the dependence of this response upon PKC and Src kinase activities. Here we showed, however, that PKC␦ may exert a negative effect upon dense granule secretion, because rottlerin potentiated the response to alboaggregin A. This is in agreement with a recent finding by Leitges et al. (14) who show a similar negative regulation of mast cell degranulation by PKC␦. This demonstrates a complex role for PKC isotypes in the regulation of secretion. The potentiation of secretion by rottlerin is paralleled by a potentiation of platelet aggregation and the cytosolic calcium response, the latter of which may therefore underlie the enhanced secretion and aggregation responses in the presence of this inhibitor. Finally, the platelet shape change response is inhibited only by PP1 but not by inhibition of PKC isoforms by BIM or rottlerin (data not shown). This is in agreement with other reports showing that Src kinases play an early signaling role downstream of GP VI and GP Ib-V-IX receptors (44,52,55) but that PKC isoforms play no significant role in the regulation of shape change (67).
In conclusion, it is clear that many PKC isoforms become phosphorylated on tyrosine upon cell activation and that PKC␦ at least associates with a member of the Src kinases, Fyn (see Fig. 8 for summary diagram). Activity of PKC␦ and its translocation to the plasma membrane are dependent upon Src kinase activation, as would be predicted from the early signaling role played by Src kinases in adhesion signaling in platelets. In addition however, translocation of PKC␦ is regulated by a PKC activity, which is not PKC␦ itself. Fyn kinase, however, is phosphorylated not only on tyrosine residues but also on serine in a PKC-dependent manner. Fyn has a high basal activity, which is not dependent upon phosphorylation by PKC, but its activity can be increased upon stimulation of platelets in a manner dependent upon PKC activity, although this activity is not likely to involve the associating PKC␦. It may be concluded that another member or members of the PKC family therefore regulate the localization of both PKC␦ and Fyn to the plasma membrane, and this activity is also required for agonistdependent activation of Fyn. It is possible that either Fyn or PKC␦ acts as a scaffold protein to permit translocation of the other kinase to the plasma membrane as part of its activation process. Interestingly, PKC activity is required for translocation of the PKC␦⅐Fyn complex to the plasma membrane, but not for the activation-dependent association of the two kinases. Finally, the role of PKC␦ in functional control of platelet activity may primarily be as a negative regulator of platelet aggregation and secretion, because rottlerin markedly potentiates these functional responses.