The EMBO Journal vol. 1 1 no.3 pp.855 - 861, 1992

Translocation of pp6Oc-src to the cytoskeleton during platelet aggregation

A.R.Horvath, L.Muszbek and S.Kelfiel Department of Clinical Chemistry, University School of Medicine, P.O. Box 40, H-4012 Debrecen, Hungary, and 'Yamanouchi Research Institute (UK) Littlemore Hospital, Oxford OX4 4XN, UK Communicated by S.Courtneidge

The high amount of pp60-csrc in platelets has led to speculation that this kinase is responsible for tyrosinespecific phosphorylation of cellular proteins during platelet activation by different agonists, and is, therefore, implicated in signal transduction of these cells. Unlike pp6OV-src, the association of which with the cytoskeleton appears to be a prerequisite for transformation, pp60(-sn is detergent-soluble in fibroblasts overexpressing the c-src gene, and its role in normal cellular function remains elusive. To gain a better understanding of the function of pp60'-s51 we have investigated the subcellular distribution of pp60c-src and its relationship to the cytoskeleton during platelet activation. Quantitative immunoblotting and immunoprecipitation have revealed that pp60CS-rC is detergent-soluble in resting platelets, while 40% of total platelet pp60C-src becomes associated with the cytoskeletal fraction upon platelet activation. We have also shown that a small pool of pp60(-'SI is associated with the membrane skeletal fraction which remains unchanged during the activation process. The interaction of pp60(-SIr with cytoskeletal proteins strongly correlates with aggregatioi and is mediated by GPIIb/IIIa receptor- fibrinogen binding. We suggest that the translocation of pp60c-' to the cytoskeleton and its association with cytoskeletal proteins may regulate tyrosine phosphorylation in platelets. Key words: cytoskeleton/GPIIblla/phosphorylation/tyrosine kinase

Introduction Tyrosine kinases have been identified in a wide variety of cells. Most of the known tyrosine kinases are receptors for growth factors or products of viral and cellular oncogenes (Bishop, 1983). The src gene product of Rous sarcoma virus (pp60-sr') is probably the best characterized transforming tyrosine kinase (Hunter and Cooper, 1985). pp6Ovsrc is closely related, both structurally and functionally, to a cellular homologue pp60f-src, however, this cellular protooncogene product has lower specific activity than pp6(I-src and possesses no transforming potential (Shalloway et al., 1984; Coussens et al., 1985). The kinase and transforming activities of pp6c-src are regulated by phosphorylation on its Tyr527 residue, and the kinase responsible for this phosphorylation has now been identified and cloned (Courtneidge, 1985; Cooper et al., 1986; Nada et al., 1991). Little is known about the function of pp6c-srcs within Oxford University Press

cells. Changes in pp6Oc-src kinase activity occur during mitosis, and it has been suggested that this may initiate certain cellular responses (Bagrodia et al., 1991). Despite this putative role in the stimulation of cell division, significant amount of pp60csrc can be found in terminally differentiated cells such as neurons and platelets (Cotton and Brugge, 1983; Tuy et al., 1983; Presek et al., 1988) suggesting a role in normal cell function unrelated to cell proliferation. Since pp60-src can also be found associated with chromaffin granules in adrenal medulla cells (Parsons and Creutz, 1986), pp60-src and its tyrosine kinase activity has been implicated in secretory processes. In accordance with this hypothesis Rendu et al. (1989) reported condensation of pp60csrc in association with membranes of dense bodies which are involved in platelet secretion. Further studies, however, using high-resolution morphological techniques have concluded that the bulk of platelet pp60csrc is associated with the plasma membrane and surface-connected canalicular system (Ferrel et al., 1990) suggesting a role for pp60csrc in transducing signals across the plasma membrane rather than in the regulation of secretion. Platelet activation results in a series of morphological and biochemical events: shape change, extension of filopodia, secretion of granular contents, aggregation and contraction, all of which are dependent on the formation and correct function of the cytoskeleton (Tuszynski, 1987). It has been recently reported that platelet activation by different agonists is accompanied by a rapid elevation in the amount of tyrosine-phosphorylated proteins due to the stimulation of pp60csrc or other tyrosine kinases (Presek et al., 1988; Golden and Brugge, 1989). The biochemical mechanisms of the processes leading to such a stimulation of pp60csrc in platelets remain elusive and have not been examined in detail. In the v-src transformed cells pp6O is concentrated in areas of cytoskeleton -plasma membrane interaction and there is mounting evidence that interaction with the cytoskeleton is essential for transformation-related cellular changes induced by pp60v-src (Hamaguchi and Hanafusa, 1987). One fundamental difference between the viral and cellular src products is that while pp6Jvsrc is tightly associated with the Triton-insoluble cytoskeletal matrix (Burr et al., 1980) pp60csrc is readily solubilized by non-ionic detergent treatment of cells (Loeb et al., 1987). Fukui et al. (1991) have mapped the region of v-src association with the cytoskeleton to the SH2 domain. Since c-src also contains an identical SH2 region, the molecular mechanism for this differential association with the cytoskeleton is unknown. These studies were, however, performed in cells which overexpress c-src, and it is possible that this could lead to aberrant localization by forcing an overabundant protein into a different subcellular compartment. Since platelets have constitutively high levels of pp60csrc these cells provide an ideal tool for examining the relationship between the localization of pp60csrc and cellular activation. In this report we demonstrate that pp60csrc is mostly

855

A.R.Horvath, L.Muszbek and S.Kellie

detergent soluble in resting unactivated platelets, however, a significant amount of pp60csrc associates to the Tritoninsoluble core material upon platelet activation. This interaction of pp6Ocsrc with cytoskeletal proteins is dependent on platelet aggregation and is mediated by GPIIb/IIIa receptor occupancy. Our findings suggest a functional role for pp6oc-src_cytoskeleton association in platelet physiology.

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Results Association of pp6O-src with the cytoskeleton of platelets activated by thrombin In contrast to pp60(-src it has been shown that pp60c-src does not bind to the detergent-insoluble matrix in cell lines which overexpress the c-src gene (Loeb et al., 1987). To investigate the detergent solubility of pp60c-src in platelets cytoskeletons were prepared from an equal number of washed human platelets before and after activation induced by 1 U/ml thrombin. Compared with resting platelets there is a 5-fold increase in the amount of pp6Oc-src retained in the Triton-insoluble residue of activated platelets (Figure IA, lanes 1 and 4). Quantitative immunoblotting revealed that cytoskeleton-associated pp6Ocsrc amounts to 7.7% (+ SD = 2.2, n = 9) of total platelet pp6Ocsrc in resting platelets and to 41% ( SD= 4.9, n = 9) in cells at 5 min of 1 U/ml thrombin-induced activation. To investigate whether the lack of a significant amount of pp6Ocsrc in the cytoskeleton of resting platelets was due to proteolysis, detergent extraction was also performed in the presence of leupeptin and benzamidine. As shown in Figure IA inclusion of proteolysis inhibitors did not change the amount of Triton-resistant pp60c-src in resting platelets (lanes 2 and 3) and only a slightly increased amount of pp6Ocsrc could be recovered in the cytoskeleton of activated ones (lanes 5 and 6). The duration of detergent treatment (10-45 min) and the type of non-ionic detergents used (Triton X-100 or Nonidet P40) did not influence retention of pp6oc-src in the cytoskeletal fraction (not shown). To verify further the specificity of pp6Oc-src cytoskeleton interaction Triton-insoluble residues of activated platelets were prepared in the presence of different concentrations of KCl. The association of pp6Ocsrc to the cytoskeleton was reduced at high ionic strength (Figure 1B). Since pp6oc-src has been localized to the plasma membrane in platelets (Ferrel et al., 1990) we investigated whether it is associated with the submembranous filamentous network termed membrane skeleton. 10-15% of total platelet pp60c-src (12%, mean of three experiments) could be detected in the membrane-skeletal fraction, the amount of which did not change during platelet activation (Figure IC, lanes 4 and 5). DNase I, which is known to depolymerize actin filaments, dramatically reduced the amount of both cytoskeleton-, and membrane skeletonassociated pp6Oc-src (Figure 1C, lanes 3 and 6).

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Distribution of pp6O-src in different fractions of detergent extracts during platelet activation In the next experiments we studied the temporal relationship of pp6oc-src to the detergent-insoluble and soluble fractions during the process of activation. Platelet aggregation induced by 1 U/ml thrombin was stopped by the addition of cytoskeleton extraction buffer at different intervals after activation. Stimulation of platelets by 1 U/ml thrombin resulted in 856

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Fig. 1. Association of pp60C-src with the detergent-insoluble fraction of platelets. The recovery of pp60csrc in the Triton-resistant matrix was studied by quantitative immunoblotting (see Materials and methods) in the presence of (A) proteolysis inhibitors, (B) high ionic strength and (C) the actin depolymerizing agent DNase I. Platelets were activated by 1 U/ml thrombin for 5 min at 37°C. (A) Cytoskeletons obtained from equal number (5 x 108 cells) of unstimulated (lanes 1-3, UNSTIM) and thrombin-stimulated platelets (lanes 4-6, STIM) were prepared without (lanes 1 and 4) or with (lanes 2 and 5) 1 mg/ml leupeptin and 50 mM benzamidine (lanes 3 and 6). The following amounts of proteins were applied to the gel: (1) 11 jg; (2) 26 JLg; (3) 32.5 Ag; (4) 32 jig; (5) 38.8 Atg; and (6) 45 Ag. (B) After aggregation detergent extraction of platelets was performed with cytoskeleton buffer containing different concentrations of KCI. Protein content of samples representing equal cell number are as follows: (1) 36 yg; (2) 34 Ag; (3) 31 jig; (4) 26 jg; (5) 19 rig; (6) 17 1tg. (C) Cytoskeletal (CSK, lanes 1-3) and membrane skeletal (MSK, lanes 4-6) fractions were prepared from equal number of resting (lanes 1 and 4) and thrombinactivated platelets in the absence (lanes 2 and 5) or presence (lanes 3 and 6) of 1 mg/mi DNase I. The amount of proteins applied to the gel: (1) 12 zg; (2) 40.5 jig; (3) 32.5 ytg; (4) 17 sg; (5) 8.5 itg; and (6) 7.5 Ag.

shape change within a few seconds and a rapid release of ATP from the dense granules which reached its maximum (2.62 + 0.44 jtmol ATP/I0" platelets n = 8) within 0.5 min. Aggregation response became maximal in the second to third minute (ATmax = 65 9 %, n = 8). Fractionation studies have revealed that the association of pp6Oc-src with the cytoskeleton gradually increased upon platelet activation, plateauing around 2.5 min after activation. In contrast, no changes could be observed in the amount of membrane skeleton-associated pp60c-src during activation (Figure 2A). Immunoprecipitation of pp60csrc from lysates of all

pp6Ocsc- cytoskeleton

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Fig. 3. Detection of pp60COS-f tyrosine kinase activity in cytoskeletal fractions of platelets. Platelets were activated by 1 U/ml thrombin for 5 min before cytoskeleton extraction. pp60C-src kinase activity was detected by in vitro phosphorylation of pp6Ocsrc immunoprecipitates prepared from lysates of cytoskeletal fractions of equal number of resting (1) and thrombin-activated (2) platelets. 50 yig of cytoskeletal preparations of both unstimulated (3 and 5) and thrombin-activated platelets (4 and 6) were also phosphorylated 'in situ' as described in Materials and methods. Autoradiographs of gels before (3 and 4) and after (5 and 6) treatment with 1 M KOH are compared. A phosphorylated band -60 kDa was apparent in the cytoskeletons of activated cells (4) and was resistant to alkali treatment (6). Arrows indicate phosphorylated bands of pp60`-sr' (1 and 2) and that of the 60 kDa protein (4 and 6) and phosphorylated IgG chains underneath (2).

66mass of 60 kDa in activated but not was resistant to alkali treatment

in resting platelets which (Figure 3).

45-

Fig. 2. Distribution of pp60C sr in the detergent-insoluble and soluble fractions during platelet activation. Platelet activation induced by I U/ml thrombin was terminated by the addition of cytoskeleton buffer at different intervals of the activation process. Cytoskeletal (CSK), membrane skeletal (MSK, *) and soluble (SOL, * *) fractions were prepared from 6 x 108 platelets as described in Materials and methods. (A) Aliquots of Triton-insoluble fractions (CSK, MSK), representing equal cell number, were separated by SDS-PAGE and analysed with immunoblotting for pp6O-src. pp6f0C-sr content of 10 yg total platelet homogenate (1) was compared with that of Tritoninsoluble fractions prepared at 0 min (2), 0.5 min (3), 1 min (4), 2.5 min (5) and 5 min (6) of platelet activation. The following amounts of proteins were applied to the gel: CSK: (2) 10 yg; (3) 24.9 ytg; (4) 36 /tg; (5) 37.8 ytg; (6) 37.5 lg; MSK: (2 .) 10 jig; (3 .) 9 ug; (4 .) 7.5 jig; (5 .) 7.5 jig; and (6 .) 10 gg. (B) Tritonsoluble fractions (SOL, * *) were immunoprecipitated with anti-pp60cO 5" then phosphorylated in vitro. Arrows indicate the autophosphorylated bands of pp60-src and phosphorylated IgG chains underneath.

fractions followed by in vitro phosphorylation demonstrated that pp6o-src retained its tyrosine kinase activity after detergent extraction of the cells (Figures 2B and 3). Enzyme activities measured in all fractions correlated with and reflected the amount of pp60csrc recovered in both Tritonsoluble (Figure 2B) and insoluble residues (Figure 3). In situ phosphorylation of cytoskeletal proteins resulted in the phosphorylation of a protein band with an apparent molecular

Association of pp60C-src with the cytoskeleton at different phases of platelet activation To investigate further the phenomenon of pp6Ocsrc cytoskeleton interaction Triton-insoluble residues were prepared from platelets activated by different concentrations of thrombin. The dependence of platelet aggregation, dense body secretion and cytoskeletal assembly on thrombin concentration is described in Table I. The slope of aggregation curve (tga) and the extent of aggregation (ATmax) as well as the amount of proteins incorporated into the cytoskeleton reached a plateau at 0.75 U/ml thrombin, while ATP secretion showed a gradual elevation by increasing thrombin concentration. The pp60csrc contents of these cytoskeletons are compared in Figure 4. We have found that the amount of cytoskeletal pp60csrc (Figure 4) increased gradually and became maximal at 0.75 U/ml thrombin concentration. Both in time course and thrombin concentration dependence studies the association of pp60csrc correlated with aggregation rather than secretion. To test this hypothesis further different agonists and conditions were used to stimulate platelets. Platelet aggregation induced by thrombin occurs only if the cell suspension is stirred intensively. When platelets were activated with thrombin in the absence of stirring, they changed shape and ATP release remained unaltered, however, they did not aggregate (not shown) (Fox et al., 1983; Ferrel and Martin, 1989). The opposite response could be observed if platelets were activated with PMA which induced full aggregation (A Tmax = 60% at 10 min) but only a slight release of granular components (0.42 ,umol ATP/10l platelets) (Carrol et al.., 1982). Figure 5A demonstrates that in the absence of aggregation the incorporation of pp60cSr' into the detergent-resistant fraction did not increase (Figure 5A, lane 3) and was 857

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A.R.Horvath, L.Muszbek and S.Kellie

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Table I. Dependence of platelet responses on thrombin concentration Thrombin concentration (U/ml) 0 0.25 0.5 0.75 1.0 1.5

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Fig. 4. Incorporation of pp60-src into the cytoskeleton of platelets activated by different concentrations of thrombin. Cytoskeletons were prepared from 6 x 108 platelets activated by 0 U/mi (1), 0.25 U/ml (2), 0.5 U/mi (3), 0.75 U/mi (4), 1 U/ml (5), 1.5 U/mi (6) and 2 U/mi (7) thrombin for 5 min. pp6Oc-src contents of cytoskeletons obtained from equal number of cells were determined by immunoblotting. The following amounts of proteins were applied to the gel: (1) 15 4g; (2) 61 ,tg; (3) 63 Ag; (4) 58 Ag; (5) 78 4tg; (6) 78 yg; and (7) 75 yg.

before cytoskeleton extraction (4). Aliquots of cytoskeleton proteins representing equal cell number were loaded on the gel: (1) 8 ltg; (2) 59.4 Aig; (3) 26.3 yg; and (4) 43.8 /Ag. (B) Cytoskeletons were prepared from unactivated platelets (1) or platelets activated by 1 U/ml thrombin for 5 min (2) in the presence of 200 ,ug/mi RGDS peptide (3), or 10 mM EDTA (4), or after pretreatment with 10 ,ug/ml cytochalasin B (5). The following amounts of cytoskeleton proteins, prepared from equal number of cells, were applied to the gel: (1) 18.5 A4g; (2) 46 Ag; (3) 38 tig; (4) 37.5 yg; and (5) 50 yg.

comparable with that of unactivated platelets. However, the same amount of pp60csrc was recovered in the cytoskeleton of PMA-activated, fully aggregated platelets as was found for thrombin aggregated ones (Figure 5A, lane 4). Calcium ions are necessary for the formation of GPlb/HIa complex, a heterodimer competent to bind fibrogen. Since the integrity of GPIIb/IIIa receptor and the binding of fibrogen to this complex is a prerequisite of platelet aggregation (Bennet and Vilaire, 1979) we asked whether pp6Ocsrc-cytoskeleton interaction was dependent on GPIIb/Illa receptor occupancy. To address this question, platelets were activated by thrombin in the presence of the Ca-chelating agent EDTA, and RGDS peptide, a tetrapeptide analogue of the receptor binding site of fibrinogen (Gartner and Bennett, 1985). Both treatments inhibited aggregation but had no effect on shape change and secretion induced by thrombin (not shown) (Ferrel and Martin, 1989), and resulted in only 20-30% reduction in the amount of proteins recovered in the cytoskeletal fraction. The association of pp6c`src with the cytoskeleton matrix was strongly inhibited in both of the above mentioned cases (Figure SB, lanes 3 and 4). Finally, when the formation of pseudopodia was prevented by cytochalasin B pretreatment but neither aggregation nor secretion induced by thrombin were inhibited (Carrol et al., 1982; Wheeler et al., 1984) the retention of pp6Ocsrc in the cytoskeleton matrix was comparable with non-pretreated, thrombin-activated cells (Figure 5B, lane 5). The relationship between different phases of platelet activation and

pp60C-srC cytoskeleton interaction in summarized in

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858

Table II. Discussion

pp6ov-src associates with the cytoskeleton via the aminoterminal region of its SH2 domain, and this interaction is required for the transforming potential of this tyrosine kinase (Burr et al., 1980; Hamaguchi and Hanafusa, 1987; Fukui al., 1991). Although the SH2 region is conserved in pp6Oc-src, this cellular proto-oncogene product does not associate with the detergent-insoluble matrix in untransformed cells which overexpress the c-src gene (Loeb et al., 1987). Since pp6o-src is the most abundant tyrosine kinase in platelets and activation by different agonists results in a rapid reorganization of cytoskeletal proteins which coincides with a stimulation of tyrosine kinase activity (Tuszynski,

et

1987; Ferrel and Martin, 1988; Golden and Brugge, 1989), studied the interaction of pp6c`src with the cytoskeleton during platelet activation. This report describes an inducible translocation of the proto-oncogene product pp6c-src, in that pp6Ocsrc, which is detergent-soluble in resting platelets, associates with the cytoskeletal matrix during platelet activation. We have also shown that there is a small pool of pp6c`src associated with the membrane skeletal fraction which is unchanged during platelet activation. The association of pp6ocsrc to the cytoskeleton was related to different phases of the activation process and the involvement of a GPIIb/IIIa mediated mechanism was also investigated. In a we

pp6Ocsc- cytoskeleton interaction in platelets Table II. Correlation of pp6Ocrc-cytoskeleton interaction with different phases of platelet activation Agonists

0 Thrombin + stirring Thrombin + no stirring Thrombin + EDTA Thrombin + RGDS Thrombin + cytochalasin B PMA

pp6Ocsrc'-cytoskeleton association

Platelet response

shape change

aggregation

secretion

+

+

+

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+

+

+

+

+

+

+

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11

+

preliminary study recently, Grondin et al. (1991) have described pp6oc-sr association with the cytoskeleton in activated platelets, however, no functional relationships were investigated. Although the inclusion of protease inhibitors in the lysis buffer has resulted in a better recovery of certain membraneassociated glycoproteins such as GPIb-IX in the cytoskeleton (Fox et al., 1988), the low amount of pp6Oc-src in the cytoskeleton of resting platelets was not due to proteolysis since the retention of pp6Oc-sr both in cytoskeletal and membrane skeletal fractions of unstimulated cells was not influenced by protease inhibitors and there was no evidence of pp60csrc breakdown. pp60c-src was specifically linked to the cytoskeleton since the association could be abolished in the presence of the actin depolymerizing agent DNase I (Figure 1). The association of pp6Oc-sr with the cytoskeleton was decreased at high ionic strength, however, the amount of proteins recovered in the cytoskeleton fraction was also reduced. Time course studies indicated that pp6Oc-sr' associated with the cytoskeleton at a distinct phase (-2.5 min) of platelet activation. Since shape change and pseudopodia extension occurred within a few seconds and the release of ATP from dense bodies was maximal in