Differential Regulation of Adapter Proteins Dok2 and Dok1 in Platelets, Leading to an Association of Dok2 with Integrin Aiibb3

Journal of Thrombosis and Haemostasis, 5: 387–394

ORIGINAL ARTICLE Differential regulation of adapter proteins Dok2 and Dok1 in platelets, leading to an association of Dok2 with integrin aIIbb3

S. C. HUGHAN* andS. P. WATSON* *Centre for Cardiovascular Sciences, Institute of Biomedical Research, University of Birmingham, Birmingham, UK; and Australian Centre for Blood Diseases, Monash University, Melbourne, Vic., Australia

To cite this article: Hughan SC, Watson SP. Differential regulation of adapter proteins Dok2 and Dok1 in platelets, leading to an association of

Dok2 with integrin aIIbb3. J Thromb Haemost 2007; 5: 387–94.

been described in platelets, including linker for activation of T Summary. Background: We previously demonstrated that cells (LAT) adapter protein, the adaptor protein Src homology Dok2 is rapidly phosphorylated on tyrosine residues in platelets 2 (SH2) domain-containing leukocyte phosphoprotein of in response to thrombin, the immunoreceptor tyrosine-based 76 kDa (SLP-76), Grb2, Gads, adhesion and degranulation- activation motif-coupled collagen receptor glycoprotein (GP) promoting adapter protein (ADAP, formerly known as SLAP- VI, and by integrin aIIbb3. Objectives and methods: In this study 130), Shc and C-Cbl, with LAT and SLP-76 being amongst the we further delineate the regulation of phosphorylation of Dok2 best characterized [1]. and compare this to the related adapter Dok1. Results: We We recently identified the presence of a new family of demonstrate expression of Dok1 in platelets and the unexpected adapter proteins in platelets, using proteomics, namely Dok observation that the adapter protein undergoes tyrosine (downstream of kinase) adapter proteins, with expression of phosphorylation in response to thrombin but not to GPVI or Dok2 being confirmed by immunoblotting techniques [2]. Dok integrin aIIbb3. Furthermore, Dok1 phosphorylation is tran- proteins share a common structural arrangement consisting of sient, peaking at 30 s and returning to basal by 5 min, whereas pleckstrin homology and phosphotyrosine binding (PTB) Dok2 phosphorylation is delayed but sustained. Dok2 phos- domains, and multiple sites of tyrosine phosphorylation and phorylation, but not that of Dok1, is inhibited by Src kinase proline-rich motifs, which may confer binding to SH2 and SH3 inhibitors and by chelation of intracellular calcium. Further, domains, respectively [3]. They are believed to play important phosphorylation of Dok2 by thrombin and integrin aIIbb3 in signaling roles in a wide range of pathways and in disease [3]. mouse platelets is independent of Syk and phospholipase Cc2. Indeed, Dok1 was first identified as a 62 kDa RasGAP-binding Additionally, Dok2 coimmunoprecipitates with integrin aIIbb3 protein in v-Abl transformed B cells and in Bcr-Abl expressing downstream of Src kinases. Conclusions: These results demon- cells [4], while Dok2 was identified as one of several tyrosine strate differential modes of regulation of Dok1 and Dok2 in phosphorylated proteins derived from bone marrow taken platelets. Further, they raise the interesting possibility that Dok2 from patients with chronic myeloid leukemia [3]. More plays an important role in integrin outside-in signaling through recently, Dok family proteins have been shown to interact a physical and functional interaction with integrin aIIbb3. with a broad range of signaling proteins, including SH2- containing inositol 5¢-phosphatase 1 (SHIP-1) [5–7], Csk [6,7],

Keywords: Dok2, integrin aIIbb3, platelets. phosphatidylinositol (PI) 3-kinase [8] and integrin b3-tail [9]. Importantly, Dok1 and Dok2 are implicated in the regulation Introduction of a wide range of physiological responses, including negative regulation of cytokines, enhanced cell migration, ﬁlopodia Adapter proteins play an important role in the formation and extension and tumor suppression [10–15]. maintenance of signaling complexes [1]. Broadly speaking, We have recently shown that Dok2 undergoes tyrosine adapter proteins lack intrinsic activity, yet possess a range of phosphorylation in platelets in response to stimulation by modular domains that facilitate protein–protein or protein– thrombin, the collagen receptor glycoprotein (GP) VI and the lipid interactions. A large number of adapter proteins have integrin aIIbb3 [2]. In the present study, we identiﬁed a second member of the Dok family of adapter proteins in platelets, Correspondence: Sascha C. Hughan, Australian Centre for Blood Dok1, and demonstrated that it is regulated differentially to Diseases, Monash University, Alfred Medical Research and Education Precinct, Commercial Road, Melbourne, Vic. 3004, Australia. Dok2. In particular, immunoprecipitation studies demonstrate Tel.: + 61 3 9903 0143; fax: + 61 3 9903 0228; e-mail: that Dok2, but not Dok1, is regulated directly by binding to [email protected] integrin aIIbb3. These studies therefore suggest that Dok1 and Dok2 adapter proteins play distinct roles in mediating platelet Received 11 September 2006, accepted 1 November 2006 activation in hemostasis.

2006 International Society on Thrombosis and Haemostasis 388 S. C. Hughan and S. P. Watson

concentration determined. For control studies, Petri dishes Materials and methods were coated with BSA alone, and platelets were allowed to adhere as above. Protein concentration was determined using a Reagents RC DC BioRad kit assay (BioRad, Hemel Hempsted, UK) and PD0173952 was from Pﬁzer (Ann Arbor, MI, USA). Anti- 100 lg of protein was used in immunoprecipitation studies. phosphotyrosine (4G10) agarose-conjugate and anti-Dok2 (H- 192) polyclonal antibodies (pAbs) were described previously Immunoprecipitation and Western blotting [2]; antirabbit Dok1(M-276), antimouse Dok1(A-3), antigoat b3 integrin (N-20) and antirabbit immunoglobulin G (IgG) Immunoprecipitation studies were performed as described antibodies were from Santa Cruz Biotechnology (Santa Cruz, previously [2]. In some studies, lysates were precleared, and

CA, USA). Monoclonal antibodies (mAbs) anti-aIIb (SZ22) then either 2 lg of Dok2 pAb or antirabbit IgG control was and b3 integrin (SZ21) were from Immunotech (Marseilles, added and incubated with beads overnight at 4 C. Proteins/ France) and integrin aIIbb3 inhibitor, Aggrastat, was from beads were washed three times with Tris-buffered saline- Merck and Co. Inc. (Whitehouse Station, NJ, USA). Complete Tween, reduced using 2 · reducing buffer, and boiled for protease inhibitor cocktail tablets were from Roche (Penzberg, 10 min prior to separation by sodium dodecyl sulfate- Germany). All other reagents were from Sigma (St Louis, MO, polyacrylamide gel (SDS–PAGE) (4–12% precast Novex gels) USA) or have been described [16–19]. and electrical transfer to polyvinylidene difluoride (PVDF) membranes. Immunoblotting proceeded as described [2]. Mouse strains ) ) Statistical analysis Phospholipase Cc2 knockout (PLCc2 / ) mice [20] were bred as heterozygotes. Syk-deficient and wild-type radiation chime- Statistical significance of results was determined using one-way ras were generated according to Moćsai et al. [21]. All mice ANOVA,usingthePRISM software program (GRAPHPAD software were treated in accordance with the Animals (Scientific for Science, San Diego, CA, USA) The P-values are indicated. Procedures) Act 1986, UK. Results Preparation of platelets Dok1 and Dok2 display distinct patterns of phosphorylation in Human blood was collected in citrate anticoagulant from platelets following activation of G protein-coupled receptors healthy volunteers who had not taken any antiplatelet medi- cation in the preceding 2 weeks, according to protocols Using a specific antibody, which detected Dok1 expression in approved by the Universities of Birmingham and Monash. murine splenocytes but not in mouse thymus [3], we demon- Donors had provided informed written consent, according to strated the presence of Dok1 in both murine and human the Declaration of Helsinki. Human and murine platelets were platelets (Fig. 1A). The latter finding was in agreement with obtained and washed as previously described [18], before results from a recent proteomics screen [23]. Thus, human and ) resuspension at 2–5 · 108 mL 1. For some immunoprecipita- mouse platelets express both Dok1 and Dok2. tion studies, platelets were washed according to Goncalves [22]. Studies were designed to investigate whether Dok1 is regulated by tyrosine phosphorylation in platelets, as is the case for Dok2 [2]. Fig. 1B demonstrates that Dok1 undergoes Platelet adhesion and phosphorylation studies tyrosine phosphorylation upon stimulation by thrombin, but Glass Petri dishes were coated with von Willebrand factor not by collagen, despite platelets undergoing full aggregation. (VWF; 10 lgmL)1), fibrinogen (200 lgmL)1) or collagen Thrombin-induced Dok1 phosphorylation reaches a maximal (100 lgmL)1) for 120 min at room temperature. Non-adher- response between 30 and 90 s before declining (Fig. 1B, upper ent proteins were removed and the surface washed with panel). Further, tyrosine phosphorylation of Dok1 by throm-

Tyrode’s buffer. Uncoated glass was blocked with heat- bin was maintained in the presence of an aIIbb3 blocker, inactivated bovine serum albumin (BSA; 5 mg mL)1)for1h lotrafiban, demonstrating that it was independent of integrin at room temperature, prior to removal and washing. Washed engagement (Fig. 1C, right panel). These results contrast ) human platelets (2 · 108 mL 1) were loaded onto the glass markedly with those for phosphorylation of Dok2 by thrombin dishesandplateletsallowedtospreadfor1hat37C. Dishes and collagen, as determined in the same experiment. For both were placed on ice and unbound platelets removed by agonists, Dok2 phosphorylation was delayed for approxi- aspiration. Lysis buffer (·1) [150 mM NaCl2,10mM Tris, mately 30 s and peaked between 90 and 180 s (Fig. 1B, lower 1mM EGTA, 1 mM EDTA, pH7.5, 1% NP-40 detergent (v/v), panel). However, Dok2 phosphorylation occurred predomin- )1 2.5 mM Na3VO4,1mM AEBSF, 5 lgmL leupeptin, antly through integrin aIIbb3, as phosphorylation was blocked 5 lgmL)1 aprotinin, and 0.5 lgmL)1 pepstatin] was added in the presence of lotrafiban (Fig. 1C and [2]). Importantly, the and spread platelets were lyzed and scraped from the glass dependence on integrin aIIbb3 for tyrosine phosphorylation of surface. The platelet lysates were collected and protein Dok2 was confirmed in collagen- and thrombin-stimulated

2006 International Society on Thrombosis and Haemostasis Regulation of Dok2 and Dok1 in platelets 389

A platelets by direct immunoprecipitation of Dok2 and probing for tyrosine phosphorylation (Fig. 1D,E). A similar set of studies was not possible for Dok1 because of the lack of availability of suitable commercial antibodies for direct immu- Murine Murinethymus Murinespleen Humanplatelets Murineplatelets platelets noprecipitation, a result consistent with others [12,14,24]. IB: Dok1 These results therefore demonstrate distinct mechanisms of regulation of tyrosine phosphorylation of Dok1 and Dok2, B Thrombin Collagen with phosphorylation of Dok1 being independent of GPVI 0” 30” 90” 180” 30” 90” 180” and integrin aIIbb3, and that of Dok2 being predominantly IP: pTyr mediated downstream of the integrin. IB: Dok1

IP: pTyr Dok2 tyrosine phosphorylation is critically dependent on Src IB: Dok2 kinases The contribution of Src kinases to tyrosine phosphorylation of C Dok1 and Dok2 was evaluated in view of their proximal role in platelet signaling [25,26], and because they been shown to Basal Basal + Thrlotra Thr + lotra Basal Thr Thr + lotra mediate regulation of Dok proteins in other cell types [8,24,27]. IP: pTyr IP: pTyr Using two structurally distinct Src kinase inhibitors, PP2 [28] IB: Dok2 IB: Dok1 and PD0173952, we demonstrate a critical role for Src kinases in thrombin- and collagen-induced Dok2 phosphorylation fol- DE lowing immunoprecipitation with 4G10 (Fig. 2A) or anti-Dok2 (Fig. 2B). Importantly, thrombin is able to promote platelet Thr Coll P+Aggra. aggregation in the presence of PP2, demonstrating that the loss 0” 180” 180” Basal TRA TRAP TRAP of phosphorylation of Dok2 is not due simply to abolition of this IB: pTyr IB: pTyr response [29]. Significantly, both PP2 and PD0173952 reduced the background level of Dok2 phosphorylation (Fig. 2A,B), IB: Dok2 IB: Dok2 suggesting that Src kinase activity regulates basal phosphorylation of the adapter protein. In marked contrast, blockade of IP:Dok2 IP:rlgG Src kinases had no significant effect on tyrosine phosphorylation Dok2 Dok2 RlgG Dok2 RlgG of Dok1 (Fig. 2C). These data further distinguish the regulation IP of the two adapter proteins by demonstrating that phosphory- Fig. 1. Dok1 and Dok2 are present in platelets and are differentially lation of Dok2, but not Dok1, is dependent on Src kinases. phosphorylated following platelet activation. (A) Whole cell lysates of murine thymus, murine spleen, murine platelets and human platelets were obtained and separated by sodium dodecyl sulfate-polyacrylamide gel Src kinase-dependent platelet spreading promotes Dok2 (SDS–PAGE) and immunoblotted for Dok1 using an anti-Dok1 poly- phosphorylation clonal antibody (pAb). (B) Human platelets were aggregated by thrombin (1.0 U mL)1) or collagen (10 lgmL)1) for various time points indicated. The above studies demonstrate that phosphorylation of Dok2, (C) Human platelets were aggregated by thrombin (30 or 90 s) in the but not Dok1, is mediated through a Src kinase-dependent presence or absence of the integrin aIIbb3 inhibitor, lotrafiban. (B, C) pathway in activated platelets. Because phosphorylation of Samples were lyzed and precleared with PAS beads. Tyrosine phosphor- Dok2 in these studies is also mediated through integrin aIIbb3, ylated proteins were immunoprecipitated using 4G10-conjugated PAS these results are consistent with a critical role for Src kinases in beads (IP:pTyr), overnight at 4 C. Samples were separated using SDS– PAGE, transferred and membranes were immunoblotted with anti-Dok2 outside-in signaling by the integrin [25]. To further examine the or anti-Dok1 antibodies as indicated. (D) Human platelets were allowed to relationship between Dok2, Src kinases and aIIbb3 outside-in remain resting (0Õ) or were stimulated with thrombin (180s) or collagen signaling, platelets were allowed to adhere and spread on (180s). Samples were lyzed, precleared with PAS beads and immunopre- immobilized fibrinogen, VWF and collagen, while BSA-coated cipitated using a Dok2 pAb (2 lg) or antirabbit immunoglobulin G surfaces were used as a negative control. As demonstrated in control antibody (RIgG), overnight (4 C). Membranes were immunoblotted with an antiphosphotyrosine antibody (pTyr, upper panels) and Fig. 3, Dok2 underwent tyrosine phosphorylation following reprobed with an anti-Dok2 (lower panels). (E) Human platelets were platelet adhesion to all immobilized proteins through a aggregated to thrombin activating peptide (TRAP) in the presence or pathway that was blocked by PP2 (Fig. 3A). In addition, absence of the integrin aIIbb3 inhibitor, Aggrastat (Aggra). Samples were PP2 blocked platelet spreading on all matrix proteins (Fig. 3B treated as per (D). Vertical lines contained within some blots indicate and [16,25,26]), consistent with a role for Src kinases in a b grouping of images from different parts of the same gel. Representative IIb 3 immunoblots are shown, n P 3. signaling.

2006 International Society on Thrombosis and Haemostasis 390 S. C. Hughan and S. P. Watson

A A

lagen BSA VWF FGN COLL Basal ThrombinCollagen Basal ThrombinCollagenBasal ThrombinCol –+–+–+ ––– +++ –––PP2 (20 μM) PP2 ––– ––– + ++PD017395 (5 μM)

IP: pTyr IB: Dok2 B Control PP2

Basal Thr Thr + PP2Thr IB: pTyr FGN

IB: Dok2

IP: Dok2 IP: rlgG

C VWF

PP2 PD017395

Basal Thr Thr + Thr + IP: pTyr IB: Dok1

Fig. 2. Phosphorylation of Dok2 is critically dependent on Src kinases. Collagen Washed human platelets were incubated with DMSO vehicle, PP2 or PD0173952 (10 min) prior to aggregation with thrombin [90s (A, B), or 20 μm 30s (C)] or collagen [2.5 min (A)]. Unstimulated samples (basal) and aggregated samples were lyzed and precleared with PAS beads (1 h, 4 C). Fig. 3. Src kinase-dependent platelet spreading is critical for Dok2 (A, C) Tyrosine phosphorylated proteins were immunoprecipitated using phosphorylation. Washed human platelets were allowed to adhere to )1 4G10-conjugated PAS beads, overnight (4 C). Samples were then immobilized bovine serum albumin (5 mg mL ), von Willebrand factor separated using sodium dodecyl sulfate-polyacrylamide gel (SDS–PAGE), (VWF), fibrinogen (Fgn) or collagen (Coll) (1 h, 37 C) in the absence ()) transferred, and membranes were immunoblotted using anti-Dok2 poly- or presence (+) of the Src kinase inhibitor, PP2. (A) Non-adherent clonal antibody (pAb) (A) or anti-Dok1 pAb (C). (B) Aggregated samples platelets were removed and adherent platelets lyzed with 1 · lysis buffer. were generated as described and immunoprecipitated with anti-Dok2 pAb The concentration of protein was determined using a DC BioRad Kit or antirabbit immunoglobulin G control (RIgG), immunoblotted using an (BioRad, Hemel Hempsted, UK) and 100 lg of protein was precleared antiphosphotyrosine antibody (pTyr), and reprobed with anti-Dok2 pAb. with PAS, then immunoprecipitated using 4G10-conjugated PAS beads. Representative immunoblots are shown, n ¼ 4. Immunoprecipitated proteins were separated using sodium dodecyl sulfate-polyacrylamide gel (SDS–PAGE), transferred onto polyvinylidene difluoride (PVDF) membrane and immunoblotted for Dok2. Represen- Thrombin-dependent Dok2 phosphorylation is independent tative immunoblots are shown, n ¼ 3. (B) Adherent platelets were fixed and mounted, prior to imaging using differential interference contrast of Syk and PLCc2 microscopy [17]. These cropped images are from one experiment, To further investigate the regulation of Dok2 by thrombin and representative of three. integrin aIIbb3,weusedmicedeficientineitherSykorPLCc2, as both Syk and PLCc2 are known to play an important role in Dok2 tyrosine phosphorylation is regulated by intracellular integrin a b outside-in signaling [16,25,26,30]. In both cases, IIb 3 calcium and actin polymerization Dok2 phosphorylation was retained following thrombin activation in the absence of the two signaling molecules, whereas We further investigated the regulation of tyrosine phosphory- phosphorylation induced by the GPVI agonist, CRP, was lation of Dok1 and Dok2 by examining the roles of the abolished (Fig. 4), consistent with the critical role of Syk/ secondary feedback agonists, adenosine diphosphate and

PLCc2 in GPVI-induced platelet activation [31]. These data thromboxane A2, the second messenger pathway, PI 3-kinase, therefore demonstrate that phosphorylation of Dok2, by aIIbb3 intracellular calcium elevation and actin polymerization, in and Src kinases, is either upstream or independent of Syk and the phosphorylation events. Pretreatment of platelets with PLCc2. Dok1 phosphorylation by thrombin was also inde- apyrase in combination with the cyclooxygenase inhibitor, pendent of Syk and PLCc2 (not shown). indomethacin, had a negligible inhibitory effect on Dok2

2006 International Society on Thrombosis and Haemostasis Regulation of Dok2 and Dok1 in platelets 391

Basal Thr (low) Thr (high) CRP

+ ––+ + ––+ Syk Basal Thr Thr + Apy/IndoColl Coll + Apy/Indo Thr Thr + Apy/Indo IP: pTyr IP:pTyr IP:pTyr IB: Dok2 IB:Dok2 IB:Dok1

B C Basal Thr Basal CRP 7.5 + ––+ + ––+ PLCγ2 * ** IP: pTyr 5.0 ns Basal Thr Thr + LY294002Thr + BAPTA-AMThr + Cyto D ns IB: Dok2 IB:pTyr Fig. 4. Thrombin-induced Dok2 phosphorylation occurs independently IP:Dok2 2.5 of Syk and phospholipase Cc2(PLCc2). Murine platelets were obtained +/+ from Syk-deficient radiation chimeric mice reconstituted with Syk (+) D Dok2 phosphorylation or Syk)/)()) deficient fetal liver cells or wild-type (+) or PLCc2)/) ()) basal) increase over (fold 0 mice. Platelets were left unstimulated (basal) or aggregated with thrombin Thr [0.1 U mL)1 (low) and 1.0 U mL)1 (high), 90 s] or collagen-related pep- Basal tide (CRP, 2.5 min). Aggregations were stopped by addition of 2 · lysis BasalThr Thr +Thr LY294002 +Thr BAPTA-AM + Cyto D Thr + Cyto D bufferandlysateswerepreclearedwithPASbeads(1h,4C). Tyrosine IP:pTyr Thr + LY294002 phosphorylated proteins were immunoprecipitated using 4G10-conju- Thr + BAPTA-AM IB:Dok1 gated PAS beads, overnight (4 C). Samples were separated using sodium dodecyl sulfate-polyacrylamide gel (SDS–PAGE) and transferred onto Fig. 5. Thrombin-induced Dok2 phosphorylation is independent of polyvinylidene difluoride (PVDF) membranes. Membranes were adenosine diphosphate (ADP) and thromboxane A2, yet critically immunoblotted with anti-Dok2 polyclonal antibody. Representative dependent on intracellular calcium. (A) Human platelets were aggregated immunoblots are shown, n P 2. Vertical lines contained within some blots through addition of thrombin (90 or 30s) or collagen (2.5 min) in the indicate grouping of images from different parts of the same gel. presence or absence of the broad-spectrum ADP inhibitor, apyrase, in

combination with indomethacin, an inhibitor of thromboxane A2 (+Apy/ Indo). (B–D) Human platelets were allowed to rest (basal) or aggregated phosphorylation induced by thrombin (Fig. 5A). Conversely, through addition of thrombin (90 or 30s) in the presence or absence of the combination of inhibitors markedly reduced the response LY294002, BAPTA-AM or cytochalasin D. In all cases, aggregation was stopped by addition of 2 · lysis buffer and lysates were precleared with to collagen, suggesting that collagen-mediated phosphorylation PAS beads. Tyrosine phosphorylated proteins were immunoprecipitated is primarily mediated downstream of the two G protein- using 4G10-conjugated PAS beads, overnight (4 C). Samples were sep- coupled receptor agonists (Fig. 5A), a result that reflects the arated using sodium dodecyl sulfate-polyacrylamide gel (SDS–PAGE), critical role of these second messengers in GPVI signaling transferred, and membranes were immunoblotted with antibodies, as [32,33]. Chelation of intracellular calcium, through treatment indicated. (C) Histogram shows the relative quantitation of Dok2 phosphorylation, in the presence of inhibitors, relative to basal samples. Data with BAPTA-AM, completely blocked Dok2 phosphorylation are represented in arbitrary units, as a fold increase over basal. Vertical (Fig. 5B,C). In contrast, inhibition of PI 3-kinase with the lines contained within some blots indicate grouping of images from dif- broad-spectrum inhibitor LY294002, and blockade of actin ferent parts of the same gel. Representative immunoblots are shown, polymerization, using cytochalasin D, had a weak inhibitory n P 3. (*P < 0.05; **P < 0.01; NS, not significant). effect on Dok2 phosphorylation (P > 0.05), suggesting a minor role for PI 3-kinase and cytoskeletal reorganization in opti- mal Dok2 phosphorylation (Fig. 5B,C). Similar results were phorylation in response to outside-in signaling through the obtained when Dok2 was directly immunoprecipitated (not integrin. This possibility is further indicated by the observa- shown). Strikingly, apyrase plus indomethacin, BAPTA-AM, tion of Calderwood et al. [9], wherein Dok1 was shown to

LY294002 and cytochalasin D treatment had no effect on associate with the cytoplasmic tail of b3 [9]. Dok2, like Dok1, phosphorylation of Dok1 (Fig. 5A, right panel; Fig. 5D), has a PTB domain and bears the same residues that mediate further distinguishing the regulation of Dok1 from Dok2. the interaction with the b3 tail [3,9]. Therefore, an interaction between Dok2 and integrin aIIbb3 was investigated by immunoprecipitation of the adapter protein and immuno- Dok2 associates with integrin a b in thrombin-aggregated IIb 3 blotting with a mAb against a , SZ22. As demonstrated in platelets. IIb Fig. 6, an association between Dok2 and aIIb was observed The above studies demonstrate a critical role for integrin in platelets aggregated to thrombin, but was prevented by an aIIbb3 in mediating Dok2 phosphorylation in platelets that is inhibitor of aIIbb3 (7E3), and by the Src kinase inhibitor independent of Syk and PLCc2. This therefore raises the (PP2). In contrast to thrombin, collagen-induced aggregation possibility that Dok2 may be regulated through an interac- did not result in a signiﬁcant interaction of Dok2 with tion with one or both of the conserved tyrosine residues in integrin aIIbb3 (Fig. 6I). We also determined that the the integrin tail that have been shown to undergo phos- association of integrin aIIbb3 with Dok2 could be detected

2006 International Society on Thrombosis and Haemostasis 392 S. C. Hughan and S. P. Watson

(I) (II) (III) remains unclear, there is precedence within the family of Dok proteins for different members to demonstrate distinct phosphorylation kinetics and to require distinct binding partners. Basal Thr Coll BasalThr Thr + 7E3 Basal Thr Thr + ThrPP2 For example, Dok4 and Dok5 have been shown to undergo IB: pTyr tyrosine phosphorylation in response to insulin and insulin-like growth factor-1 in transfected HEK cells [34]. However, the α IB: IIb kinetics of their phosphorylation differed, with Dok4 phosphorylation reaching a maximum over 40 min, while Dok5 IB: Dok2 phosphorylation occurred rapidly, reaching a half-maximum IP: Dok2 IP: Dok2 IP: Dok2 rlgG after 5 min [34]. The different requirements for phosphorylation between Dok1 and Dok2 for Src kinases was another Fig. 6. Thrombin-induced aggregation promotes an interaction between unexpected observation, as other published studies have Dok2 and integrin aIIbb3. Washed human platelets were aggregated by described a critical role for Src kinases in mediating phos- thrombin (I–III) or collagen (I) in the presence of 7E3 or PP2. Platelet suspensions were lyzed, precleared using PAS beads (1 h) and Dok2 was phorylation of Dok1 [8,14,24]. The reason for the independence immunoprecipitated overnight (4 C). Immunoprecipitates were washed of Dok1 phosphorylation from Src kinases in platelets and the three times and samples separated using sodium dodecyl sulfate-poly- nature of the kinase mediating the phosphorylation are unclear. acrylamide gel (SDS–PAGE) and transferred onto polyvinylidene Nonetheless, it appears that although Dok proteins share difluoride (PVDF) membranes. Membranes were immunoblotted with common structural similarities, they do not necessarily perform an anti-aIIb mAb (SZ22). An isotype-matched antirabbit immunoglobulin G antibody (rIgG) was used as a control and all blots were immunoblotted thesameroleinthesamecell. with anti-Dok2 polyclonal antibody to show equal loading. Representa- The finding that Dok2 tyrosine phosphorylation occurs tive immunoblots from at least two experiments are shown. downstream of Src kinases, yet is independent of Syk and PLCc2, has important implications for the potential role of using two alternative integrin antibodies, b3 pAb (N20) and Dok2 in integrin aIIbb3-mediated signaling. Adapter proteins b3 mAb (SZ21) (not shown). Taken together, these data previously identified to be important for integrin and therefore suggest that Dok2 coprecipitates with aIIbb3 in a immunoreceptor tyrosine-based activation motif (ITAM) manner that is dependent on integrin outside-in signaling and signaling, like SLP-76, are phosphorylated downstream of Src kinases. Syk; however, Dok2 appears to be unique in platelets for its independence from Syk. This may suggest either that Dok2 plays a novel role in signaling between Src and Syk, or that Discussion Dok2 mediates Src kinasesÕ effects, independently of Syk. It is The studies presented here provide new insight into the noteworthy that in Cos-1 cells, transiently cotransfected with identification and characterization of the Dok family of Dok3 and Src and Syk kinases, Dok3 can also undergo adapter proteins in platelets. We have found both Dok1 and tyrosine phosphorylation in response to Src-related tyrosine Dok2 to be present and regulated by tyrosine phosphorylation kinase enzymes, independent of Syk and ZAP-70 [7]. in human and mouse platelets following platelet activation. Another important finding is our observation that Dok2

While Dok2 tyrosine phosphorylation appears to be a general coprecipitates with the aIIb and b3 subunits of integrin aIIbb3 feature of platelet activation, Dok1 phosphorylation is selec- following platelet aggregation by thrombin. While this finding tively regulated by thrombin. Interestingly, Dok2 phosphory- is perhaps not surprising given the demonstration by Calder- lation is critically dependent on Src kinases and occurs wood et al. [9] of Dok1 binding to recombinant b3, our study predominantly, but not exclusively, downstream of integrin is the first to show an association between Dok2 and aIIbb3, aIIbb3. We also demonstrate that integrin aIIbb3-induced Dok2 and moreover it is the first demonstration in platelets. The phosphorylation is independent of Syk and PLCc2andthat observed interaction appears to be dependent on outside-in

Dok2 can associate with aIIbb3, possibly through direct binding signaling of integrin aIIbb3 and Src kinases, and may be to the NPXY motif in the integrin b3 tail. In contrast, the mediated by phosphorylation of Dok2 and/or b3. Our data to regulation of Dok1 by thrombin is independent of these date suggest that Dok2 phosphorylation is necessary for the signaling components. Further studies are required to establish association with the integrin, given that both events are the mechanism of regulation of Dok1 by thrombin, and to governed by Src kinases and integrin aIIbb3 activation. confirm whether this is mediated by direct tyrosine phosphory- However, there are two lines of evidence that suggest that lation of the adapter or assembled into signaling complexes Dok2 phosphorylation is necessary, but not sufficient, for the that are regulated through tyrosine phosphorylation, in view of association. Firstly, limited Dok2 phosphorylation occurs in our inability to demonstrate tyrosine phosphorylation of Dok1 thepresenceofintegrinaIIbb3 inhibitors, yet the association is by direct immunoprecipitation because of the lack of suitable inhibited. Secondly, we have evidence that collagen can evoke antibodies. robust Dok2 phosphorylation, yet only a very weak integrin– A clear finding herein is the demonstration that Dok1 and Dok2 association. Dok2 are regulated in distinct ways and with distinct kinetics. The other explanation for the interaction of Dok2 and

While the explanation and signiﬁcance of these differences integrin aIIbb3 may relate to the phosphorylation status of

2006 International Society on Thrombosis and Haemostasis Regulation of Dok2 and Dok1 in platelets 393 tyrosine residues 747 and 759 in the b3 tail, which have Disclosure of Conflict of Interests consensus sequences for binding to the PTB domain contained The authors state that they have no conflicts of interest. within Dok proteins [3,9]. Studies in the literature have established that phosphorylation of the b3 tail may either promote or inhibit b3 interactions and subsequent signaling References events (reviewed in [35]). As such, the phosphorylation of the 1 Leo A, Schraven B. Networks in signal transduction: the role of cytoplasmic tail of b3 may be critical in mediating the observed adaptor proteins in platelet activation. Platelets 2000; 11: 429–45. interaction. While this has not been formerly investigated, it 2 Garcia A, Prabhakar S, Hughan S, Anderson TW, Brock CJ, would be of great interest to establish if Dok2 and aIIbb3 can Pearce AC, Dwek RA, Watson SP, Hebestreit HF, Zitzmann N. coassociate in the diYF mouse model [35] in which tyrosine Differential proteome analysis of TRAP-activated platelets: involve- 747 759 ment of DOK-2 and phosphorylation of RGS proteins. Blood 2004; residues Y and Y have been mutated to phenylalanine, 103: 2088–95. thereby compromising b3 phosphorylation and a number of 3 Di Cristofano A, Carpino N, Dunant N, Friedland G, Kobayashi platelet responses. R, Strife A, Wisniewski D, Clarkson B, Pandolfi PP, Resh MD. Further studies are required to establish the functional role Molecular cloning and characterization of p56dok-2 defines a of Dok1 and Dok2 in platelets. However, in the absence of new family of RasGAP-binding proteins. JBiolChem1998; 273: 4827– 30. specific inhibitors, it will be necessary to perform these 4 Carpino N, Wisniewski D, Strife A, Marshak D, Kobayashi R, Still- studies in mice deficient in Dok1 and Dok2, and in mice manB,ClarksonB.p62(dok):aconstitutively tyrosine-phosphoryl- deficient in both Dok isoforms. The generation of Dok1- and ated, GAP-associated protein in chronic myelogenous leukemia Dok2-deficient mice has been described [10,11,15,36], but progenitor cells. Cell 1997; 88: 197–204. functional studies on platelets from these mice have not been 5 SattlerM,VermaS,PrideYB,SalgiaR,RohrschneiderLR,Griffin JD. SHIP1, an SH2 domain containing polyinositol-5-phosphatase, reported. Significantly, the mice do not have a major regulates migration through two critical tyrosine residues and forms a bleeding phenotype. This observation does not argue against novel signaling complex with DOK1 and CRKL. JBiolChem2001; a platelet phenotype, however, because the vast majority of 276: 2451–8. mice that are deficient in proteins that are known to play a 6 Robson JD, Davidson D, Veillette A. Inhibition of the Jun N-terminal role in platelet function do not have a bleeding phenotype, protein kinase pathway by SHIP-1, a lipid phosphatase that interacts with the adaptor molecule Dok-3. Mol Cell Biol 2004; 24: 2332–43. e.g. GPVI-deficient mice, most probably because of the 7 Lemay S, Davidson D, Latour S, Veillette A. Dok-3, a novel adapter redundancy in the pathways of platelet activation and the molecule involved in the negative regulation of immunoreceptor absence of a suitable challenge that gives rise to bleeding. signaling. MolCellBiol2000; 20: 2743–54. Importantly, studies in other cells have demonstrated 8 Liang X, Wisniewski D, Strife A, Shivakrupa, Clarkson B, Resh MD. important roles for Dok1 and Dok2 in both the positive Phosphatidylinositol 3-kinase and Src family kinases are required for phosphorylation and membrane recruitment of Dok-1 in c-Kit signa- and negative regulation of signaling [8,10,11,36], which raises ling. JBiolChem2002; 277: 13732–8. the possibility that Dok1 and Dok2 may serve to either 9 Calderwood DA, Fujioka Y, de Pereda JM, Garcia-Alvarez B, hamper or facilitate platelet function. While this remains to Nakamoto T, Margolis B, McGlade CJ, Liddington RC, Ginsberg be determined, the present study is the first to reveal the MH. Integrin beta cytoplasmic domain interactions with phospho- differential phosphorylation of adapter proteins Dok2 and tyrosine-binding domains: a structural prototype for diversity in integrin signaling. Proc Natl Acad Sci USA 2003; 100: 2272–7. Dok1 downstream of G-protein, integrin and/or ITAM 10 Niki M, Di Cristofano A, Zhao M, Honda H, Hirai H, Van Aelst L, receptor pathways in platelets. Furthermore, the data raise Cordon-Cardo C, Pandolfi PP. Role of Dok-1 and Dok-2 in leukemia the intriguing possibility that these adapter proteins serve suppression. JExpMed2004; 200: 1689–95. distinct functions within the platelet, which for Dok2 may 11 Yasuda T, Shirakata M, Iwama A, Ishii A, Ebihara Y, Osawa M, Honda K, Shinohara H, Sudo K, Tsuji K, Nakauchi H, Iwakura Y, involve regulating integrin aIIbb3 outside-in signaling through Hirai H, Oda H, Yamamoto T, Yamanashi Y. Role of Dok-1 and direct functional and physical interactions with integrin Dok-2 in myeloid homeostasis and suppression of leukemia. JExp aIIbb3. Studies on Dok1- and Dok2-deficient mice will be Med 2004; 200: 1681–7. performed to address these important questions. 12 Woodring PJ, Meisenhelder J, Johnson SA, Zhou GL, Field J, Shah K,BladtF,PawsonT,NikiM,PandolfiPP,WangJY,HunterT.c- Abl phosphorylates Dok1 to promote filopodia during cell spreading. Acknowledgements JCellBiol2004; 165: 493–503. 13 Gerard A, Favre C, Garcon F, Nemorin JG, Duplay P, Pastor S, S. C. Hughan holds a National Health and Medical Collette Y, Olive D, Nunes JA. Functional interaction of RasGAP- Research Council (Australia) C. J. Martin Fellowship. S. binding proteins Dok-1 and Dok-2 with the Tec protein tyrosine P. Watson holds a British Heart Foundation Chair. The kinase. Oncogene 2004; 23: 1594–8. authors wish to thank S. Jackson (Australian Centre for 14 Noguchi T, Matozaki T, Inagaki K, Tsuda M, Fukunaga K, Kitamura Y, Kitamura T, Shii K, Yamanashi Y, Kasuga M. Tyrosine phos- Blood Diseases, Monash University, Victoria, Australia) for phorylation of p62(Dok) induced by cell adhesion and insulin: possible support and helpful discussions. The authors also wish to role in cell migration. EMBO J 1999; 18: 1748–60. express their thanks to V. Tybulewicz and E. Schweighoffer 15 Di Cristofano A, Niki M, Zhao M, Karnell FG, Clarkson B, Pear WS, from the National Institute for Medical Research, London, Van Aelst L, Pandolfi PP. p62(dok), a negative regulator of Ras and UK, for the provision of radiation chimeric Syk mutant mitogen-activated protein kinase (MAPK) activity, opposes leukem- ogenesis by p210(bcr-abl). JExpMed2001; 194: 275–84. mice.

2006 International Society on Thrombosis and Haemostasis 394 S. C. Hughan and S. P. Watson

16 Wonerow P, Pearce AC, Vaux DJ, Watson SP. A critical role for 26 Goncalves I, Hughan SC, Schoenwaelder SM, Yap CL, Yuan Y, phospholipase Cc2inaIIbb3-mediated platelet spreading. JBiolChem Jackson SP. Integrin aIIbb3-dependent calcium signals regulate 2003; 278: 37520–9. platelet-fibrinogen interactions under flow: involvement of phosphol- 17 McCarty OJ, Zhao Y, Andrew N, Machesky LM, Staunton D, ipase C c2. JBiolChem2003; 278: 34812–22. Frampton J, Watson SP. Evaluation of the role of platelet integrins in 27 Lock P, Casagranda F, Dunn AR. Independent SH2-binding sites fibronectin-dependent spreading and adhesion. J Thromb Haemost mediate interaction of Dok-related protein with RasGTPase-activating 2004; 2: 1823–33. protein and Nck. JBiolChem1999; 274: 22775–84. 18 Hughan SC, Senis Y, Best D, Thomas A, Frampton J, Vyas P, Watson 28 Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, SP. Selective impairment of platelet activation to collagen in the ab- Weringer EJ, Pollok BA, Connelly PA. Discovery of a novel, potent, sence of GATA1. Blood 2005; 105: 4369–76. and Src family-selective tyrosine kinase inhibitor. Study of Lck- and 19 Suzuki-Inoue K, Inoue O, FramptonJ,WatsonSP.MurineGPVI FynT-dependent T cell activation. JBiolChem1996; 271: 695–701. stimulates weak integrin activation in PLCgamma2-/- platelets: 29QuekLS,PasquetJM,HersI,CornallR,KnightG,BarnesM,Hibbs involvement of PLCgamma1 and PI3-kinase. Blood 2003; 102: 1367– ML,DunnAR,LowellCA,WatsonSP.FynandLynphosphorylate 73. the Fc receptor gamma chain downstream of glycoprotein VI in mu- 20 Wang D, Feng J, Wen R, Marine J-C, Sangster MY, Parganas E, rine platelets, and Lyn regulates a novel feedback pathway. Blood 2000; Hoffmeyer A, Jackson CW, Cleveland JL, Murray PJ, Ihle JN. 96: 4246–53. Phospholipase C [gamma]2 is essential in the functions of B cell and 30 Gao J, Zoller KE, Ginsberg MH, Brugge JS, Shattil SJ. Regulation of several Fc receptors. Immunity 2000; 13: 25–35. the pp72syk protein tyrosine kinase by platelet integrin alpha IIb beta 21 Mocsai A, Zhou M, Meng F, Tybulewicz VL, Lowell CA. Syk is 3. EMBO J 1997; 16: 6414–25. required for integrin signaling in neutrophils. Immunity 2002; 16: 547– 31 Watson SP, Auger JM, McCarty OJT, Pearce AC. GPVI and integrin 58. aIIbb3 signaling in platelets. J Thromb Haemost 2005; 3: 1752–62. 22 Goncalves I, Nesbitt WS, Yuan Y, Jackson SP. Importance of 32 Atkinson BT, Stafford MJ, Pears CJ, Watson SP. Signalling events temporal flow gradients and integrin alphaIIbbeta3 mechanotrans- underlying platelet aggregation induced by the glycoprotein VI agonist duction for shear activation of platelets. JBiolChem2005; 280: convulxin. Eur J Biochem 2001; 268: 5242–8. 15430–7. 33 Atkinson BT, Jarvis GE, Watson SP. Activation of GPVI by collagen 23 Garcia A, Senis YA, Antrobus R, Hughes CE, Dwek RA, Watson SP, is regulated by alpha2beta1 and secondary mediators. JThromb Zitzmann N. A global proteomics approach identifies novel phos- Haemost 2003; 1: 1278–87. phorylated signaling proteins in GPVI-activated platelets: involvement 34 Cai D, Dhe-Paganon S, Melendez PA, Lee J, Shoelson SE. Two new of G6f, a novel platelet Grb2-binding membrane adapter. Proteomics substrates in insulin signaling, IRS5/DOK4 and IRS6/DOK5. JBiol 2006; 6: 5332–43. Chem 2003; 278: 25323–30. 24 Nemorin JG, Duplay P. Evidence that Lck-mediated phosphorylation 35 Phillips DR, Nannizzi-Alaimo L, Prasad KS. Beta3 tyrosine phos- of p56dok and p62dok may play a role in CD2 signaling. JBiolChem phorylation in alphaIIbbeta3 (platelet membrane GP IIb-IIIa) outside- 2000; 275: 14590–7. in integrin signaling. Thromb Haemost 2001; 86: 246–58. 25 Obergfell A, Eto K, Mocsai A, Buensuceso C, Moores SL, Brugge JS, 36 Yamanashi Y, Tamura T, Kanamori T, Yamane H, Nariuchi H, Lowell CA, Shattil SJ. Coordinate interactions of Csk, Src, and Syk Yamamoto T, Baltimore D. Role of the rasGAP-associated docking kinases with [alpha]IIb [beta]3 initiate integrin signaling to the cyto- protein p62(dok) in negative regulation of B cell receptor-mediated skeleton. JCellBiol2002; 157: 265–75. signaling. Genes Dev 2000; 14: 11–6.

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