Orthovanadate decreases the leptin content in isolated mouse fat pads via proteasome activation

In a physiological milieu platelets continue to be exposed to agonists long after clot formation. We studied the regulation of postaggregation events consequent on protease-activated receptor (PAR) 1 ligation with either thrombin or the thrombin receptor-activating peptide (TRAP). Stimulation with TRAP (20 microM) but not with thrombin (1 U/ml) for 15 min evoked platelet disaggregation by about 30% and downregulation of high-affinity fibrinogen binding sites on integrin alpha(IIb)beta(3) to nearly prestimulation levels. Concurrently, only TRAP disorganized the actin-based cytoskeleton, with decrease in the cytoskeletal content of focal contact-associated proteins like integrin alpha(IIb)beta(3), Src, and focal adhesion kinase (FAK). While protein tyrosine kinases were activated during the initial period of platelet aggregation with either agonist, stimulation of protein tyrosine phosphatases determined the successive phase of reduced phosphotyrosine content. SHP-1, an abundant protein tyrosine phosphatase in the platelets, was tyrosine phosphorylated on challenge of PAR-1 and coprecipitated with two unidentified tyrosine phosphorylated proteins of 140 and 60 kDa; in addition, SHP-1 tyrosine phosphorylation (which is associated with enhanced phosphatase activity) was sustained until 15 min. Activity of calpain was upregulated following incubation with thrombin and not with TRAP. Collectively, these data suggest that signaling pathways elicited by PAR-1 agonists thrombin and TRAP are markedly different, which could have important implications on late platelet responses.     

Enhanced phosphorylation and enzymatic activity of phosphoglucomutase by the Btk29A tyrosine kinase in Drosophila

The Drosophila Btk29A tyrosine kinase is suggested to be involved in diverse processes, although its target proteins are unknown. In the present study, we investigated substrates of Btk29A tyrosine kinase by expressing a catalytically activated form of Btk29A-P1 (Btk-EG) in Drosophila compound eyes. Expression in eye disks led to the development of the rough-eye phenotype and increased tyrosine phosphorylation of a 65-kDa protein. Partial amino acid sequence analysis of this protein showed that it was phosphoglucomutase. Phosphoglucomutase activity in heads from Btk-EG-expressing flies was higher than that in controls, suggesting that the levels of tyrosine phosphorylation and activity of the enzyme are associated with Btk29A tyrosine kinase activity.     

Phosphorylation of Bruton's tyrosine kinase by c-Abl

Bruton's tyrosine kinase (Btk) is necessary for B-lymphocyte development. Mutation in the gene coding for Btk causes X-linked agammaglobulinemia (XLA) in humans. Similar to Btk, c-Abl is a tyrosine kinase shuttling between the cytoplasm and the nucleus where it is involved in different functions depending on the localization. In this report we describe for the first time that c-Abl and Btk physically interact and that c-Abl can phosphorylate tyrosine 223 in the SH3 domain of Btk. Interestingly, the Btk sequence matched a v-Abl substrate [correction] identified from a randomized peptide library and was also highly related to a number of previously found c-Abl substrates.     

Bruton酪氨酸激酶在昆虫细胞中表达的初步研究

用杆状病毒表达载体系统表达小鼠Ｂｒｕｔｏｎ酪氨酸激酶（Ｂｒｕｔｏｎｔｙｒｏｓｉｎｅｋｉｎａｓｅ,Ｂｔｋ）．构建重组转染载体时,于Ｂｔｋ起始码的上游插入了一段Ｈ９０２序列．用重组转染载体、苜蓿夜蛾核型多角体病毒线性ＤＮＡ和质脂体共转染Ｓｆ９昆虫细胞,经过对重组杆状病毒三轮扩增后,用Ｈ９０２抗体检测表明,昆虫细胞中Ｂｔｋ的表达已达最高水平．对表达后Ｂｔｋ自身磷酸化检测表明,该激酶具有自身磷酸化活性．从而证实Ｂｔｋ在昆虫细胞中的表达获得了成功     

Tec homology (TH) adjacent to the PH domain

The pleckstrin homology (PH) domain is extended in the Btk kinase family by a region designated the TH (Tec homology) domain, which consists of about 80 residues preceding the SH3 domain. The TH domain contains a conserved 27 amino acid stretch designated the Btk motif and a proline-rich region. Sequence similarity was found to a putative Ras GTPase activating protein and a human interferon-γ binding protein both in the PH domain and the Btk motif region. SLK1/SSP31 protein kinase and a non-catalytic p85 subunit of PI-3 kinase had similarity only with the proline rich region. The identification of a PH domain extension in some signal transduction proteins in different species suggests that this region is involved in protein—protein interactions.     

Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation.     

Cytoskeletal changes in platelets induced by thrombin and phorbol myristate acetate (PMA).

Changes in shape, and aggregation that accompanies platelet activation, are dependent on the assembly and reorganization of the cytoskeleton. To assess the changes in cytoskeleton induced by thrombin and PMA, suspensions of aspirin-treated,32P-prelabeled, washed pig platelets in Hepes buffer containing ADP scavengers were activated with thrombin, and with PMA, an activator of protein kinase C. The cytoskeletal fraction was prepared by adding Triton extraction buffer. The Triton-insoluble (cytoskeletal) fraction isolated by centrifugation was analysed by SDS-PAGE and autoradiography. Incorporation of actin into the Triton-insoluble fraction was used to quantify the formation of F-actin. Thrombin-stimulated platelet cytoskeletal composition was different from PMA-stimulated cytoskeletal composition. Thrombin-stimulated platelets contained not only the three major proteins: actin (43 kDa), myosin (200 kDa) and an actin-binding protein (250 kDa), but three additional proteins of Mr56 kDa, 80 kDa and 85 kDa in the cytoskeleton, which were induced in by thrombin dose-response relationship. In contrast, PMA-stimulated platelets only induced actin assembly, and the 56 kDa, 80 kDa and 85 kDa proteins were not found in the cytoskeletal fraction. Exposure of platelets to thrombin or PMA induced phosphorylation of pleckstrin parallel to actin assembly. Staurosporine, an inhibitor of protein kinase C, inhibited actin assembly and platelet aggregation induced by thrombin or PMA, but did not inhibit the incorporation of 56 kDa, 80 kDa and 85 kDa into the cytoskeletal fraction induced by thrombin. These three extra proteins seem to be unrelated to the induction of protein kinase C. We conclude that actin polymerization and platelet aggregation were induced by a mechanism dependent on protein kinase C, and suggest that thrombin-activated platelets aggregation could involve additional cytoskeletal components (56 kDa, 80 kDa, 85 kDa) of the cytoskeleton, which made stronger actin polymerization and platelet aggregation more.     

Pleckstrin homology domains interact with filamentous actin.

A fraction of Bruton's tyrosine kinase (Btk) co-localizes with actin fibers upon stimulation of mast cells via the high affinity IgE receptor (FcepsilonRI). In this study, a molecular basis of the Btk co-localization with actin fibers is presented. Btk and other Tec family tyrosine kinases have a pleckstrin homology (PH) domain at their N termini. The PH domain is a short peptide module frequently found in signal-transducing proteins and cytoskeletal proteins. Filamentous actin (F-actin) is shown to be a novel ligand for a subset of PH domains, including that of Btk. The actin-binding site was mapped to a 10-residue region of the N-terminal region of Btk. Basic residues in this short stretch are demonstrated to be involved in actin binding. Isolated PH domains induced actin filament bundle formation. Consistent with these observations, Btk binds F-actin in vitro and in vivo. Wild-type Btk protein is in part translocated to the cytoskeleton upon FcepsilonRI cross-linking, whereas Btk containing a mutated PH domain is not. Phosphatidylinositol 3,4, 5-trisphosphate-mediated membrane translocation of Btk was enhanced in cytochalasin D-pretreated, FcepsilonRI-stimulated mast cells. These data indicate that PH domain-mediated F-actin binding plays a role in Btk co-localization with actin filaments.     

Coordinate interactions of Csk, Src, and Syk kinases with [alpha]IIb[beta]3 initiate integrin signaling to the cytoskeleton

Integrins regulate cell adhesion and motility through tyrosine kinases, but initiation of this process is poorly understood. We find here that Src associates constitutively with integrin alphaIIbbeta3 in platelets. Platelet adhesion to fibrinogen caused a rapid increase in alphaIIbbeta3-associated Src activity, and active Src localized to filopodia and cell edges. Csk, which negatively regulates Src by phosphorylating Tyr-529, was also constitutively associated with alphaIIbbeta3. However, fibrinogen binding caused Csk to dissociate from alphaIIbbeta3, concomitant with dephosphorylation of Src Tyr-529 and phosphorylation of Src activation loop Tyr-418. In contrast to the behavior of Src and Csk, Syk was associated with alphaIIbbeta3 only after fibrinogen binding. Platelets multiply deficient in Src, Hck, Fgr, and Lyn, or normal platelets treated with Src kinase inhibitors failed to spread on fibrinogen. Inhibition of Src kinases blocked Syk activation and inhibited phosphorylation of Syk substrates (Vav1, Vav3, SLP-76) implicated in cytoskeletal regulation. Syk-deficient platelets exhibited Src activation upon adhesion to fibrinogen, but no spreading or phosphorylation of Vav1, Vav3, and SLP-76. These studies establish that platelet spreading on fibrinogen requires sequential activation of Src and Syk in proximity to alphaIIbbeta3, thus providing a paradigm for initiation of integrin signaling to the actin cytoskeleton.     

Interaction of Bruton's tyrosine kinase and protein kinase Ctheta in platelets. Cross-talk between tyrosine and serine/threonine kinases.

The nonreceptor Bruton's tyrosine kinase (Btk) has been previously shown to associate physically and functionally with members of the protein kinase C (PKC) family of serine/threonine kinases in a variety of cell types. Here we show evidence for a novel interaction between Btk and PKCtheta; in platelets activated through the adhesion receptors GP Ib-V-IX and GP VI. Alboaggregin A, a snake venom component capable of activating both receptors in combination, leads to tyrosine phosphorylation of Btk downstream of Src family kinases. Inhibition of Btk by the selective antagonist LFM-A13 causes a reduction in calcium entry, although secretion of 5-hydroxytryptamine is potentiated. Btk is also phosphorylated on threonine residues in a PKC-dependent manner and associates with PKCtheta; upon platelet activation by either alboaggregin A or activation of GP Ib-V-IX alone by von Willebrand factor/ristocetin. PKCtheta; in turn becomes tyrosine-phosphorylated in a manner dependent upon Src family and Btk kinase activity. Inhibition of Btk activity by LFM-A13 leads to enhancement of PKCtheta; activity, whereas nonselective inhibition of PKC activity by bisindolylmaleimide I leads to reduction in Btk activity. We propose a reciprocal feedback interaction between Btk and PKCtheta; in platelets, in which PKCtheta; positively modulates activity of Btk, which in turn feeds back negatively upon PKCtheta;.     

Tyrosin dephosphorylation and concurrent inactivation of protein kinase FA/GSK-3α by genistein in A431 cells

Modulation of protein Kinase _F/GSK-3α by tyrosine phosphorylation in A431 cells was investigated. Kinase F _A/GSK-3α was found to exist in a highly tyrosine-phosphorylated/activated state in resting cells but could become tyrosine-dephosphorylated and inactivated down to less than 30% of control values in concentration dependent manner by 50-400 μM genistein( a Specific tyrosine kinase inhibitor), as demonstrated by metobolic ³²p-labeling of the cells followed by immunoprecipitation and two-dimensional phosphoamino acid analysis and byimmunodetection in an antikinase F_A/GSK-3α immunoprecipitate kinase assay. Taken together, the results provide evidence that Kinase F_A/GSK-3α may exist in a highly tyrosine-phosphorylated/activated state in resting cells which can by tyrosine-dephosphorylated and nactivated by extracellular stimulus and that tyrosine kinase(s) and /or tyrosine phosphatase(s) may play a role in the modulation of kinse F_A/GSK-3α activity in cells.     

Selective association of the tyrosine kinases Src, Fyn, and Lyn with integrin-rich actin cytoskeletons of activated, nonaggregated platelets.

Integrin-mediated interactions between cytoskeletal proteins and extracellular fibrinogen are required for platelet adhesion. We have previously demonstrated that the major platelet integrin, alpha(IIb)beta(3), becomes incorporated into the actin cytoskeleton of platelets in an activation-dependent, aggregation-independent manner. To determine if regulatory molecules are also associated with these integrin-rich cytoskeletal complexes, we examined actin cytoskeletons for the presence of kinases and phosphoproteins. Western immunoblot analysis revealed that the tyrosine kinases Src, Fyn, and Lyn are specifically associated with actin cytoskeletons of activated, nonaggregated platelets. However, as noted by others, the cytoskeletal association of focal adhesion kinase depends on platelet aggregation. Actin cytoskeletons isolated from (32)P-labeled platelets also contain a number of phosphorylated proteins. Interestingly, an approximately 18-kDa phosphoprotein was uniquely present in activated platelet cytoskeletons. Collectively, our results demonstrate that actin cytoskeletons of activated, nonaggregated platelets contain not only integrins, but also kinases and phosphoproteins that could regulate platelet adhesion and transmembrane communication.     

Effects of BTK signalling in pathogenic microorganism infections

As a cytoplasmic protein tyrosine kinase, Bruton's tyrosine kinase (Btk) is widely considered as a vital kinase in many aspects of different physiologic processes. It is engaged in many important signalling pathways related to the immune response, such as the B cell receptor pathway, pattern‐recognition receptor pathway, and triggering receptor expressed on myeloid cell pathway. Recent studies have increasingly focused on the important role of Btk in various inflammatory diseases, which are related to Btk expression in myeloid innate immune cells, such as macrophages, dendritic cells and neutrophils. Although some investigations have explored the role of Btk in microbial infections, many aspects remain elusive, and some of the results are opposite and controversial. Considering the complicated and multiple roles of Btk in the immune system, we summarized the engagement of Btk signalling in various pathogenic microorganism infections, the possible mechanisms involved and its therapeutic potential in the control of infectious diseases.     

The effect of glycoprotein IIb-IIIa receptor occupancy on the cytoskeleton of resting and activated platelets

The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), serves as the receptor for fibrinogen. This study examined what effect GPIIb-IIIa receptor occupancy had on the cytoskeleton of resting and activated platelets. Triton X-100-insoluble residues (cytoskeletons) were isolated from resting washed platelets incubated with either 500 microM RGDS or 500 microM RGES and examined for protein content. RGDS did not increase the amount of GPIIb-IIIa associated with the cytoskeletal residues which sedimented at either 15,800 x g or 100,000 x g. To determine the effect of receptor occupancy on the formation of the activated platelet cytoskeleton, stirred and nonstirred RGDS-treated platelets in plasma were activated with ADP. Triton X-100-insoluble residues were isolated and examined for both protein content and retention of GPIIb-IIIa. Further, morphological studies were performed on the RGDS-ADP-stimulated platelets. The results of this study suggest that 1) RGDS peptide receptor occupancy does not lead to GPIIb-IIIa linkage to the cytoskeleton, 2) ADP-stimulated platelet shape change, polymerization of actin, and association of myosin with the cytoskeleton are unaffected by RGDS peptide receptor occupancy. 3) RGDS inhibits an aggregation-dependent incorporation of ABP, alpha-actinin, talin, and GPIIb-IIIa into the Triton-insoluble residue.     

Ca2+-independent activation of Bruton's tyrosine kinase is required for store-mediated Ca2+ entry in human platelets

Store-mediated Ca(2+) entry (SMCE), which is rapidly activated by depletion of the intracellular Ca(2+) stores, is a major mechanism for Ca(2+) influx. Several studies have involved tyrosine kinases in the activation of SMCE, such as pp60(src), although at present those involved in the early activation steps are unknown. Here we report the involvement of Bruton's tyrosine kinase (Btk) in the early stages of SMCE in human platelets. Cell treatment with thrombin or thapsigargin (TG) plus ionomycin (Iono) results in rapid activation of Btk, which was independent of rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) but dependent on H(2)O(2) generation. Platelet treatment with Btk inhibitors, LFM-A13 or terreic acid, significantly reduced TG+Iono- and thrombin-evoked SMCE. Btk was rapidly activated by addition of low concentrations of H(2)O(2), whose effect on Ca(2+) entry was prevented by Btk inhibitors. Our results indicate that pp60(src) and Btk co-immunoprecipitate after platelet stimulation with TG+Iono, thrombin or H(2)O(2). In addition, we have found that LFM-A13 impaired actin filament reorganization after store depletion and agonist-induced activation of pp60(src), while the inhibitor of pp60(src), a protein that requires actin reorganization for its activation, did not modify Btk activation, suggesting that Btk is upstream of pp60(src). We propose a role for Btk in the early steps of activation of SMCE in human platelets.     

Differential Regulation of Focal Adhesion Kinase and Mitogen-Activated Protein Kinase Tyrosine Phosphorylation During Insulin-Like Growth Factor-I-Mediated Cytoskeletal Reorganization

Abstract: In SH-SY5Y human neuroblastoma cells, insulin-like growth factor (IGF)-I mediates membrane ruffling and growth cone extension. We have previously shown that IGF-I activates the tyrosine phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK) 2. In the current study, we examined which signaling pathway underlies IGF-I-mediated FAK phosphorylation and cytoskeletal changes and determined if an intact cytoskeleton was required for IGF-I signaling. Treatment of SH-SY5Y cells with cytochalasin D disrupted the actin cytoskeleton and prevented any morphological changes induced by IGF-I. Inhibitors of phosphatidylinositol 3-kinase (PI 3-K) blocked IGF-I-mediated changes in the actin cytoskeleton as measured by membrane ruffling. In contrast, PD98059, a selective inhibitor of ERK kinase, had no effect on IGF-I-induced membrane ruffling. In parallel with effects on the actin cytoskeleton, cytochalasin D and PI 3-K inhibitors blocked IGF-I-induced FAK tyrosine phosphorylation, whereas PD98059 had no effect. It is interesting that cytochalasin D did not block IGF-I-induced ERK2 tyrosine phosphorylation. Therefore, it is likely that FAK and ERK2 tyrosine phosphorylations are regulated by separate pathways during IGF-I signaling. Our study suggests that integrity as well as dynamic motility of the actin cytoskeleton mediated by PI 3-K is required for IGF-I-induced FAK tyrosine phosphorylation, but not for ERK2 activation.     

A cyclic adenosine 3',5'-monophosphate-induced tyrosine phosphorylation of Syk protein tyrosine kinase in the flagella of boar spermatozoa

A cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein tyrosine phosphorylation is involved in the expression of fertilizing ability in mammalian spermatozoa. However, there are only limited data concerning the identification of protein tyrosine kinase (PTK) that is activated by the cAMP signaling. In this study, we have shown data supporting that boar sperm flagellum possesses a unique cAMP-protein kinase A (PKA) signaling cascade leading to phosphorylation of Syk PTK at the tyrosine residues of the activation loop. Ejaculated spermatozoa were washed and then incubated in a modified Krebs-Ringer HEPES medium (mKRH) containing polyvinyl alcohol (PVA) plus 0.1 mM cBiMPS (a cell-permeable cAMP analog), 0.25 mM sodium orthovanadate (Na3VO4) (a protein tyrosine phosphatase (PTP) inhibitor) or both at 38.5 degrees C for 180 min. Aliquots of the sperm suspensions were recovered before and after incubation and then used to detect sperm tyrosine-phosphorylated proteins by Western blotting and indirect immunofluorescence. In the Western blotting, the anti-phosphotyrosine monoclonal antibody (4G10) recognized several bands including 72-kDa protein in the protein extracts from spermatozoa that were incubated solely with cBiMPS. The tyrosine phosphorylation in these sperm proteins was dependent on cBiMPS and enhanced by the addition of Na3VO4. The 72-kDa tyrosine-phosphorylated protein was apparently reacted with the anti-phospho-Syk antibody (Tyr525/526). Indirect immunofluorescence revealed that the connecting and principal pieces of spermatozoa incubated with cBiMPS and Na3VO4 were stained with the anti-phospho-Syk antibody. However, the reactivity of the 72-kDa protein with the anti-phospho-Syk antibody was reduced by the addition of H-89 (a PKA inhibitor, 0.01-0.1 mM) to the sperm suspensions but not affected by the pretreatment of spermatozoa with BAPTA-AM (an intracellular Ca2+ chelator, 0.1 mM). Fractionation of phosphorylated proteins from the spermatozoa with a detergent Nonidet P-40 suggested that the 72-kDa tyrosine-phosphorylated protein might be a cytoskeletal component. Based on these findings, we have concluded that the cAMP-PKA signaling is linked to the Ca2+-independent tyrosine phosphorylation of Syk in the connecting and principal pieces of boar spermatozoa.     

Protein tyrosine phosphatase activity modulation by endothelin-1 in rabbit platelets

Protein tyrosine phosphorylation, modulated by the rate of both protein tyrosine kinase and protein tyrosine phosphatase activities, is critical for cellular signal transduction cascades. We report that endothelin-1 stimulation of rabbit platelets resulted in a dose- and time-dependent tyrosine phosphorylation of four groups of proteins in the molecular mass ranges of 50, 60, 70–100 and 100–200 kDa and that one of these corresponds to focal adhesion kinase. This effect is also related to the approximately 60% decrease in protein tyrosine phosphatase activity. Moreover, this inhibited activity was less sensitive to orthovanadate. In the presence of forskolin that increases the cAMP level a dose-dependent inhibition of the endothelin-stimulated tyrosine phosphorylation of different protein substrates and a correlation with an increase in the protein tyrosine phosphatase activity (11.6-fold compared to control) have been found. Further studies by immunoblotting of immunoprecipitated soluble fraction with anti-protein tyrosine phosphatase-1C from endothelin-stimulated platelets have demonstrated that the tyrosine phosphorylation of platelet protein tyrosine phosphatase-1C is correlated with the decrease in its phosphatase activity. As a consequence, modulation and regulation by endothelin-1 in rabbit platelets can be proposed through a cAMP-dependent pathway and a tyrosine phosphorylation process that may affect some relevant proteins such as focal adhesion kinase.     

Interaction between Btk TH and SH3 domain

Several mechanisms are involved in the regulation of cellular signaling. Bruton tyrosine kinase (Btk) of the Tec family contains in the Tec homology (TH) domain a proline-rich region (PRR) capable of interacting with several SH3 domains. The Btk has the SH3 domain adjacent to the TH domain. CD and fluorescence spectroscopy were used to study the binding of two peptides corresponding to segments in the PRR to the Btk SH3 domain. The peptide for the N-terminal half of the PRR binds specifically, whereas the other peptide had hardly any affinity. The TH domain has about four times lower affinity to the SH3 domain than the peptide, 17.0 vs 3.9 microM. The interaction was further tested with an SH3 domain construct that contained the PRR. The two peptides cannot compete for the binding to the extended protein and the TH domain has two times lower affinity to the extended SH3 domain. The intra- or intermolecular interaction between the TH and SH3 domain might have regulatory function also in the other Tec family members.     

Translocation of pp60c-src to the cytoskeleton during platelet aggregation.

The high amount of pp60c-src in platelets has led to speculation that this kinase is responsible for tyrosine-specific phosphorylation of cellular proteins during platelet activation by different agonists, and is, therefore, implicated in signal transduction of these cells. Unlike pp60v-src, the association of which with the cytoskeleton appears to be a prerequisite for transformation, pp60c-src 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 pp60c-src 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 pp60c-src 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 pp60c-src is associated with the membrane skeletal fraction which remains unchanged during the activation process. The interaction of pp60c-src with cytoskeletal proteins strongly correlates with aggregation and is mediated by GPIIb/IIIa receptor-fibrinogen binding. We suggest that the translocation of pp60c-src to the cytoskeleton and its association with cytoskeletal proteins may regulate tyrosine phosphorylation in platelets.