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.     

Two thrombin-activated Ca2+ channels in human platelets.

The regulation of extracellular Ca2+ entry into fura-2-loaded human platelets was examined following stimulation with thrombin. In the presence of external Ca2+, stimulation of platelets with thrombin resulted in a rapid increase, followed by a plateau, in intracellular Ca2+ concentration ([Ca2+]i). Pretreatment with wortmannin, a specific inhibitor of myosin light chain kinase, suppressed only the plateau phase and had no effect on the initial rapid increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thrombin induced a transient and relatively small increase in [Ca2+]i caused by Ca2+ release from internal stores. When Ca2+ was added subsequently to the Ca(2+)-free medium within 10 min after thrombin activation, a marked increase in [Ca2+]i was seen, reflecting thrombin-stimulated external Ca2+ entry. With the Ca(2+)-free medium, wortmannin did not affect either the Ca2+ mobilization from the internal stores or the rapid external Ca2+ entry at early time points (within 5 s) after thrombin stimulation, whereas it significantly inhibited Ca2+ entry when Ca2+ was added later (at 3 min). Wortmannin inhibition of this late Ca2+ entry and that of 20-kDa myosin light chain phosphorylation after thrombin stimulation were dose- and preincubation time-dependent and correlated well with each other. These results suggest that two different channels are responsible for Ca2+ entry in human platelets at the early and late phases of thrombin stimulation and that the channel responsible for the late phase of Ca2+ entry may be activated by a mechanism involving myosin light chain kinase.     

Evidence for the selective association of a subpopulation of GPIIb-IIIa with the actin cytoskeletons of thrombin-activated platelets

Activation of blood platelets triggers a series of responses leading to the formation and retraction of blood clots. Among these responses is the establishment of integrin-mediated transmembrane connections between extracellular matrix components and the actin cytoskeleton of the platelet. Here we report that a specific subpopulation of the major platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa) (also referred to as alpha IIb beta 3 integrin), becomes incorporated into the detergent- insoluble actin cytoskeleton of platelets during the platelet activation response. The cytoskeletal association of GPIIb-IIIa is independent of platelet aggregation and fibrin sedimentation and is sensitive to cytochalasin D treatment. As determined by Western immunoblot analysis, approximately 22% of the total cellular GPIIb-IIIa becomes associated with the actin cytoskeleton upon thrombin activation in a manner that is independent of the detection of talin, alpha- actinin, or vinculin in the complex. We found that the cytoskeleton- associated GPIIb-IIIa is derived from an intracellular source since it is not available for lactoperoxidase-catalyzed radioiodination before platelet activation. Two intracellular sources of GPIIb-IIIa are present in resting platelets: GPIIb-IIIa associated with the alpha- granule secretory compartment as well as surface-inaccessible domains of the surface-connected canalicular system. Interestingly, alpha- granule secretion, which occurs in thrombin-activated platelets and results in the translocation of intracellular GPIIb-IIIa to the plasma membrane, appears to be required for the cytoskeleton incorporation of GPIIb-IIIa that we observe. Collectively, our data provide evidence that a subpopulation of GPIIb-IIIa derived from an intracellular source is selectively linked to the actin cytoskeleton of platelets upon thrombin activation in the absence of platelet aggregation.     

Heat-shock protein 90 complexes in resting and thrombin-activated platelets

The orexins are peptides which were recently isolated from the rat hypothlamus. They play a role in energy homeostasis and regulation of feeding as well as in other functions such as the sleep-wake cycle. The involvement of glucocorticoids in stress processes as well as in body weight regulation is well known. In the present paper, we investigated the role of glucocorticoids on hypocretin (Hcrt)/orexin (OX) pathway in Sprague-Dawley rats. We confirmed by in situ hybridization that prepro-Hcrt/OX mRNA expression is restricted to the lateral hypothalamus area with extension to the perifornical nucleus and the posterior hypothalamic area. Lateral hypothalamic prepro-Hcrt/OX mRNA expression was decreased by 50% after adrenalectomy (99.8+/-5.0 vs 49.2+/-4.4 nCi/g, p<0.01). Peripheral glucocorticoid treatment (dexamethasone) restored its expression to normal levels (105.4+/-6.1 nCi/g). The present data provide direct evidence that Hcrt/OX expression in the lateral hypothalamus is modulated by the glucocorticoids status. As the Hcrt/Ox system is closely interactive with the corticotropin-releasing hormone and neuropeptide Y systems, we propose that hypocretin/orexins peptides constitute a very sensitive key relay for mediating both stress and feeding behavior.     

Thrombin-dependent association of phosphatidylinositol-3 kinase with p60c-src and p59fyn in human platelets.

Recent studies have shown that ligand-activated growth factor receptors as well as transforming versions of nonreceptor protein-tyrosine kinases physically associate with phosphatidylinositol-3 kinase (PI-3 kinase). Reasoning that PI-3 kinase might also play a role in the normal functions of nonreceptor kinases, we sought to determine whether association with PI-3 kinase might serve as a measure of nonreceptor protein-tyrosine kinase activation under physiological conditions. We found that p60c-src as well as p59fyn, the product of another member of the src family of proto-oncogenes, physically associated with a PI kinase activity within 5 s after exposure to thrombin. Furthermore, PI kinase reaction products generated in p60v-src, p60c-src or p59fyn containing immunoprecipitates were indistinguishable, demonstrating the identity of the associated enzyme as PI-3 kinase. These findings demonstrate a thrombin-dependent interaction between p60c-src or p59fyn and PI-3 kinase and suggest a role for nonreceptor protein-tyrosine kinases in human platelet signal transduction.     

Essential role for Csk upstream of Fyn and Yes in zebrafish gastrulation

Morphogenetic cell movements during gastrulation shape the vertebrate embryo bodyplan. Non-canonical Wnt signaling has been established to regulate convergence and extension cell movements that mediate anterior-posterior axis elongation. In recent years, many other factors have been implicated in the process by modulation of non-canonical Wnt signaling or by different, unknown mechanisms. We have found that the Src family kinases, Fyn and Yes, are required for normal convergence and extension cell movements in zebrafish embryonic development and they signal in parallel to non-canonical Wnts, eventually converging on a common downstream factor, RhoA. Here, we report that Csk, a negative regulator of Src family kinases has a role in gastrulation cell movements as well. Csk knock down induced a phenotype that was similar to the defects observed after knock down of Fyn and Yes, in that gastrulation cell movements were impaired, without affecting cell fate. The Csk knock down phenotype was rescued by simultaneous partial knock down of Fyn and Yes. We conclude that Csk acts upstream of Fyn and Yes to control vertebrate gastrulation cell movements.     

Association of Fyn and Lyn with the proline-rich domain of glycoprotein VI regulates intracellular signaling

The glycoprotein VI (GPVI)-Fc receptor (FcR) gamma-chain complex, a key activatory receptor for collagen on platelet surface membranes, is constitutively associated with the Src family kinases Fyn and Lyn. Molecular cloning of GPVI has revealed the presence of a proline-rich domain in the sequence of GPVI cytoplasmic tail which has the consensus for interaction with the Src homology 3 (SH3) domains of Fyn and Lyn. A series of in vitro experiments demonstrated the ability of the SH3 domains of both Src kinases to bind the proline-rich domain of GPVI. Furthermore, depletion of the proline-rich domain in GPVI (Pro(-)-GPVI) prevented binding of Fyn and Lyn and markedly reduced phosphorylation of FcR gamma-chain in transiently transfected COS-7 cells, but did not affect the association of the gamma-chain with GPVI. Jurkat cells stably transfected with wild type GPVI show robust increases in tyrosine phosphorylation and intracellular Ca2+ in response to the snake venom convulxin that targets GPVI. Importantly, convulxin is not able to activate cells transfected with Pro(-)-GPVI, even though the association with the immunoreceptor tyrosine-based activation motif-containing chains is maintained. These findings demonstrate that the proline-rich domain of GPVI mediates the association with Fyn/Lyn via their SH3 domain and that this interaction initiates activation signals through GPVI.     

Protein tyrosine phosphatase epsilon activates Yes and Fyn in Neu-induced mammary tumor cells

The receptor-type form of protein tyrosine phosphatase epsilon (RPTP) is among the few tyrosine phosphatases that can support the transformed phenotype of tumor cells. Accordingly, cells from mammary epithelial tumors induced by activated Neu in mice genetically lacking RPTP appear morphologically less transformed and exhibit reduced proliferation. The effect of RPTP in these cells is mediated at least in part by its ability to activate Src, the prototypic member of a family of related kinases. We show here that RPTP is a physiological activator of two additional Src family kinases, Yes and Fyn. Activities of both kinases are inhibited in mammary tumor cells lacking RPTP, and phosphorylation at their C-terminal inhibitory tyrosines is increased. In agreement, opposite effects on activities and phosphorylation of Yes and Fyn are observed following increased expression of PTP. RPTP also forms stable complexes with either kinase, providing physical opportunity for their activation by RPTP. Surprisingly, expression of Yes or of Fyn does not rescue the morphological phenotype of RPTP-deficient tumor cells in contrast with the strong ability of Src to do so. We conclude that RPTP activates Src, Yes, and Fyn, but that these related kinases play distinct roles in Neu-induced mammary tumor cells.     

Identification of Src, Fyn, Lyn, PI3K and Abl SH3 domain ligands using phage display libraries.

Many proteins involved in intracellular signal transduction contain a small, 50-60 amino acid domain, termed the Src homology 3 (SH3) domain. This domain appears to mediate critical protein-protein interactions that are involved in responses to extracellular signals. Previous studies have shown that the SH3 domains from several proteins recognize short, contiguous amino acid sequences that are rich in proline residues. While all SH3 recognition sequences identified to date share a conserved P-X-X-P motif, the sequence recognition specificity of individual SH3 domains is poorly understood. We have employed a novel modification of phage display involving biased libraries to identify peptide ligands of the Src, Fyn, Lyn, PI3K and Abl SH3 domains. With biased libraries, we probed SH3 recognition over a 12 amino acid window. The Src SH3 domain prefers the sequence XXXRPLPPLPXP, Fyn prefers XXXRPLPP(I/L)PXX, Lyn prefers RXXRPLPPLPXP, PI3K prefers RXXRPLPPLPP while the Abl SH3 domain selects phage containing the sequence PPPYPPPP(I/V)PXX. We have also analysed the binding properties of Abl and Src SH3 ligands. We find that although the phage-displayed Abl and Src SH3 ligands are proline rich, they are distinct. In surface plasmon resonance binding assays, these SH3 domains displayed highly selective binding to their cognate ligands when the sequences were displayed on the surface of the phage or as synthetic peptides. The selection of these high affinity SH3 peptide ligands provides valuable information on the recognition motifs of SH3 domains, serve as new tools to interfere with the cellular functions of SH3 domain-mediated processes and form the basis for the design of SH3-specific inhibitors of disease pathways.     

Differential activation and redistribution of c-Src and Fyn in platelets, assessed by MoAb specific for C-terminal tyrosine-dephosphorylated c-Src and Fyn

Tyrosine kinases, c-Src and Fyn, in their active form, have their C-terminal tyrosine residue dephosphorylated. In this study, we used clone 28, a monoclonal antibody (MoAb) that recognizes dephosphorylated C-terminal tyrosine of c-Src and Fyn, to investigate the mode of activation and mobilization of these kinases. Independently of integrin alphaIIbbeta3 signaling, the Fyn activity increased by 8.3-fold 5 s after stimulation with 20 microM TRAP (thrombin receptor agonist peptide), while that of c-Src increased only by 2.9-fold 15 s after stimulation. Both c-Src and Fyn translocated to the Triton-insoluble cytoskeletal fraction in an aggregation-dependent manner. Five minutes after TRAP-stimulation, 85% of Fyn translocated to the cytoskeleton, while only about 20% of c-Src was recovered in this fraction. The Triton-insoluble fraction was further fractionated by RIPA (radioimmunoprecipitation assay) buffer containing 0.1% SDS. While active c-Src was predominantly present in the Triton-insoluble/RIPA-insoluble fraction, clone 28-negative c-Src was present in the Triton-insoluble/RIPA-soluble fraction. On the other hand, Fyn was present only in the Triton-insoluble/RIPA-insoluble fraction. These findings suggest that the mode of activation and redistribution into the cytoskeleton differs between c-Src and Fyn, and that clone 28 provides a useful tool for investigating the activation and mobilization of Src family tyrosine kinases.     

Lupus-like kidney disease in mice deficient in the Src family tyrosine kinases Lyn and Fyn

Systemic lupus erythematosus (SLE) is an autoimmune disease whose cause is poorly understood. Mice rendered deficient in specific genes have served as useful animal models in deciphering the genetic control of the disease [1]. We [2] and others [3, 4] previously demonstrated that mice deficient in the Src family tyrosine kinase Lyn developed a mild lupus-like disease with high survival rates. During the course of investigating the functional interaction of Src family kinases, we generated a mouse strain deficient in both Lyn and Fyn. The double-mutant mice died at relatively young ages and developed a severe lupus-like kidney disease. Unlike the double-mutant mice, single mutants deficient in either Lyn or Fyn lived longer and had distinct subsets of the symptoms found in the former. Lyn deficiency led to high levels of autoantibody production and glomerulonephritis, as previously reported [2--4], whereas loss of Fyn contributed to proteinuria by a B and T lymphocyte-independent mechanism. Our data suggest that the severe kidney disease in the double-mutant mice results from a combination of immunological and kidney-intrinsic defects. This new animal model may be informative about the causes of human SLE.     

Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains.

Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.     

Molecular cloning of smg p21B and identification of smg p21 purified from bovine brain and human platelets as smg p21B

We have previously purified smg p21 from bovine brain membranes and isolated its cDNA from a bovine brain cDNA library. In the present studies, we have performed extensive screening of the bovine brain cDNA library with the cloned smg p21 cDNA as a probe and isolated another cDNA encoding a protein highly homologous to smg p21. The proteins encoded by the previously and newly isolated cDNAs are designated as smg p21A and -B, respectively. Since the partial amino acid sequences determined previously from the smg p21 purified from bovine brain were identical with the common amino acid sequences between smg p21A and -B, we have further sequenced smg p21 and identified it as smg p21B. We have also further sequenced the smg p21 purified from human platelet membranes and identified it as smg p21B. Amino acid sequence analysis indicates that smg p21A is identical with the rap1A and Krev-1 proteins and smg p21B is identical with the rap1B protein.     

Agonist-induced association of the p21ras GTPase-activating protein with phosphatidylinositol 3-kinase.

The signal transduction properties of the 21-kDa GTP-binding proteins, encoded by the ras genes, are only partly known. In a recent report, we demonstrated that the signaling pathway of p21ras, like that of several growth factors, is closely associated with phosphatidylinositol 3-kinase (PtdIns 3-kinase) activity. We showed that insulin-like growth factor-1 (IGF-1) and insulin increased the phosphatidylinositol 3-kinase activity in immunoprecipitates obtained with anti-phosphotyrosine and anti-ras antibodies in Ha-ras-transformed epithelial cells. Several findings in this previous study suggested that an additional protein was likely to be associated with the PtdIns 3-kinase. The suggestion that p21ras GTPase-activating protein (GAP) acts not only as a regulator of p21ras activity but also as a direct downstream target in the signaling pathway of p21ras led us to investigate the possible association of PtdIns 3-kinase with GAP. The stimulation of Ha-ras-transformed epithelial cells with IGF-1 caused an increased association of PtdIns 3-kinase activity with GAP, as seen by immunoprecipitation with anti-p21ras and anti-GAP antibodies. The 85-kDa regulatory subunit of PtdIns 3-kinase was present in immunoprecipitates obtained with antibodies against GAP and p21ras of IGF-1 stimulated cells. These data suggest that GAP acts as a downstream target for p21ras via its association with PtdIns 3-kinase.     

Evidence for coupling of phospatidic acid formation and calcium influx in thrombin-activated human platelets

The time-sequential relationship between Ca²⁺ flux, phospholipid metabolism and platelet activation have been examined. Thrombin-activation caused a marked enhancement in ⁴⁵Ca²⁺ influx and a decrease in extracellular Ca²⁺ concentration measured by murexide dye, which occurred in parallel with the conversion of 1,2-diacylglycerol (DG) to phosphatidic acid (PA). The incorporated ⁴⁵Ca²⁺ was located mainly in cytosolic fraction. The influx of Ca²⁺ was observed to commence prior to the onset of lysophospholipids formation and subsequent liberation of arachidonic acid. These data provide evidence which indicates a coupling between the rapid PI-turnover and the active Ca²⁺ influx, in which phosphatidic acid (PA) may serve as a Ca²⁺ ionophore.     

B-cell antigen receptor motifs have redundant signalling capabilities and bind the tyrosine kinases PTK72, Lyn and Fyn

BACKGROUND: The 13 cell antigen receptor (BCR) is a multimeric protein complex consisting of an antigen recognition structure (membrane immunoglobulin) and two associated proteins, lg-alpha and Ig-beta It has been proposed that signalling through the BCR involves Ig-alpha and Ig-beta. Both of these proteins contain within their cytoplasmic domains an amino-acid motif that is present in a number of immune recognition receptors, including the BCR, T-cell antigen receptor and Fc receptor complexes. This motif, termed the antigen-receptor homology motif (ARH1), appears to have signal transduction ability. RESULTS: We now show that the presence of cytoplasmic regions containing the ARM motif from either Ig-alpha or Ig-beta is sufficient to confer signalling capability on an otherwise non-functional fusion protein. Both Ig-alpha- and Ig-beta-containing chimeras induced, in an apparently redundant fashion, signalling events seen upon membrane immunoglobulin crosslinking, including tyrosine phosphorylation of particular proteins, phosphoinositicle breakdown and calcium mobilization. Furthermore, crosslinking of the chimeras resulted in tyrosine phosphorylation of the Ig-alpha and Tg-beta tails and their association with the tyrosine kinases PTK72, p53/56(lyn) and p59(fyn). CONCLUSIONS: These observations indicate that Ig-alpha and Ig-beta are responsible for coupling membrane immunoglobulin to intracellular signalling components. Moreover, they demonstrate that a number of tyrosine kinases associate directly with the cytoplasmic domains of both Ig-alpha and Ig-beta. Stimulation of the chimeras, which results in tyrosine phosphorylation of the ig-alpha and Ig-beta tails, is a prerequisite for some of these associations. The implications of these findings for the mechanism by which the BCR initiates the signalling reactions are discussed.     

Fyn/Yes and non-canonical Wnt signalling converge on RhoA in vertebrate gastrulation cell movements

Convergent extension (CE) cell movements during gastrulation mediate extension of the anterior-posterior body axis of vertebrate embryos. Non-canonical Wnt5 and Wnt11 signalling is essential for normal CE movements in vertebrate gastrulation. Here, we show that morpholino (MO)-mediated double knock-down of the Fyn and Yes tyrosine kinases in zebrafish embryos impaired normal CE cell movements, resembling the silberblick and pipetail mutants, caused by mutations in wnt11 and wnt5, respectively. Co-injection of Fyn/Yes- and Wnt11- or Wnt5-MO was synergistic, but wnt11 or wnt5 RNA did not rescue the Fyn/Yes knockdown or vice versa. Remarkably, active RhoA rescued the Fyn/Yes knockdown as well as the Wnt11 knockdown, indicating that Fyn/Yes and Wnt11 signalling converged on RhoA. Our results show that Fyn and Yes act together with non-canonical Wnt signalling via RhoA in CE cell movements during gastrulation.