scholarly journals Platelet αIIb-β3 integrin engagement induces the tyrosine phosphorylation of Cbl and its association with phosphoinositide 3-kinase and Syk

2000 ◽  
Vol 351 (3) ◽  
pp. 669-676 ◽  
Author(s):  
Abdelhafid SACI ◽  
Francine RENDU ◽  
Christilla BACHELOT-LOZA
Keyword(s):  
Signal Transduction ◽  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Src Family Kinases ◽  
Β3 Integrin ◽  
Phosphoinositide 3 Kinase ◽  
Src Family Tyrosine Kinases

Agonist-induced platelet activation triggers ‘inside-out’signalling which activates αIIb-β3, the most abundant integrin in platelet membranes. The engagement of activated αIIb-β3 integrin by linking fibrinogen is necessary for platelet aggregation, and this induces subsequent outside-in signalling, which enhances platelet activation. Here we studied the involvement of Cbl during αIIb-β3-integrin-mediated signal transduction. During thrombin-induced platelet activation, Cbl was tyrosine phosphorylated, and phosphoinositide 3-kinase (PI 3-kinase) activity measured in Cbl immunoprecipitates was increased. Both Cbl phosphorylation and its association with PI 3-kinase were dependent on αIIb-β3 engagement by linking fibrinogen. The P256 and anti-LIBS6 (ligand-induced binding site 6) antibodies, which activate platelets directly through αIIb-β3, induced Cbl phosphorylation and increased the PI 3-kinase activity associated with Cbl. Both thrombin and antibodies to αIIb-β3 induced association of Cbl with the tyrosine kinase, Syk. Experiments performed with inhibitors of tyrosine kinases indicated that both Src-family kinases and Syk contribute to phosphorylation of Cbl and its consequent association with PI 3-kinase. The results show that, following integrin αIIb-β3 engagement, Cbl is tyrosine phosphorylated, recruits PI 3-kinase to this integrin signalling pathway and possibly enhances PI 3-kinase activity, downstream of Src-family tyrosine kinases and Syk activation.

Streptococcus sanguis-induced platelet activation involves two waves of tyrosine phosphorylation mediated by FcγRIIA and αIIbβ3

2005 ◽  
Vol 93 (05) ◽  
pp. 932-939 ◽  
Author(s):  
Caroline Pampolina ◽  
Archibald McNicol
Keyword(s):  
Signal Transduction ◽  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Lag Phase ◽  
Dependent Manner ◽  
Protein Tyrosine ◽  
Streptococcus Sanguis

SummaryThe low-affinity IgG receptor, FcγRIIA, has been implicated in Streptococcus sanguis-induced platelet aggregation. Therefore, it is likely that signal transduction is at least partly mediated by FcγRIIA activation and a tyrosine kinase-dependent pathway. In this study the signal transduction mechanisms associated with platelet activation in response to the oral bacterium, S. sanguis were characterised. In the presence of IgG, S. sanguis strain 2017–78 caused the tyrosine phosphorylation of FcγRIIA 30s following stimulation, which led to the phosphorylation of Syk, LAT, and PLCγ2. These early events were dependent on Src family kinases but independent of either TxA2 or the engagement of the αIIbβ3 integrin. During the lag phase prior to platelet aggregation, FcγRIIA, Syk, LAT, and PLCγ2 were each dephosphorylated, but were re-phosphorylated as aggregation occurred. Platelet stimulation by 2017–78 also induced the tyrosine phosphorylation of PECAM-1, an ITIM-containing receptor that recruits protein tyrosine phosphatases. PECAM-1 co-precipitated with the protein tyrosine phosphatase SHP-1 in the lag phase. SHP-1 was also maximally tyrosine phosphorylated during this phase, suggesting a possible role for SHP-1 in the observed dephosphorylation events. As aggregation occurred, SHP-1 was dephosphorylated, while FcγRIIA, Syk, LAT, and PLCγ2 were rephosphorylated in an RGDS-sensitive, and therefore αIIbβ3-dependent, manner. Additionally, TxA2 release, 5-hydro-xytryptamine secretion and phosphatidic acid formation were all blocked by RGDS. Aspirin also abolished these events, but only partially inhibited αIIbβ3-mediated re-phosphorylation. Therefore, S.sanguis-bound IgG cross links FcγRIIA and initiates a signaling pathway that is down-regulated by PECAM-1-bound SHP-1. Subsequent engagement of αIIbβ3 leads to SHP-1 dephosphorylation permiting a second wave of signaling leading to TxA2 release and consequent platelet aggregation.

scholarly journals p42 mitogen-activated protein kinase and p90 ribosomal S6 kinase are selectively phosphorylated and activated during thrombin-induced platelet activation and aggregation.

1994 ◽  
Vol 14 (1) ◽  
pp. 463-472 ◽  
Author(s):  
J Papkoff ◽  
R H Chen ◽  
J Blenis ◽  
J Forsman
Keyword(s):  
Signal Transduction ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Immune Complex ◽  
Platelet Activation ◽  
Kinase Activity ◽  
Mitogen Activated Protein Kinase ◽  
S6 Kinase ◽  
Mitogen Activated Protein ◽  
Ribosomal S6 Kinase

Human platelets provide an excellent model system for the study of phosphorylation events during signal transduction and cell adhesion. Platelets are terminally differentiated cells that exhibit rapid phosphorylation of many proteins upon agonist-induced activation and aggregation. We have sought to identify the kinases as well as the phosphorylated substrates that participate in thrombin-induced signal transduction and platelet aggregation. In this study, we have identified two forms of mitogen-activated protein kinase (MAPK), p42mapk and p44mapk, in platelets. The data demonstrate that p42mapk but not p44mapk becomes phosphorylated on serine, threonine, and tyrosine during platelet activation. Immune complex kinase assays, gel renaturation assays, and a direct assay for MAPK activity in platelet extracts all support the conclusion that p42mapk but not p44mapk shows increased kinase activity during platelet activation. The activation of p42mapk, independently of p44mapk, in platelets is unique since in other systems, both kinases are coactivated by a variety of stimuli. We also show that platelets express p90rsk, a ribosomal S6 kinase that has previously been characterized as a substrate for MAPK. p90rsk is phosphorylated on serine in resting platelets, and this phosphorylation is enhanced upon thrombin-induced platelet activation. Immune complex kinase assays demonstrate that the activity of p90rsk is markedly increased during platelet activation. Another ribosomal S6 protein kinase, p70S6K, is expressed by platelets but shows no change in kinase activity upon platelet activation with thrombin. Finally, we show that the increased phosphorylation and activity of both p42mapk and p90rsk does not require integrin-mediated platelet aggregation. Since platelets are nonproliferative cells, the signal transduction pathways that include p42mapk and p90rsk cannot lead to a mitogenic signal and instead may regulate cytoskeletal or secretory changes during platelet activation.

scholarly journals Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1.

1995 ◽  
Vol 15 (1) ◽  
pp. 186-197 ◽  
Author(s):  
S Richard ◽  
D Yu ◽  
K J Blumer ◽  
D Hausladen ◽  
M W Olszowy ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Src Family Kinases ◽  
Src Family Kinase ◽  
Sh3 Domains ◽  
Sh2 Domains ◽  
Src Family Tyrosine Kinases

src family tyrosine kinases contain two noncatalytic domains termed src homology 3 (SH3) and SH2 domains. Although several other signal transduction molecules also contain tandemly occurring SH3 and SH2 domains, the function of these closely spaced domains is not well understood. To identify the role of the SH3 domains of src family tyrosine kinases, we sought to identify proteins that interacted with this domain. By using the yeast two-hybrid system, we identified p62, a tyrosine-phosphorylated protein that associates with p21ras GTPase-activating protein, as a src family kinase SH3-domain-binding protein. Reconstitution of complexes containing p62 and the src family kinase p59fyn in HeLa cells demonstrated that complex formation resulted in tyrosine phosphorylation of p62 and was mediated by both the SH3 and SH2 domains of p59fyn. The phosphorylation of p62 by p59fyn required an intact SH3 domain, demonstrating that one function of the src family kinase SH3 domains is to bind and present certain substrates to the kinase. As p62 contains at least five SH3-domain-binding motifs and multiple tyrosine phosphorylation sites, p62 may interact with other signalling molecules via SH3 and SH2 domain interactions. Here we show that the SH3 and/or SH2 domains of the signalling proteins Grb2 and phospholipase C gamma-1 can interact with p62 both in vitro and in vivo. Thus, we propose that one function of the tandemly occurring SH3 and SH2 domains of src family kinases is to bind p62, a multifunctional SH3 and SH2 domain adapter protein, linking src family kinases to downstream effector and regulatory molecules.

p42 mitogen-activated protein kinase and p90 ribosomal S6 kinase are selectively phosphorylated and activated during thrombin-induced platelet activation and aggregation

1994 ◽  
Vol 14 (1) ◽  
pp. 463-472
Author(s):  
J Papkoff ◽  
R H Chen ◽  
J Blenis ◽  
J Forsman
Keyword(s):  
Signal Transduction ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Immune Complex ◽  
Platelet Activation ◽  
Kinase Activity ◽  
Mitogen Activated Protein Kinase ◽  
S6 Kinase ◽  
Mitogen Activated Protein ◽  
Ribosomal S6 Kinase

Human platelets provide an excellent model system for the study of phosphorylation events during signal transduction and cell adhesion. Platelets are terminally differentiated cells that exhibit rapid phosphorylation of many proteins upon agonist-induced activation and aggregation. We have sought to identify the kinases as well as the phosphorylated substrates that participate in thrombin-induced signal transduction and platelet aggregation. In this study, we have identified two forms of mitogen-activated protein kinase (MAPK), p42mapk and p44mapk, in platelets. The data demonstrate that p42mapk but not p44mapk becomes phosphorylated on serine, threonine, and tyrosine during platelet activation. Immune complex kinase assays, gel renaturation assays, and a direct assay for MAPK activity in platelet extracts all support the conclusion that p42mapk but not p44mapk shows increased kinase activity during platelet activation. The activation of p42mapk, independently of p44mapk, in platelets is unique since in other systems, both kinases are coactivated by a variety of stimuli. We also show that platelets express p90rsk, a ribosomal S6 kinase that has previously been characterized as a substrate for MAPK. p90rsk is phosphorylated on serine in resting platelets, and this phosphorylation is enhanced upon thrombin-induced platelet activation. Immune complex kinase assays demonstrate that the activity of p90rsk is markedly increased during platelet activation. Another ribosomal S6 protein kinase, p70S6K, is expressed by platelets but shows no change in kinase activity upon platelet activation with thrombin. Finally, we show that the increased phosphorylation and activity of both p42mapk and p90rsk does not require integrin-mediated platelet aggregation. Since platelets are nonproliferative cells, the signal transduction pathways that include p42mapk and p90rsk cannot lead to a mitogenic signal and instead may regulate cytoskeletal or secretory changes during platelet activation.

scholarly journals Regulation of the renal Na-HCO3 cotransporter. XI. Signal transduction underlying CO2stimulation

1999 ◽  
Vol 277 (4) ◽  
pp. F580-F586 ◽  
Author(s):  
Ofelia S. Ruiz ◽  
R. Brooks Robey ◽  
Yi-Yong Qiu ◽  
Long Jiang Wang ◽  
Cheng Jin Li ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Mapk Pathway ◽  
Selective Inhibitor ◽  
Src Family Kinases ◽  
Negative Regulator ◽  
Protein Tyrosine Kinases ◽  
Renal Epithelial Cells ◽  
Stimulation Of

We have previously shown that CO2 stimulation of the renal Na-HCO3 cotransporter (NBC) activity is abrogated by general inhibitors of protein tyrosine kinases. The more selective inhibitor herbimycin also blocked this effect at concentrations known to preferentially inhibit Src family kinases (SFKs). We therefore examined a role for SFKs in CO2-stimulated NBC activity. To this end, we engineered OK cells to express the COOH-terminal Src kinase (Csk), a negative regulator of SFKs. CO2 stimulated NBC activity normally in β-galactosidase-expressing and untransfected control cells. In contrast, Csk-expressing cells had normal baseline NBC activity that was not stimulated by CO2. CO2 stimulation increased both total SFK activity and specific tyrosine phosphorylation of Src. The specific MEK1/2 inhibitor PD-98059 completely inhibited the CO2 stimulation of NBC activity as well as the accompanying phosphorylation and activation of ERK1/2. Our data suggest the involvement of both SFKs, probably Src, and the “classic” MAPK pathway in mediating CO2-stimulated NBC activity in renal epithelial cells.

Impaired integrin-mediated signal transduction, altered cytoskeletal structure and reduced motility in Hck/Fgr deficient macrophages

1999 ◽  
Vol 112 (22) ◽  
pp. 4067-4078 ◽  
Author(s):  
P.W. Suen ◽  
D. Ilic ◽  
E. Caveggion ◽  
G. Berton ◽  
C.H. Damsky ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Subcellular Localization ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Double Mutant ◽  
Src Family Kinases ◽  
Wild Type ◽  
Associated Proteins ◽  
Coated Surfaces ◽  
Mutant Cells

Integrin-mediated adhesion of monocytes and macrophages initiates a signal transduction pathway that leads to actin cytoskeletal reorganization, cell migration and immunologic activation. This signaling pathway is critically dependent on tyrosine kinases. To investigate the role of the Src-family of tyrosine kinases in integrin signal transduction, we have examined the adhesive properties of macrophages isolated from hck-/-fgr-/- double knockout mice which lack two of the three predominant Src-family kinases expressed in myeloid cells. Previous examination of polymorphonuclear leukocytes from these animals indicated that these kinases were critical in initiating the actin cytoskeletal rearrangements that lead to respiratory burst and granule secretion following integrin ligation. Double mutant peritoneal exudate macrophages demonstrated markedly reduced tyrosine phosphorylation responses compared to wild-type cells following plating on fibronectin, collagen or vitronectin-coated surfaces. Tyrosine phosphorylation of several actin-associated proteins (cortactin, paxillin, and tensin), as well as the Syk and Pyk2 tyrosine kinases, were all significantly reduced in double mutant cells. The subcellular localization of focal-adhesion associated proteins was also dramatically altered in mutant macrophages cultured on fibronectin-coated surfaces. In wild-type cells, filamentous actin, paxillin, and talin were concentrated along leading edges of the plasma membrane, suggesting that these proteins contribute to cellular polarization during migration in culture. Double mutant cells failed to show the polarized subcellular localization of these proteins. Likewise, double mutant macrophages failed to form normal filopodia under standard culture conditions. Together, these signaling and cytoskeletal defects may contribute to the reduced motility observed in in vitro assays. These data provide biochemical and morphological evidence that the Src-family kinases Hck and Fgr are required for normal integrin-mediated signal transduction in murine macrophages.

Distinct Domains of αIIbβ3 Support Different Aspects of Outside-In Signal Transduction and Platelet Activation Induced by LSARLAF, an αIIbβ3 Interacting Peptide

2001 ◽  
Vol 86 (09) ◽  
pp. 894-901 ◽  
Author(s):  
Jerry Derrick ◽  
Sanford Shattil ◽  
Mortimer Poncz ◽  
Ralph Gruppo ◽  
Kent Gartner
Keyword(s):  
Signal Transduction ◽  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Cytoplasmic Tail ◽  
Cytoplasmic Domain ◽  
Cytoskeletal Reorganization ◽  
Integrin Β3 ◽  
Platelet Spreading

SummaryThe peptide LSARLAF causes αIIbβ3-dependent platelet activation exemplified by secretion, aggregation, spreading and adhesion on fibrinogen, and tyrosine phosphorylation. αIIbβ3-dependent outside-in signal transduction induced by LSARLAF was investigated in variant thrombasthenic platelets which lack most of the cytoplasmic domain of the integrin β3 subunit (αIIbβ3 Δ724). These studies revealed that only certain aspects of this αIIbβ3-dependent outside-in signaling were affected by the β3 truncation. Specifically, αIIbβ3 724 supported LSARLAF-induced platelet aggregation, agglutination and secretion, but failed to trigger cytoskeletal reorganization and platelet spreading on fibrinogen, despite the fact that PMA-induced non αIIbβ3 mediated signaling caused spreading of these platelets on fibrinogen. Thus, distinct domains of αIIbβ3 are required to support different aspects of LSARLAF-induced platelet activation. Furthermore, these studies suggest that not all αIIbβ3-dependent platelet responses require an intact β3 cytoplasmic tail.

Collagen Induces Normal Signal Transduction in Platelets Deficient in CD36 (Platelet Glycoprotein IV)

1994 ◽  
Vol 71 (03) ◽  
pp. 353-356 ◽  
Author(s):  
James L Daniel ◽  
Carol Dangelmaier ◽  
Robert Strouse ◽  
J Bryan Smith
Keyword(s):  
Signal Transduction ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Tyrosine Kinases ◽  
Platelet Glycoprotein ◽  
Serotonin Secretion

SummaryThe receptor involved in platelet activation by collagen has not been identified. Platelet glycoprotein IV, now known as CD36, has been implicated in interaction with collagen and also been shown to be associated with intracellular tyrosine kinases. In order to investigate the possible role of collagen-mediated signal transduction via CD36, platelets were obtained from a donor that were deficient in CD36. The collagen-induced intracellular mobilization of Ca2+ in the CD36 deficient cells was of the same magnitude as that seen in platelets from normal donors. In addition, serotonin secretion did not appear to be impaired. Tyrosine phosphorylation was also comparable between the CD36-deficient and normal platelets. Thus, it is unlikely that CD36 plays a major role in collagen-dependent platelet signal transduction.

Src-family tyrosine kinases, phosphoinositide 3-kinase and Gab1 regulate extracellular signal-regulated kinase 1 activation induced by the type A endothelin-1 G-protein-coupled receptor

2001 ◽  
Vol 360 (1) ◽  
pp. 77-85 ◽  
Author(s):  
Sandra BISOTTO ◽  
Elizabeth D. FIXMAN
Keyword(s):  
Tyrosine Phosphorylation ◽  
G Protein ◽  
Tyrosine Kinases ◽  
Type A ◽  
Extracellular Signal Regulated Kinase ◽  
Endothelin 1 ◽  
Extracellular Signal ◽  
Phosphoinositide 3 Kinase ◽  
Src Family Tyrosine Kinases ◽  
G Protein Coupled

The multisubstrate docking protein, growth-factor-receptor-bound protein 2-associated binder 1 (Gab1), which is phosphorylated on tyrosine residues following activation of receptor tyrosine kinases and cytokine receptors, regulates cell proliferation, survival and epithelial morphogenesis. Gab1 is also tyrosine phosphorylated following activation of G-protein-coupled receptors (GPCRs) where its function is poorly understood. To elucidate the role of Gab1 in GPCR signalling, we investigated the mechanism by which the type A endothelin-1 (ET-1) GPCR induced tyrosine phosphorylation of Gab1. Tyrosine phosphorylation of Gab1 induced by endothelin-1 was inhibited by PP1, a pharmacological inhibitor of Src-family tyrosine kinases. ET-1-induced Gab1 tyrosine phosphorylation was also inhibited by LY294002, which inhibits phosphoinositide 3-kinase (PI 3-kinase) enzymes. Inhibition of Src-family tyrosine kinases or PI 3-kinase also inhibited ET-1-induced activation of the mitogen activated protein kinase family member, extracellular signal-regulated kinase (ERK) 1. Thus we determined whether Gab1 regulated ET-1-induced ERK1 activation. Overexpression of wild-type Gab1 potentiated ET-1-induced activation of ERK1. Structure–function analyses of Gab1 indicated that mutant forms of Gab1 that do not bind the Src homology (SH) 2 domains of the p85 adapter subunit of PI 3-kinase or the SH2-domain-containing protein tyrosine phosphatase 2 (SHP-2) were impaired in their ability to potentiate ET-1-induced ERK1 activation. Taken together, our data indicate that PI 3-kinase and Src-family tyrosine kinases regulate ET-1-induced Gab1 tyrosine phosphorylation, which, in turn, induces ERK1 activation via PI 3-kinase- and SHP-2-dependent pathways.

scholarly journals Cysteine sulfenylation by CD36 signaling promotes arterial thrombosis in dyslipidemia

2020 ◽  
Vol 4 (18) ◽  
pp. 4494-4507 ◽  
Author(s):  
Moua Yang ◽  
Wei Li ◽  
Calvin Harberg ◽  
Wenjing Chen ◽  
Hong Yue ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Hydrogen Peroxide ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Arterial Thrombosis ◽  
Reactive Oxygen ◽  
Src Family Kinases ◽  
Oxygen Species ◽  
Carbon Nucleophiles ◽  
Platelet Proteins

Abstract Arterial thrombosis in the setting of dyslipidemia promotes clinically significant events, including myocardial infarction and stroke. Oxidized lipids in low-density lipoproteins (oxLDL) are a risk factor for athero-thrombosis and are recognized by platelet scavenger receptor CD36. oxLDL binding to CD36 promotes platelet activation and thrombosis by promoting generation of reactive oxygen species. The downstream signaling events initiated by reactive oxygen species in this setting are poorly understood. In this study, we report that CD36 signaling promotes hydrogen peroxide flux in platelets. Using carbon nucleophiles that selectively and covalently modify cysteine sulfenic acids, we found that hydrogen peroxide generated through CD36 signaling promotes cysteine sulfenylation of platelet proteins. Specifically, cysteines were sulfenylated on Src family kinases, which are signaling transducers that are recruited to CD36 upon recognition of its ligands. Cysteine sulfenylation promoted activation of Src family kinases and was prevented by using a blocking antibody to CD36 or by enzymatic degradation of hydrogen peroxide. CD36-mediated platelet aggregation and procoagulant phosphatidylserine externalization were inhibited in a concentration-dependent manner by a panel of sulfenic acid–selective carbon nucleophiles. At the same concentrations, these probes did not inhibit platelet aggregation induced by the purinergic receptor agonist adenosine diphosphate or the collagen receptor glycoprotein VI agonist collagen-related peptide. Selective modification of cysteine sulfenylation in vivo with a benzothiazine-based nucleophile rescued the enhanced arterial thrombosis seen in dyslipidemic mice back to control levels. These findings suggest that CD36 signaling generates hydrogen peroxide to oxidize cysteines within platelet proteins, including Src family kinases, and lowers the threshold for platelet activation in dyslipidemia.

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