p60src and p72Syk Tyrosine Kinases Activate Both Lw and CD44 to Mediate Sickle Red Blood Cell Adhesion to Endothelium

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3223-3223
Author(s):  
Edward Chiou ◽  
Rahima Zennadi
Keyword(s):  
Shear Stress ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Conflicts Of Interest ◽  
Selective Inhibitor ◽  
Intermittent Flow ◽  
Αvβ3 Integrin ◽  
Src Tyrosine Kinase

Abstract Abstract 3223 The vaso-occlusive process in patients with sickle cell disease is complex and involves interactions between hemoglobin S red blood cells (SSRBCs) and vascular endothelial cells (ECs). However, the pathophysiologic triggers inducing SSRBC adhesion and vaso-occlusion are poorly understood. Elucidation of these mechanisms at the molecular level would allow development of new preventive and treatment strategies to abrogate vaso-occlusive events. Because non-receptor tyrosine kinases in non-erythroid cells are known to mediate cell-cell interactions, we have now investigated the role of non-receptor tyrosine kinases in modulating SSRBC adherence to cultured ECs, identified the kinases involved, and defined both the receptors on activated SSRBCs and the ligands on ECs involved in these interactions. Less than 20% of non-treated SSRBCs were able to adhere to non-stimulated ECs in vitro in intermittent flow conditions at a shear stress of 2 dynes/cm2. However, treatment of SSRBCs with sodium orthovanadate (Na3VO4), a potent broad spectrum inhibitor of protein tyrosine phosphatases, was able to significantly up-regulate RBC adhesion to cultured ECs by 4.6±1-fold over baseline adhesion in vitro. Na3VO4 in contrast, completely failed to increase adhesion of normal RBCs to cultured ECs. The increased SSRBC adhesion induced by Na3VO4 was significantly inhibited with piceatannol (p<0.001), which inhibits non-receptor tyrosine kinases p72Syk and p56Lck, and PP1 (p<0.001), an src-selective tyrosine p56Lck, p59Fyn and p60src kinase inhibitor. However, genistein, a broad range inhibitor of tyrosine kinases, and damnacanthal, a highly potent and selective inhibitor of p56Lck, completely failed to inhibit the effect of Na3VO4 on SSRBC adhesion. In addition, phenylarsine oxide, which specifically activates p56Lck tyrosine kinase, did not affect SSRBC adhesion to ECs. Together, these data suggest that SSRBC adhesion to ECs can be upregulated via activation of at least p72Syk and p60src tyrosine kinases, but not via the src-tyrosine kinase p56Lck. We further confirmed that increased SSRBC adhesion by Na3VO4 treatment is indeed a result of the activation of p72Syk and p60src tyrosine kinases. Slight basal phosphorylation of p72Syk and p60src was detected in all SSRBC samples tested. However, Na3VO4-treatment of SSRBCs significantly enhanced phosphorylation of both p60src and p72Syk kinases over basal phosphorylation (p<0.05), and this effect induced by Na3VO4 was completely blocked with the src-selective inhibitor PP1 or PP2, and piceatannol, respectively, suggesting that p72Syk and p60src undergo enhanced activation and are involved in up-regulation of SSRBC adhesion to endothelium. Moreover, at a shear stress of 2 dynes/cm2, both anti-LW (ICAM-4) and anti-CD44 antibodies individually inhibited adhesion of activated SSRBCs to ECs, by up to 83% and 78%, respectively. Recombinant soluble LW (srLW) and CD44 (srCD44) proteins also completely abolished adhesion of activated SSRBCs to ECs, identifying LW and CD44 as the RBC receptors involved in this interaction. The EC ligands for activated SSRBCs were also identified using antibody inhibition studies, as the αvβ3 integrin, a ligand previously shown to be important to SSRBC adhesion to activated ECs in vivo, and the endothelial CD44. These data demonstrate that activation of p72Syk and p60src-dependent pathways can act to activate LW- and CD44-mediated SSRBC adhesion to endothelial αvβ3 integrin and CD44, respectively, suggesting that this mechanism may initiate or exacerbate vaso-occlusion by increasing SSRBC adhesion to the endothelium. RBC CD44 is also the first adhesion molecule shown to be involved in SSRBC adhesion to endothelium. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The v-Src SH3 domain binds phosphatidylinositol 3'-kinase.

1993 ◽  
Vol 13 (9) ◽  
pp. 5225-5232 ◽  
Author(s):  
X Liu ◽  
L E Marengere ◽  
C A Koch ◽  
T Pawson
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Chicken Embryo ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Src Tyrosine Kinase ◽  
Amino Terminal ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Fibroblasts transformed by v-src or by related oncogenes encoding activated tyrosine kinases contain elevated levels of polyphosphoinositides with phosphate at the D-3 position of the inositol ring, as a result of the activation of phosphatidylinositol (PI) 3'-kinase. v-src-transformed cells also contain increased levels of PI 3'-kinase activity immunoprecipitable with anti-phosphotyrosine antibodies; furthermore, PI 3'-kinase can be detected in association with the v-Src tyrosine kinase. To identify regions of v-Src that can interact with PI 3'-kinase, the v-Src SH2 and SH3 domains were expressed in bacteria and incubated with lysates of normal chicken embryo fibroblasts. In vitro, the v-Src SH3 domain, but not the SH2 domain, bound PI 3'-kinase in lysates of uninfected chicken embryo fibroblasts. Substitutions of two highly conserved SH3 residues implicated in ligand binding abolished the ability of the v-Src SH3 domain to associate with PI 3'-kinase. Furthermore, the v-Src SH3 domain bound in vitro to the amino-terminal region of the p85 alpha subunit of PI 3'-kinase. These results suggest that the v-Src SH3 domain may mediate an interaction between the v-Src tyrosine kinase and PI 3'-kinase, by direct binding to p85.

Tyrosine 842 Residue in the Activation Loop of FLT3-ITD Is Indespensible for Oncogenic Transformation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1561-1561
Author(s):  
Julhash U. Kazi ◽  
Lars Rönnstrand
Keyword(s):  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Conflicts Of Interest ◽  
Point Mutations ◽  
Cellular Transformation ◽  
Receptor Activation ◽  
Tumor Formation ◽  
Activation Loop ◽  
Tyrosine Residues

Abstract The receptor tyrosine kinase FLT3 is mutated in around 30% of acute myeloid leukemia patients. The so-called internal tandem duplication (ITD) mutation in the juxtamembrane domain is the most common type of mutation in FLT3. Other oncogenic mutations include point mutations in the kinase domain. One of the hallmark of receptor activation is phosphorylation on several tyrosine residues in the receptor. Oncogenic FLT3 mutants display constitutive activity leading to aberrant cell proliferation and survival. Phosphorylation on several critical tyrosine residues is known to be essential for FLT3 signaling. The tyrosine residue Y842, which is located in the activation loop of FLT3, and is well conserved in all receptor tyrosine kinases. It has been demonstrated that tyrosine phosphorylation of the activation loop is essential for the activity of some but not all receptor tyrosine kinases. In this report we show that phosphorylation on Y842 residue is dispensable for receptor activity and stability. However, Y842 plays critical roles in oncogenic FLT3-ITD mediated transformation. We have shown that a Y-to-F mutation (Y842F) results in reduced cell viability and proliferation. This mutation also led to dramatic decrease in in vitro colony formation in semi-solid medium. Furthermore, cells carrying the Y842F mutant of FLT3-ITD displayed significant delay in tumor formation in nude mice. Gene expression analysis, using microarrays, showed that mutation of Y842 causes suppression of anti-apoptotic genes. Finally, we showed that phosphorylated Y842 is a binding site for SHP2 and mutation of this site results in impaired activation of signaling through the RAS/ERK pathway. Collectively, our data suggest that Y842 in FLT3 plays an important role in FLT3-mediated RAS/ERK signaling and cellular transformation. Disclosures No relevant conflicts of interest to declare.

The v-Src SH3 domain binds phosphatidylinositol 3'-kinase

1993 ◽  
Vol 13 (9) ◽  
pp. 5225-5232
Author(s):  
X Liu ◽  
L E Marengere ◽  
C A Koch ◽  
T Pawson
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Chicken Embryo ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Src Tyrosine Kinase ◽  
Amino Terminal ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Fibroblasts transformed by v-src or by related oncogenes encoding activated tyrosine kinases contain elevated levels of polyphosphoinositides with phosphate at the D-3 position of the inositol ring, as a result of the activation of phosphatidylinositol (PI) 3'-kinase. v-src-transformed cells also contain increased levels of PI 3'-kinase activity immunoprecipitable with anti-phosphotyrosine antibodies; furthermore, PI 3'-kinase can be detected in association with the v-Src tyrosine kinase. To identify regions of v-Src that can interact with PI 3'-kinase, the v-Src SH2 and SH3 domains were expressed in bacteria and incubated with lysates of normal chicken embryo fibroblasts. In vitro, the v-Src SH3 domain, but not the SH2 domain, bound PI 3'-kinase in lysates of uninfected chicken embryo fibroblasts. Substitutions of two highly conserved SH3 residues implicated in ligand binding abolished the ability of the v-Src SH3 domain to associate with PI 3'-kinase. Furthermore, the v-Src SH3 domain bound in vitro to the amino-terminal region of the p85 alpha subunit of PI 3'-kinase. These results suggest that the v-Src SH3 domain may mediate an interaction between the v-Src tyrosine kinase and PI 3'-kinase, by direct binding to p85.

ACh and adenosine activate PI3-kinase in rabbit hearts through transactivation of receptor tyrosine kinases

2002 ◽  
Vol 283 (6) ◽  
pp. H2322-H2330 ◽  
Author(s):  
Thomas Krieg ◽  
Qining Qin ◽  
Elizabeth C. McIntosh ◽  
Michael V. Cohen ◽  
James M. Downey
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Growth Factor Receptor ◽  
Pi3 Kinase ◽  
Dependent Manner ◽  
Src Tyrosine Kinase ◽  
Akt Phosphorylation ◽  
Downstream Target

Adenosine and acetylcholine (ACh) trigger preconditioning through different signaling pathways. We tested whether either could activate myocardial phosphatidylinositol 3-kinase (PI3-kinase), a putative signaling protein in ischemic preconditioning. We used phosphorylation of Akt, a downstream target of PI3-kinase, as a reporter. Exposure of isolated rabbit hearts to ACh increased Akt phosphorylation 2.62 ± 0.33 fold ( P = 0.001), whereas adenosine caused a significantly smaller increase (1.52 ± 0.08 fold). ACh-induced activation of Akt was abolished by the tyrosine kinase blocker genistein indicating at least one tyrosine kinase between the muscarinic receptor and Akt. ACh-induced Akt activation was blocked by the Src tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine (PP2) and by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478), an epidermal growth factor receptor (EGFR) inhibitor, suggesting phosphorylation of a receptor tyrosine kinase in an Src tyrosine kinase-dependent manner. ACh caused tyrosine phosphorylation of the EGFR, which could be blocked by PP2, thus supporting this receptor hypothesis. AG-1478 failed to block the cardioprotection of ACh, however, suggesting that other receptor tyrosine kinases might be involved. Therefore, Gi protein-coupled receptors can activate PI3-kinase/Akt through transactivation of receptor tyrosine kinases in an Src tyrosine kinase-dependent manner.

scholarly journals The role of tyrosine kinases in the pathogenesis of idiopathic pulmonary fibrosis

2015 ◽  
Vol 45 (5) ◽  
pp. 1426-1433 ◽  
Author(s):  
Friedrich Grimminger ◽  
Andreas Günther ◽  
Carlo Vancheri
Keyword(s):  
Growth Factor ◽  
Animal Models ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Growth Factor Receptor ◽  
Phase Iii

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a median survival time from diagnosis of 2–3 years. Although the pathogenic pathways have not been fully elucidated, IPF is believed to be caused by persistent epithelial injury in genetically susceptible individuals. Tyrosine kinases are involved in a range of signalling pathways that are essential for cellular homeostasis. However, there is substantial evidence from in vitro studies and animal models that receptor tyrosine kinases, such as the platelet-derived growth factor receptor, vascular endothelial growth factor receptor and fibroblast growth factor receptor, and non-receptor tyrosine kinases, such as the Src family, play critical roles in the pathogenesis of pulmonary fibrosis. For example, the expression and release of tyrosine kinases are altered in patients with IPF, while specific tyrosine kinases stimulate the proliferation of lung fibroblasts in vitro. Agents that inhibit tyrosine kinases have shown anti-fibrotic and anti-inflammatory effects in animal models of pulmonary fibrosis. Recently, the tyrosine kinase inhibitor nintedanib has shown positive results in two phase III trials in patients with IPF. Here, we summarise the evidence for involvement of specific tyrosine kinases in the pathogenesis of IPF and the development of tyrosine kinase inhibitors as treatments for IPF.

scholarly journals Thrombin and thrombin receptor agonist peptide induce tyrosine phosphorylation and tyrosine kinases in the platelet cytoskeleton. Translocation of pp60c-src and integrin αIIbβ3 (glycoprotein IIb/IIIa) is not required for aggregation, but is dependent on formation of large aggregate structures

1993 ◽  
Vol 294 (1) ◽  
pp. 253-260 ◽  
Author(s):  
K M Pumiglia ◽  
M B Feinstein
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Receptor Agonist ◽  
Kinase Activity ◽  
Shear Stresses ◽  
Tyrosine Kinase Activity ◽  
Src Tyrosine Kinase ◽  
Agonist Peptide

The maximal aggregation of platelets induced by alpha-thrombin or by the receptor agonist peptide thrombin-(42-47)-peptide (TRP42/47) rapidly increased the pp60c-src associated with the cytoskeleton fraction. There was good correlation between the tyrosine kinase activity and the mass of pp60c-src. Tyrosine kinase activity associated with the cytoskeleton phosphorylated several endogenous cytoskeleton-associated proteins, as revealed by immunoblotting with anti-phosphotyrosine antibody following incubation with ATP in vitro. However, with the exception of pp60c-src, few phosphotyrosine-containing proteins were retained in the cytoskeleton in intact platelets when compared with total platelet lysates. Translocation of pp60c-src to the cytoskeleton induced by alpha-thrombin and TRP42/47 is dependent on glycoprotein IIb/IIIa (GPIIb/IIIa)-fibrinogen-mediated aggregation, but does not occur when ristocetin/von Willebrand factor produces GPIb-mediated platelet aggregation. The translocation of GPIIb/IIIa and pp60c-src to the cytoskeleton is not necessary for aggregation, as it is not seen when clearly visible small to moderate-sized aggregates are initially formed after exposure to thrombin. The linkage of these proteins to the cytoskeleton occurs only after later extensive formation of large aggregates. Translocation of GPIIa/IIIa to the cytoskeleton is not sufficient for the cytoskeletal association of pp60c-src, as the former occurs independently in platelets stimulated with concanavalin A in the absence of aggregation. Linkage of the integrin GPIIb/IIIa and pp60c-src to the internal cytoskeleton structure, and the corresponding tyrosine phosphorylation of certain proteins upon formation of large aggregates, may be an example of mechanochemical transduction by integrin receptors and may represent a structure with the requisite tensile strength to stabilize large platelet aggregates against high shear stresses.

scholarly journals In vitro guidance of retinal ganglion cell axons by RAGS, a 25 kDa tectal protein related to ligands for Eph receptor tyrosine kinases

Cell ◽  
1995 ◽  
Vol 82 (3) ◽  
pp. 359-370 ◽  
Author(s):  
Uwe Drescher ◽  
Claus Kremoser ◽  
Claudia Handwerker ◽  
Jürgen Löschinger ◽  
Masaharu Noda ◽  
...  
Keyword(s):  
Ganglion Cell ◽  
Retinal Ganglion Cell ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Retinal Ganglion ◽  
Eph Receptor

scholarly journals G-CSF receptor activation of the Src kinase Lyn is mediated by Gab2 recruitment of the Shp2 phosphatase

Blood ◽  
2011 ◽  
Vol 118 (4) ◽  
pp. 1077-1086 ◽  
Author(s):  
Muneyoshi Futami ◽  
Quan-sheng Zhu ◽  
Zakary L. Whichard ◽  
Ling Xia ◽  
Yuehai Ke ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Src Kinase ◽  
Receptor Activation ◽  
Phosphorylation Sites ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Stimulating Factor ◽  
In Cells

Abstract Src activation involves the coordinated regulation of positive and negative tyrosine phosphorylation sites. The mechanism whereby receptor tyrosine kinases, cytokine receptors, and integrins activate Src is not known. Here, we demonstrate that granulocyte colony-stimulating factor (G-CSF) activates Lyn, the predominant Src kinase in myeloid cells, through Gab2-mediated recruitment of Shp2. After G-CSF stimulation, Lyn dynamically associates with Gab2 in a spatiotemporal manner. The dephosphorylation of phospho-Lyn Tyr507 was abrogated in Shp2-deficient cells transfected with the G-CSF receptor but intact in cells expressing phosphatase-defective Shp2. Auto-phosphorylation of Lyn Tyr396 was impaired in cells treated with Gab2 siRNA. The constitutively activated Shp2E76A directed the dephosphorylation of phospho-Lyn Tyr507 in vitro. Tyr507 did not undergo dephosphorylation in G-CSF–stimulated cells expressing a mutant Gab2 unable to bind Shp2. We propose that Gab2 forms a complex with Lyn and after G-CSF stimulation, Gab2 recruits Shp2, which dephosphorylates phospho-Lyn Tyr507, leading to Lyn activation.

Signaling in new directions

1995 ◽  
Vol 73 (3-4) ◽  
pp. 133-136 ◽  
Author(s):  
Haleh Vahidi Samiei
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Tyrosine Kinase ◽  
Map Kinase ◽  
Receptor Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Molecular Level ◽  
Clear Cut ◽  
New Directions

Many laboratories, using a variety of organisms, have contributed to deciphering the identity and the order of the components leading from ligand-bound receptor tyrosine kinases to various intracellular events, including changes in gene expression. The gaps have only been filled recently. This minireview summarizes the findings and points out the degree of conservation of the same pathway in distant organisms, both at the molecular level and in terms of the consecutive steps. The review also looks at points at which this pathway might be diverging and points onto which other pathways might be converging. These interactions are not always clear cut, and understanding them will be the challenge for the future.Key words: signal transduction, receptor tyrosine kinase, RAS, RAF, MAP kinase.

Eph/Ephrin signalling in skeletal muscle development and regeneration

2014 ◽  
Author(s):  
◽  
Danny A. Stark
Keyword(s):  
Skeletal Muscle ◽  
Satellite Cells ◽  
Tyrosine Kinases ◽  
Muscle Development ◽  
Receptor Tyrosine Kinases ◽  
Repulsive Interaction ◽  
Type I ◽  
Ephrin Ligands

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Skeletal muscle can be isolated into 642 individual muscles and makes up to one third to one half of the mass of the human body. Each of these muscles is specified and patterned prenatally and after birth they will increase in size and take on characteristics suited to each muscle's unique function. To make the muscles functional, each muscle cell must be innervated by a motor neuron, which will also affect the characteristics of the mature muscle. In a healthy adult, muscles will maintain their specialized pattern and function during physiological homeostasis, and will also recapitulate them if the integrity or health of the muscle is disrupted. This repair and regeneration is dependent satellite cells, the skeletal muscle stem cells. In this dissertation, we study a family of receptor tyrosine kinases, Ephs, and their juxtacrine ephrin ligands in the context of skeletal muscle specification and regeneration. First, using a classical ephrin 'stripe' assay to test for contact-mediated repulsion, we found that satellite cells respond to a subset of ephrins with repulsive motility in vitro and that these forward signals through Ephs also promote patterning of differentiating myotubes parallel to ephrin stripes. This pattering can be replicated in a heterologous in vivo system (the hindbrain of the developing quail, where neural crest cells migrate in streams to the branchial arches, and in the forelimb of the developing quail, where presumptive limb myoblasts emigrate from the somite). Second, we present evidence that specific pairwise interactions between Eph receptor tyrosine kinases and ephrin ligands are required to ensure appropriate muscle innervation when it is originally set during postnatal development and when it is recapitulated after muscle or nerve trauma during adulthood. We show expression of a single ephrin, ephrin-A3, exclusively on type I (slow) myofibers shortly after birth, while its receptor EphA8 is only localized to fast motor endplates, suggesting a functional repulsive interaction for motor axon guidance and/or synaptogenesis. Adult EFNA3-/- mutant mice show a significant loss of slow myofibers, while misexpression of ephrin-A3 on fast myofibers results in a switch from a fast fiber type to slow in the context of sciatic nerve injury and regrowth. Third, we show that EphA7 is expressed on satellite cell derived myocytes in vitro, and marks both myocytes and regenerating myofibers in vivo. In the EPHA7 knockout mouse, we find a regeneration defect in a barium chloride injury model starting 3 days post injection in vivo, and that cultured mutant satellite cells are slow to differentiate and divide. Finally, we present other potential Ephs and ephrins that may affect skeletal muscle, such as EphB1 that is expressed on all MyHC-IIb fibers and a subset of MyHC-IIx fibers, and we show a multitude of Ephs and ephrins at the neuromuscular junction that appear to localize on specific myofibers and at different areas of the synapse. We propose that Eph/ephrin signaling, though well studied in development, continues to be important in regulating post natal development, regeneration, and homeostasis of skeletal muscle.

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