PDGF alpha-receptor mediated cellular responses are not dependent on Src family kinases in endothelial cells

1998 ◽  
Vol 111 (5) ◽  
pp. 607-614
Author(s):  
R. Hooshmand-Rad ◽  
K. Yokote ◽  
C.H. Heldin ◽  
L. Claesson-Welsh
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Kinases ◽  
Src Family Kinases ◽  
Wild Type ◽  
Actin Reorganization ◽  
Aortic Endothelial Cells ◽  
Mutant Receptor ◽  
Juxtamembrane Region ◽  
Src Family Tyrosine Kinases ◽  
Type Receptor

Two novel autophosphorylation sites in the juxtamembrane region of the PDGF alpha-receptor, Tyr-572 and Tyr-574, were identified. A Y572/574F mutant PDGF (alpha)-receptor was generated and stably expressed in porcine aortic endothelial cells. In contrast to the wild-type receptor, the mutant receptor was unable to associate with or activate Src family tyrosine kinases. Tyrosine phosphorylated synthetic peptides representing the juxtamembrane sequence of the receptor dose-dependently inhibited the binding of Src family tyrosine kinases to the autophosphorylated PDGF alpha-receptor. The mutant receptor showed similar PDGF-induced kinase activity and ability to mediate mitogenicity, actin reorganization and chemotaxis as the wild-type receptor. Thus activation of Src family kinases by the PDGF alpha-receptor is not essential for PDGF-induced mitogenicity or actin reorganization.

scholarly journals Sensitization of Human Aortic Endothelial Cells to Lipopolysaccharide via Regulation of Toll-Like Receptor 4 by Bacterial Fimbria-Dependent Invasion

2005 ◽  
Vol 73 (12) ◽  
pp. 8050-8059 ◽  
Author(s):  
Hiromichi Yumoto ◽  
Hsin-Hua Chou ◽  
Yusuke Takahashi ◽  
Michael Davey ◽  
Frank C. Gibson ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Periodontal Disease ◽  
Deficient Mutant ◽  
Wild Type ◽  
Microbial Infection ◽  
Monocyte Chemoattractant Protein 1 ◽  
Aortic Endothelial Cells ◽  
E Coli ◽  
Human Aortic Endothelial Cells ◽  
Aortic Endothelial

ABSTRACT Toll-like receptors (TLRs) are differentially up-regulated in response to microbial infection and chronic inflammatory diseases such as atherosclerosis. Epidemiological data support the idea that periodontal disease may be a risk factor for acceleration of atherosclerosis. Porphyromonas gingivalis, the etiological agent of periodontal disease, invades endothelium, has been detected in human atheromatous tissue, and accelerates atheroma formation in apolipoprotein E−/− mice with concurrent induction of TLRs in the aorta. As endothelial cells can present antigen via TLRs and play an important role in the development of atherosclerosis, we examined TLR expression in human aortic endothelial cells (HAEC) cultured with wild-type P. gingivalis, a fimbria-deficient mutant, and purified antigens. We observed increased TLR expression in HAEC infected with wild-type P. gingivalis by fluorescence-activated cell sorter, but not with noninvasive, fimbria-deficient mutant or purified P. gingivalis antigens. Following a wild-type P. gingivalis challenge, functional TLR2 and TLR4 activation was assessed by subsequent stimulation with TLR agonists Staphylococcus aureus lipoteichoic acid (SLTA; TLR2 ligand) and Escherichia coli lipopolysaccharide (LPS; TLR4 ligand). Unchallenged HAEC failed to elicit monocyte chemoattractant protein 1 (MCP-1) in response to LPS or SLTA but did so when cultured with wild-type P. gingivalis. P. gingivalis-induced TLR2 and -4 expression on HAEC functionally reacted to SLTA and E. coli LPS as measured by a further increase in MCP-1 production. Furthermore, MCP-1 expression elicited by E. coli LPS was inhibitable with TLR4-specific antibody and polymyxin B. These results indicate that invasive P. gingivalis stimulates TLR expression on the surface of endothelium and these primed cells respond to defined TLR-specific ligands.

Deletion of the kinase insert sequence of the platelet-derived growth factor beta-receptor affects receptor kinase activity and signal transduction

1990 ◽  
Vol 10 (2) ◽  
pp. 801-809
Author(s):  
L Severinsson ◽  
B Ek ◽  
K Mellström ◽  
L Claesson-Welsh ◽  
C H Heldin
Keyword(s):  
Growth Factor ◽  
Protein Tyrosine Kinase ◽  
Catalytic Efficiency ◽  
Kinase Domain ◽  
Platelet Derived Growth Factor ◽  
Tyrosine Kinase Domain ◽  
Wild Type ◽  
Mutant Receptor ◽  
Beta Receptor ◽  
Type Receptor

A characteristic feature of the platelet-derived growth factor (PDGF) beta-receptor is the presence of an insert sequence in the protein tyrosine kinase domain. A receptor mutant which lacks the entire insert of 98 amino acids was expressed in CHO cells, and its functional characteristics were compared with those of the wild-type receptor. The mutant receptor bound PDGF-BB with high affinity and mediated internalization and degradation of the ligand with efficiency similar to that of the wild-type receptor but did not transduce a mitogenic signal. It was found to display a decreased autophosphorylation after ligand stimulation and had a decreased ability to phosphorylate exogenous substrates; phosphofructokinase was not phosphorylated at all, whereas a peptide substrate was phosphorylated, albeit at a lower rate compared with phosphorylation by the wild-type receptor. Furthermore, the mutant receptor did not mediate actin reorganization but mediated an increase in c-fos expression. The data indicate that the insert in the kinase domain of the PDGF beta-receptor is important for the substrate specificity or catalytic efficiency of the kinase; the deletion of the insert interferes with the transduction of some, but not all, of the signals that arise after activation of the receptor.

The Novel Inhibitor PLX3397 Effectively Inhibits FLT3-Mutant AML,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3632-3632 ◽  
Author(s):  
Elizabeth Burton ◽  
Bernice Wong ◽  
Jiazhong Zhang ◽  
Brian West ◽  
Gideon Bollag ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Tumor Regression ◽  
Wild Type ◽  
Gut Peptides ◽  
Cellular Assays ◽  
Juxtamembrane Region ◽  
Clear Dose Response ◽  
Major Subset ◽  
Subcutaneous Xenograft

Abstract Abstract 3632 Mutations of FLT3 (FMS-like tyrosine kinase 3) are frequently present in acute myeloid leukemias (AML) and associated with poor prognosis. Such mutations alter the conformation and constitutively activate the FLT3 tyrosine kinase. PLX3397 is a novel, orally active, selective small molecule inhibitor of the FLT3-mutant as well as FMS and KIT kinases. The high selectivity of PLX3397 has been demonstrated in biochemical assays against a panel of over 200 recombinant kinases. FLT3, FMS and KIT are significantly inhibited at low nanomolar concentrations. The majority of kinases screened were not inhibited by PLX3397 (IC50=>10 μM) and the only other kinase with significant sub-micromolar inhibition was KDR. PLX3397 exhibits favorable pharmaceutical properties and demonstrated minimal off-target activity when tested in a broad array of 71 targets in 8 families (Neurotransmitter-related, Steroids, Ion Channels, Nitric Oxide, Prostaglandins, Growth Factors, Brain/Gut Peptides, and Enzymes). In cellular assays PLX3397 effectively inhibited ligand-stimulated autophosphorylation of the endogenous receptor tyrosine kinases FMS (IC50=20 nM) and KIT (IC50=120 nM), but not ligand-stimulated wild-type FLT3 (IC50=1.7 μM). In SEMK2 cells, which over-express wild type FLT3, PLX3397 inhibited FLT3 autophosphorylation with an IC50 of 240 nM. The phosphorylation of the activated FLT3-ITD protein in MV-4-11 and MOLM-14 cells harboring mutations (FLT3-ITD) of the juxtamembrane region of FLT3 was efficiently inhibited (IC50 26 nM and 30 nM respectively), indicating a possible application in this major subset of FLT3 mutated AML. In parallel with inhibition of FLT3-mutant autophosphorylation, the phosphorylation of known downstream effectors (STAT5, AKT, MEK, and ERK) is also inhibited. However, no inhibition was observed for KDR phosphorylation. PLX3397 potently inhibited of mutant-FLT3 driven proliferation in both MV-4-11 and MOLM-14 AML cells in culture (IC50=100–200 nM), but was less effective against HL60 cells expressing wild-type FLT3. MV-4-11 cells grown as subcutaneous xenograft tumors in mice were also highly sensitive to PLX3397 dosed orally at 10 or 30 mg/kg qd, with tumor regression observed at the higher dose. Primary samples collected from AML patients at relapse were tested in culture. While no significant effects were seen in FLT3 wild type samples below 1 μM, a clear dose response to PLX3397 was observed in samples with FLT3-ITD mutations. Because the estimated protein binding of PLX3397 in plasma is >99%, we also tested the drug against the MV-4-11 cell line in plasma and found the IC50 for inhibition of autophosphorylation to be 2.8 μM. In humans trough levels of PLX3397 (10 μM) capable of inhibiting FLT3-mutants can be safely achieved. A Phase I/II study is planned to evaluate the safety and explore the efficacy of PLX3397 in patients with FLT3-mutant- AML. Disclosures: Burton: Plexxikon Inc.: Employment. Wong:Plexxikon Inc.: Employment. Zhang:Plexxikon Inc.: Employment. West:Plexxikon Inc.: Employment. Bollag:Plexxikon Inc.: Employment. Habets:Plexxikon Inc.: Employment. Nguyen:Plexxikon Inc.: Employment. Levis:Plexxikon Inc.: Honoraria.

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.

scholarly journals Fcγ Receptor–Mediated Phagocytosis in Macrophages Lacking the Src Family Tyrosine Kinases Hck, Fgr, and Lyn

2000 ◽  
Vol 191 (4) ◽  
pp. 669-682 ◽  
Author(s):  
Cheryl J. Fitzer-Attas ◽  
Malcolm Lowry ◽  
Mary T. Crowley ◽  
Alexander J. Finn ◽  
Fanying Meng ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Actin Polymerization ◽  
Src Family Kinases ◽  
Phosphatidylinositol 3 Kinase ◽  
Functional Responses ◽  
Fcγ Receptors ◽  
Triple Mutant ◽  
Extracellular Signal ◽  
Src Family Tyrosine Kinases ◽  
Ig G

Macrophage Fcγ receptors (FcγRs) mediate the uptake and destruction of antibody-coated viruses, bacteria, and parasites. We examined FcγR signaling and phagocytic function in bone marrow–derived macrophages from mutant mice lacking the major Src family kinases expressed in these cells, Hck, Fgr, and Lyn. Many FcγR-induced functional responses and signaling events were diminished or delayed in these macrophages, including immunoglobulin (Ig)G-coated erythrocyte phagocytosis, respiratory burst, actin cup formation, and activation of Syk, phosphatidylinositol 3-kinase, and extracellular signal–regulated kinases 1 and 2. Significant reduction of IgG-dependent phagocytosis was not seen in hck−/−fgr−/− or lyn−/− cells, although the single mutant lyn−/− macrophages did manifest signaling defects. Thus, Src family kinases clearly have roles in two events leading to FcγR-mediated phagocytosis, one involving initiation of actin polymerization and the second involving activation of Syk and subsequent internalization. Since FcγR-mediated phagocytosis did occur at modest levels in a delayed fashion in triple mutant macrophages, these Src family kinases are not absolutely required for uptake of IgG-opsonized particles.

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.

scholarly journals Growth Hormone-induced JAK2 Signaling and GH Receptor Down-regulation: Role of GH Receptor Intracellular Domain Tyrosine Residues

Endocrinology ◽  
2012 ◽  
Vol 153 (5) ◽  
pp. 2311-2322 ◽  
Author(s):  
Luqin Deng ◽  
Jing Jiang ◽  
Stuart J. Frank
Keyword(s):  
Tyrosine Phosphorylation ◽  
Metabolic Effects ◽  
Wild Type ◽  
Down Regulation ◽  
Mutant Receptor ◽  
Tyrosine Residues ◽  
Gh Receptor ◽  
Tyrosine Kinase 2 ◽  
Activation Mechanisms ◽  
Type Receptor

GH receptor (GHR) mediates important somatogenic and metabolic effects of GH. A thorough understanding of GH action requires intimate knowledge of GHR activation mechanisms, as well as determinants of GH-induced receptor down-regulation. We previously demonstrated that a GHR mutant in which all intracellular tyrosine residues were changed to phenylalanine was defective in its ability to activate signal transducer and activator of transcription (STAT)5 and deficient in GH-induced down-regulation, but able to allow GH-induced Janus family of tyrosine kinase 2 (JAK2) activation. We now further characterize the signaling and trafficking characteristics of this receptor mutant. We find that the mutant receptor's extracellular domain conformation and its interaction with GH are indistinguishable from the wild-type receptor. Yet the mutant differs greatly from the wild-type in that GH-induced JAK2 activation is augmented and far more persistent in cells bearing the mutant receptor. Notably, unlike STAT5 tyrosine phosphorylation, GH-induced STAT1 tyrosine phosphorylation is retained and augmented in mutant GHR-expressing cells. The defective receptor down-regulation and persistent JAK2 activation of the mutant receptor do not depend on the sustained presence of GH or on the cell's ability to carry out new protein synthesis. Mutant receptors that exhibit resistance to GH-induced down-regulation are enriched in the disulfide-linked form of the receptor, which reflects the receptor's activated conformation. Furthermore, acute GH-induced internalization, a proximal step in down-regulation, is markedly impaired in the mutant receptor compared to the wild-type receptor. These findings are discussed in the context of determinants and mechanisms of regulation of GHR down-regulation.

The D816V Mutation of C-Kit Circumvents a Requirement for Src Family Kinases in C-Kit Mediated Signal Tranduction and Transformation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2849-2849
Author(s):  
Jianmin Sun ◽  
Malin Pedersen ◽  
Lars Rönnstrand
Keyword(s):  
Substrate Specificity ◽  
Cell Survival ◽  
Tyrosine Kinases ◽  
Src Family Kinases ◽  
Kinase Assay ◽  
Common Mutation ◽  
Wild Type ◽  
Constitutive Activation ◽  
Type C ◽  
Kit D816v

Abstract The receptor tyrosine kinase c-Kit plays a critical role in hematopoiesis and gain-of-function mutations of the receptor are frequently seen in several malignancies, including acute myeloid leukemia (AML), mastocytoma and sinonasal NK/T cell lymphomas. The most common mutation of c-Kit in these disorders is a substitution of the aspartic acid residue in position 816 to a valine (D816V), leading to constitutive activation of the receptor. In this study we aimed to investigate the role of Src family kinases in c-Kit/D816V signaling. Src family kinases are necessary for the phosphorylation of wild-type c-Kit as well as for activation of downstream signaling pathways including receptor ubiquitination and the Ras/MEK/Erk pathway. Tyrosine 568 of c-Kit, that is important for Src activation by wild-type c-Kit, was mutated to phenylalanine in both wild-type c-Kit and c-Kit/D816V and stably transfected into the hematopoietic cell line Ba/F3. Our data demonstrate that, unlike wild-type c-Kit, the phosphorylation of c-Kit/D816V is not dependent on Src family kinases. In addition we found that neither receptor ubiquitination nor Erk activation by c-Kit/D816V required activation of Src family kinases. In vitro kinase assay using synthetic peptides revealed that c-Kit/D816V had an altered substrate specificity resembling Src and Abl tyrosine kinases. The serine/threonine kinases Akt and Erk play important roles in cell survival and proliferation mediated by receptor tyrosine kinases. We could show constitutive activation of both PI3-kinase pathway and Erk in Ba/F3 cells expressing c-Kit/D816V, although ligand stimulation induced even stronger activation. We further present evidence that, in contrast to wild-type c-Kit, Src family kinases are dispensible for c-Kit/D816V cell survival. Taken together, we demonstrate that the signal transduction pathways mediated by c-Kit/D816V are markedly different from those activated by wild-type c-Kit and that altered substrate substrate specificity of c-Kit circumvents a need for Src family kinases in signaling of growth and survival, thereby contributing to the transforming potential of c-Kit/D816V.

scholarly journals Src family kinases mediate neutrophil adhesion to adherent platelets

Blood ◽  
2006 ◽  
Vol 109 (6) ◽  
pp. 2461-2469 ◽  
Author(s):  
Virgilio Evangelista ◽  
Zehra Pamuklar ◽  
Antonio Piccoli ◽  
Stefano Manarini ◽  
Giuseppe Dell'Elba ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Src Family Kinases ◽  
Β2 Integrin ◽  
Contact Sites ◽  
Downstream Effector ◽  
Activated Platelets ◽  
Genetic Deletion ◽  
Src Family Tyrosine Kinases

Abstract Polymorphonuclear leukocyte (PMN)–platelet interactions at sites of vascular damage contribute to local and systemic inflammation. We sought to determine the role of “outside-in” signaling by Src-family tyrosine kinases (SFKs) in the regulation of αMβ2-integrin–dependent PMN recruitment by activated platelets under (patho)physiologic conditions. Activation-dependent epitopes in β2 integrin were exposed at the contact sites between PMNs and platelets and were abolished by SFK inhibitors. PMNs from αMβ2−/−, hck−/−fgr−/−, and hck−/−fgr−/−lyn−/− mice had an impaired capacity to adhere with activated platelets in suspension. Phosphorylation of Pyk2 accompanied PMN adhesion to platelets and was blocked by inhibition as well as by genetic deletion of αMβ2 integrin and SFKs. A Pyk2 inhibitor reduced platelet-PMN adhesion, indicating that Pyk2 may be a downstream effector of SFKs. Analysis of PMN-platelet interactions under flow revealed that SFK signaling was required for αMβ2-mediated shear-resistant adhesion of PMNs to adherent platelets, but was dispensable for P-selectin–PSGL-1–mediated recruitment and rolling. Finally, SFK activity was required to support PMN accumulation along adherent platelets at the site of vascular injury, in vivo. These results definitely establish a role for SFKs in PMN recruitment by activated platelets and suggest novel targets to disrupt the pathophysiologic consequences of platelet-leukocyte interactions in vascular disease.

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