GCN2 kinase activation by ATP-competitive kinase inhibitors

The effect of the macrophage- and T-lymphocyte-derived cytokine oncostatin M (OSM) on endothelin-1 (ET-1) production in cultured human umbilical cord vein endothelial cells (HUVEC) was studied. OSM (2.5–10 ng/ml) stimulated ET-1 production and the expression of preproendothelin-1 mRNA. The stimulatory effect of OSM was reversed by anti-interleukin (IL)-6 IgG (33 μg/ml). IL-6 (10 ng/ml) was shown to stimulate ET-1 production. The tyrosine kinase inhibitors herbimycin (250–500 ng/ml) and genistein (1–4 μg/ml) suppressed basal ET-1 production and reversed the stimulatory effect of OSM, whereas daidzein (1–8 μg/ml), a less active analog of genistein, had no effect on basal ET-1 production and only partly reversed the stimulatory effect of OSM. The phorbol ester phorbol 12-myristate 13-acetate (PMA) inhibited ET-1 production. Downregulation of protein kinase C (PKC) with PMA (1 μM) preincubation potentiated OSM-induced ET-1 production. In summary, OSM stimulated ET-1 production in cultured HUVEC. The stimulation was probably mediated by IL-6. Furthermore, the present data suggest that tyrosine kinase activation was involved in ET-1 stimulation and that PKC activation leads to suppression of basal and OSM-stimulated ET-1 production.

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Transmission of growth cone traction force through apCAM–cytoskeletal linkages is regulated by Src family tyrosine kinase activity

The Journal of Cell Biology ◽

10.1083/jcb.200107063 ◽

2001 ◽

Vol 155 (3) ◽

pp. 427-438 ◽

Cited By ~ 91

Author(s):

Daniel M. Suter ◽

Paul Forscher

Keyword(s):

Tyrosine Kinase ◽

Growth Cone ◽

Kinase Activity ◽

Kinase Inhibitors ◽

Traction Force ◽

Tyrosine Kinase Activity ◽

Cellular Mechanisms ◽

Traction Forces ◽

Kinase Activation ◽

Neuronal Growth Cone

Tyrosine kinase activity is known to be important in neuronal growth cone guidance. However, underlying cellular mechanisms are largely unclear. Here, we report how Src family tyrosine kinase activity controls apCAM-mediated growth cone steering by regulating the transmission of traction forces through receptor–cytoskeletal linkages. Increased levels of tyrosine phosphorylation were detected at sites where beads coated with apCAM ligands were physically restrained to induce growth cone steering, but not at unrestrained bead binding sites. Interestingly, the rate and level of phosphotyrosine buildup near restrained beads were decreased by the myosin inhibitor 2,3-butanedione-2-monoxime, suggesting that tension promotes tyrosine kinase activation. While not affecting retrograde F-actin flow rates, genistein and the Src family selective tyrosine kinase inhibitors PP1 and PP2 strongly reduced the growth cone's ability to apply traction forces through apCAM–cytoskeletal linkages, assessed using the restrained bead interaction assay. Furthermore, increased levels of an activated Src family kinase were detected at restrained bead sites during growth cone steering events. Our results suggest a mechanism by which growth cones select pathways by sampling both the molecular nature of the substrate and its ability to withstand the application of traction forces.

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Crystal structure of the WD40 domain dimer of LRRK2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1817889116 ◽

2019 ◽

Vol 116 (5) ◽

pp. 1579-1584 ◽

Cited By ~ 15

Author(s):

Pengfei Zhang ◽

Ying Fan ◽

Heng Ru ◽

Li Wang ◽

Venkat Giri Magupalli ◽

...

Keyword(s):

Crystal Structure ◽

Kinase Activity ◽

Kinase Inhibitors ◽

Human Genetics ◽

Kinase Domain ◽

Rab Gtpases ◽

Kinase Activation ◽

Surface Patches ◽

Wd40 Domain ◽

Lrrk2 Kinase

Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein with both a Ras of complex (ROC) domain and a kinase domain (KD) and, therefore, exhibits both GTPase and kinase activities. Human genetics studies have linked LRRK2 as a major genetic contributor to familial and sporadic Parkinson’s disease (PD), a neurodegenerative movement disorder that inflicts millions worldwide. The C-terminal region of LRRK2 is a Trp-Asp-40 (WD40) domain with poorly defined biological functions but has been implicated in microtubule interaction. Here, we present the crystal structure of the WD40 domain of human LRRK2 at 2.6-Å resolution, which reveals a seven-bladed WD40 fold. The structure displays a dimeric assembly in the crystal, which we further confirm by measurements in solution. We find that structure-based and PD-associated disease mutations in the WD40 domain including the common G2385R polymorphism mainly compromise dimer formation. Assessment of full-length LRRK2 kinase activity by measuring phosphorylation of Rab10, a member of the family of Rab GTPases known to be important kinase substrates of LRRK2, shows enhancement of kinase activity by several dimerization-defective mutants including G2385R, although dimerization impairment does not always result in kinase activation. Furthermore, mapping of phylogenetically conserved residues onto the WD40 domain structure reveals surface patches that may be important for additional functions of LRRK2. Collectively, our analyses provide insights for understanding the structures and functions of LRRK2 and suggest the potential utility of LRRK2 kinase inhibitors in treating PD patients with WD40 domain mutations.

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Dasatinib (BMS-354825) is active in Philadelphia chromosome–positive chronic myelogenous leukemia after imatinib and nilotinib (AMN107) therapy failure

Blood ◽

10.1182/blood-2006-07-035493 ◽

2006 ◽

Vol 109 (2) ◽

pp. 497-499 ◽

Cited By ~ 109

Author(s):

Alfonso Quintas-Cardama ◽

Hagop Kantarjian ◽

Dan Jones ◽

Claude Nicaise ◽

Susan O'Brien ◽

...

Keyword(s):

Chronic Myelogenous Leukemia ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Philadelphia Chromosome ◽

Src Kinase ◽

Cytogenetic Response ◽

Myelogenous Leukemia ◽

Imatinib Resistance ◽

Kinase Activation ◽

Complete Hematologic Response

Abstract Developing strategies to counteract imatinib resistance constitutes a challenge in chronic myelogenous leukemia (CML). Therapy with the tyrosine kinase inhibitors nilotinib (AMN107) and dasatinib (BMS-354825) has produced high rates of hematologic and cytogenetic response. Src kinase activation has been linked to Bcr-Abl–mediated leukemogenesis and CML progression. In addition to binding Abl kinase with less stringent conformational requirements than imatinib, dasatinib is a potent Src kinase inhibitor. In the current study, we report on 23 patients with CML (19 of them in accelerated or blastic phases) treated with dasatinib after treatment failure with both imatinib and nilotinib. More than half (13; 57%) of 23 patients responded to dasatinib: 10 (43%) had a complete hematologic response (CHR), including 7 (30%) who had a cytogenetic response (2 complete, 4 partial, and 1 minor). These results suggest that dasatinib may be active in some patients after failure with both imatinib and nilotinib.

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Platelet/polymorphonuclear leukocyte interaction in dynamic conditions: evidence of adhesion cascade and cross talk between P-selectin and the beta 2 integrin CD11b/CD18

Blood ◽

10.1182/blood.v88.11.4183.bloodjournal88114183 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4183-4194 ◽

Cited By ~ 4

Author(s):

V Evangelista ◽

S Manarini ◽

S Rotondo ◽

N Martelli ◽

R Polischuk ◽

...

Keyword(s):

Tyrosine Kinase ◽

Kinase Inhibitors ◽

Inhibitory Antibody ◽

Mixed Cell ◽

Platelet Surface ◽

Kinase Activation ◽

Dynamic Conditions ◽

Activated Platelets ◽

Beta 2 ◽

Adhesion Of Platelets

Adhesion between platelets and polymorphonuclear leukocytes (PMN) is a key event in thrombosis and inflammation. Double color fluorescence- activated cell sorter (FACS) analysis was used to determine the extent and kinetics of adhesion of thrombin-activated platelets to resting or activated PMN when mixed cell populations were incubated in dynamic conditions. Activated platelets bound very rapidly to PMN. Mixed cell conjugates reached a maximum at 1 minute and were reversible within 10 minutes. Platelet/PMN adhesion required both Ca2+ and Mg2+ and was markedly increased by the presence of Mn2+. The latter made mixed cell conjugates stable up to 10 minutes. Adhesion of platelets required metabolic activity of PMN and was abolished by tyrosine kinase inhibitors. Furthermore, adhesion of platelets to PMN resulted in binding of a monoclonal antibody (MoAb 24) known as beta 2 integrins “activation reporter.” When PMN were activated by exogenous stimuli, the adhesion of platelets was markedly increased: fMLP induced a rapid and transient effect, while PMA resulted in a slower, but stable, increase in mixed conjugates formation. The hypothesis that activated PMN beta 2 integrins are able to bind a counter-receptor on platelets was directly demonstrated by the increase of mixed cell conjugates following PMN treatment with KIM127 and KIM185, two anti-CD18 antibodies able to induce the active conformation of beta 2 integrins. Consistently, two other anti-CD18, as well as an anti-CD11b inhibitory antibody abolished platelet/PMN adhesion. PMN beta 2 integrin activation was not the only mechanism for activated platelet/PMN adhesion to occur: indeed, this phenomenon could also be inhibited by two anti-P-selectin antibodies. Resting platelets did not adhere to resting PMN, but markedly adhered to fMLP-or PMA-activated PMN. Resting platelet/fMLP-activated PMN adhesion was abolished by anti-CD18 antibodies, but not by anti-P-selectin antibodies. In conclusion, activated platelet/PMN interaction can be modeled as an adhesion cascade involving a P-selectin-dependent recognition step and a functional signal. The latter proceeds through tyrosine kinase activation and enables a beta 2 integrin-dependent adhesion to a not yet identified counter-receptor constitutively expressed on platelet surface.

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Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1507329112 ◽

2015 ◽

Vol 112 (22) ◽

pp. 6955-6960 ◽

Cited By ~ 45

Author(s):

Hsin-Yung Yen ◽

Ying-Chih Liu ◽

Nai-Yu Chen ◽

Chia-Feng Tsai ◽

Yi-Ting Wang ◽

...

Keyword(s):

Lung Cancer ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Cancer Progression ◽

Conformational Changes ◽

Kinase Inhibitors ◽

Growth Factor Receptor ◽

Kinase Activation ◽

Downstream Signaling ◽

Egfr Mutant

Epidermal growth factor receptor (EGFR) is a heavily glycosylated transmembrane receptor tyrosine kinase. Upon EGF-binding, EGFR undergoes conformational changes to dimerize, resulting in kinase activation and autophosphorylation and downstream signaling. Tyrosine kinase inhibitors (TKIs) have been used to treat lung cancer by inhibiting EGFR phosphorylation. Previously, we demonstrated that EGFR sialylation suppresses its dimerization and phosphorylation. In this report, we further investigated the effect of sialylation on the phosphorylation profile of EGFR in TKI-sensitive and TKI-resistant cells. Sialylation was induced in cancer progression to inhibit the association of EGFR with EGF and the subsequent autophosphorylation. In the absence of EGF the TKI-resistant EGFR mutant (L858R/T790M) had a higher degree of sialylation and phosphorylation at Y1068, Y1086, and Y1173 than the TKI-sensitive EGFR. In addition, although sialylation in the TKI-resistant mutants suppresses EGFR tyrosine phosphorylation, with the most significant effect on the Y1173 site, the sialylation effect is not strong enough to stop cancer progression by inhibiting the phosphorylation of these three sites. These findings were supported further by the observation that the L858R/T790M EGFR mutant, when treated with sialidase or sialyltransferase inhibitor, showed an increase in tyrosine phosphorylation, and the sensitivity of the corresponding resistant lung cancer cells to gefitinib was reduced by desialylation and was enhanced by sialylation.

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Coronary arteriolar flow-induced vasodilation signals through tyrosine kinase

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1996.270.6.h1878 ◽

1996 ◽

Vol 270 (6) ◽

pp. H1878-H1884 ◽

Cited By ~ 11

Author(s):

J. M. Muller ◽

M. J. Davis ◽

W. M. Chilian

Keyword(s):

Substance P ◽

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Kinase Inhibitors ◽

Fluorescein Isothiocyanate ◽

Intraluminal Pressure ◽

Kinase Activation ◽

Genistein Treatment ◽

Dependent Flow ◽

Coronary Arterioles

Coronary arterioles demonstrate flow-dependent vasodilation that is mediated by endothelial release of nitric oxide. The signaling mechanisms for this response remain unknown. Because tyrosine kinases are an enzyme family linked to many signaling pathways, including some for mechanosensitive transduction, we hypothesized that tyrosine kinase activation is a critical step in flow-induced vasodilation. To test this hypothesis, coronary arterioles were isolated, cannulated with micropipettes, and perfused by two independent reservoir systems. Intraluminal pressure was set at 60 cmH2O, and flow was generated by changing the heights of the reservoirs in equal and opposite directions, thus establishing a pressure difference across the arteriole without altering intraluminal pressure. Vasodilatory responses to intraluminal flow and substance P (1 x 10(-12) to 1 x 10(-7) M) were evaluated before and after intraluminal application of the tyrosine kinase inhibitors genistein (5 microM) and piceatannol (10 microM). Exposure to these inhibitors did not alter spontaneous tone. Substance P caused dose-dependent vasodilation that was not affected by genistein or piceatannol. Increases in intraluminal flow (generated by pressure differences ranging from 4 to 60 cmH2O) elicited graded increases in diameter. Both genistein and piceatannol inhibited the vasodilatory responses to flow. Treatment with daidzein, an inactive analogue of genistein, had no effect on either the flow-induced responses or substance P-induced vasodilation. To further confirm that tyrosine kinase activation is involved in flow-induced vasodilation, vessels were exposed to flow in the absence or presence of genistein and subsequently stained with a fluorescein isothiocyanate-labeled phosphotyrosine antibody. Exposure to flow significantly increased fluorescence of endothelial cells. Genistein treatment reversed the flow-induced increase in tyrosine phosphorylation. These results indicate that endothelium-dependent, flow-induced vasodilation in isolated porcine coronary arterioles is accompanied by an increase in tyrosine kinase activity. We conclude that endothelium-dependent, nitroxidergic, flow-induced vasodilation is mediated, at least in part, by a signaling pathway involving a tyrosine kinase.

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Platelet/polymorphonuclear leukocyte interaction in dynamic conditions: evidence of adhesion cascade and cross talk between P-selectin and the beta 2 integrin CD11b/CD18

Blood ◽

10.1182/blood.v88.11.4183.4183 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4183-4194 ◽

Cited By ~ 161

Author(s):

V Evangelista ◽

S Manarini ◽

S Rotondo ◽

N Martelli ◽

R Polischuk ◽

...

Keyword(s):

Tyrosine Kinase ◽

Kinase Inhibitors ◽

Inhibitory Antibody ◽

Mixed Cell ◽

Platelet Surface ◽

Kinase Activation ◽

Dynamic Conditions ◽

Activated Platelets ◽

Beta 2 ◽

Adhesion Of Platelets

Abstract Adhesion between platelets and polymorphonuclear leukocytes (PMN) is a key event in thrombosis and inflammation. Double color fluorescence- activated cell sorter (FACS) analysis was used to determine the extent and kinetics of adhesion of thrombin-activated platelets to resting or activated PMN when mixed cell populations were incubated in dynamic conditions. Activated platelets bound very rapidly to PMN. Mixed cell conjugates reached a maximum at 1 minute and were reversible within 10 minutes. Platelet/PMN adhesion required both Ca2+ and Mg2+ and was markedly increased by the presence of Mn2+. The latter made mixed cell conjugates stable up to 10 minutes. Adhesion of platelets required metabolic activity of PMN and was abolished by tyrosine kinase inhibitors. Furthermore, adhesion of platelets to PMN resulted in binding of a monoclonal antibody (MoAb 24) known as beta 2 integrins “activation reporter.” When PMN were activated by exogenous stimuli, the adhesion of platelets was markedly increased: fMLP induced a rapid and transient effect, while PMA resulted in a slower, but stable, increase in mixed conjugates formation. The hypothesis that activated PMN beta 2 integrins are able to bind a counter-receptor on platelets was directly demonstrated by the increase of mixed cell conjugates following PMN treatment with KIM127 and KIM185, two anti-CD18 antibodies able to induce the active conformation of beta 2 integrins. Consistently, two other anti-CD18, as well as an anti-CD11b inhibitory antibody abolished platelet/PMN adhesion. PMN beta 2 integrin activation was not the only mechanism for activated platelet/PMN adhesion to occur: indeed, this phenomenon could also be inhibited by two anti-P-selectin antibodies. Resting platelets did not adhere to resting PMN, but markedly adhered to fMLP-or PMA-activated PMN. Resting platelet/fMLP-activated PMN adhesion was abolished by anti-CD18 antibodies, but not by anti-P-selectin antibodies. In conclusion, activated platelet/PMN interaction can be modeled as an adhesion cascade involving a P-selectin-dependent recognition step and a functional signal. The latter proceeds through tyrosine kinase activation and enables a beta 2 integrin-dependent adhesion to a not yet identified counter-receptor constitutively expressed on platelet surface.

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The UDP-sugar-sensing P2Y14receptor promotes Rho-mediated signaling and chemotaxis in human neutrophils

AJP Cell Physiology ◽

10.1152/ajpcell.00138.2012 ◽

2012 ◽

Vol 303 (5) ◽

pp. C490-C498 ◽

Cited By ~ 37

Author(s):

Juliana I. Sesma ◽

Silvia M. Kreda ◽

Natacha Steinckwich-Besancon ◽

Hong Dang ◽

Rafael García-Mata ◽

...

Keyword(s):

Cell Migration ◽

Kinase Inhibitors ◽

Rho Kinase ◽

Human Neutrophils ◽

Functional Responses ◽

Nucleotide Sugar ◽

Kinase Activation ◽

Hl60 Cells ◽

Rho Kinase Inhibitors ◽

Neutrophil Differentiation

The Gi-coupled P2Y14receptor (P2Y14-R) is potently activated by UDP-sugars and UDP. Although P2Y14-R mRNA is prominently expressed in circulating neutrophils, the signaling pathways and functional responses associated with this receptor are undefined. In this study, we illustrate that incubation of isolated human neutrophils with UDP-glucose resulted in cytoskeleton rearrangement, change of cell shape, and enhanced cell migration. We also demonstrate that UDP-glucose promotes rapid, robust, and concentration-dependent activation of RhoA in these cells. Ecto-nucleotidases expressed on neutrophils rapidly hydrolyzed extracellular ATP, but incubation with UDP-glucose for up to 1 h resulted in negligible metabolism of the nucleotide-sugar. HL60 human promyelocytic leukemia cells do not express the P2Y14-R, but neutrophil differentiation of HL60 cells with DMSO resulted in markedly enhanced P2Y14-R expression. Accordingly, UDP-glucose, UDP-galactose, and UDP- N-acetylglucosamine promoted Rho activation in differentiated but not in undifferentiated HL60 cells. Stable expression of recombinant human P2Y14-R conferred UDP-sugar-promoted responses to undifferentiated HL60 cells. UDP-glucose-promoted RhoA activation also was accompanied by enhanced cell migration in differentiated HL60 cells, and these responses were blocked by Rho kinase inhibitors. These results support the notion that UDP-glucose is a stable and potent proinflammatory mediator that promotes P2Y14-R-mediated neutrophil motility via Rho/Rho kinase activation.

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