scholarly journals Characterization of the hypertonically induced tyrosine phosphorylation of erythrocyte band 3

1998 ◽  
Vol 335 (2) ◽  
pp. 305-311 ◽  
Author(s):  
Giampaolo MINETTI ◽  
Claudio SEPPI ◽  
Annarita CIANA ◽  
Cesare BALDUINI ◽  
Philip S. LOW ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Volume ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Phosphatase ◽  
Volume Effect ◽  
Band 3 ◽  
Membrane Curvature ◽  
Cell Shrinkage ◽  
Constitutively Active

Human erythrocyte band 3 becomes rapidly phosphorylated on tyrosine residues after exposure of erythrocytes to hypertonic conditions. The driving force for this phosphorylation reaction seems to be a decrease in cell volume, because (1) changes in band 3 phosphotyrosine content accurately track repeated changes in erythrocyte volume through several cycles of swelling and shrinking; (2) the level of band 3 phosphorylation is independent of the osmolyte employed but strongly sensitive to the magnitude of cell shrinkage; and (3) exposure of erythrocytes to hypertonic buffers under conditions in which intracellular osmolarity increases but volume does not change (nystatin-treated cells) does not promote an increase in tyrosine phosphorylation. We hypothesize that shrinkage-induced tyrosine phosphorylation results either from an excluded-volume effect, stemming from an increase in intracellular crowding, or from changes in membrane curvature that accompany the decrease in cell volume. Although the net phosphorylation state of band 3 is shown to be due to a delicate balance between a constitutively active tyrosine phosphatase and constitutively active tyrosine kinase, the increase in phosphorylation during cell shrinkage was demonstrated to derive specifically from an activation of the latter. Further, a peculiar inhibition pattern of the volume-sensitive erythrocyte tyrosine kinase that matched that of p72syk, a tyrosine kinase already known to associate with band 3 in vivo, suggested the involvement of this kinase in the volume-dependent response.

Deoxygenation of sickle cells stimulates Syk tyrosine kinase and inhibits a membrane tyrosine phosphatase

Blood ◽  
2001 ◽  
Vol 98 (10) ◽  
pp. 3121-3127 ◽  
Author(s):  
Patrick Merciris ◽  
Marie-Dominique Hardy-Dessources ◽  
Françoise Giraud
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphatase ◽  
Band 3 ◽  
Red Cells ◽  
Protein Tyrosine ◽  
Sickle Cells ◽  
Cellular Dehydration ◽  
Cell Dehydration

Abstract Polymerization of hemoglobin S in sickle red cells, in deoxygenated conditions, is associated with K+ loss and cellular dehydration. It was previously reported that deoxygenation of sickle cells increases protein tyrosine kinase (PTK) activity and band 3 tyrosine phosphorylation and that PTK inhibitors reduce cell dehydration. Here, the study investigates which PTKs are involved and the mechanism of their activation. Deoxygenation of sickle cells induced a 2-fold increase in Syk activity, measured by autophosphorylation in immune complex assays, but had no effect on Lyn. Syk was not stimulated by deoxygenation of normal red cells, and stimulation was partly reversible on reoxygenation of sickle cells. Syk activation was independent of the increase in intracellular Ca++ and Mg2+ associated with deoxygenation. Lectins that promote glycophorin or band 3 aggregation did not activate Syk. In parallel to Syk stimulation, deoxygenation of sickle cells, but not of normal red cells, decreased the activity of both membrane-associated protein tyrosine phosphatase (PTPs) and membrane protein thiol content. In vitro pretreatment of Syk immune complexes with membrane PTP inhibited Syk autophosphorylation. It is suggested that Syk activation in vivo could be mediated by PTP inhibition, itself resulting from thiol oxidation, as PTPs are known to be inhibited by oxidants. Altogether these data indicate that Syk could be involved in the mechanisms leading to sickle cell dehydration.

Activation of c-Src in cells bearing v-Crk and its suppression by Csk

1992 ◽  
Vol 12 (10) ◽  
pp. 4706-4713
Author(s):  
H Sabe ◽  
M Okada ◽  
H Nakagawa ◽  
H Hanafusa
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Src Kinase ◽  
Cellular Protein ◽  
Morphological Transformation ◽  
Protein Tyrosine ◽  
In Cells

The protein product of the CT10 virus, p47gag-crk (v-Crk), which contains Src homology region 2 (SH2) and 3 (SH3) domains but lacks a kinase domain, is believed to cause an increase in cellular protein tyrosine phosphorylation. A candidate tyrosine kinase, Csk (C-terminal Src kinase), has been implicated in c-Src Tyr-527 phosphorylation, which negatively regulates the protein tyrosine kinase of pp60c-src (c-Src). To investigate how c-Src kinase activity is regulated in vivo, we first looked at whether v-Crk can activate c-Src kinase. We found that cooverexpression of v-Crk and c-Src caused elevation of c-Src kinase activity, resulting in an increase of tyrosine phosphorylation of cellular proteins and morphological transformation of rat 3Y1 fibroblasts. v-Crk and c-Src complexes were not detected, although v-Crk bound to a variety of tyrosine-phosphorylated proteins in cells overexpressing v-Crk and c-Src. Overexpression of Csk in these transformed cells caused reversion to normal phenotypes and also reduced the level of c-Src kinase activity. However, Csk did not cause reversion of cells transformed by v-Src or c-Src527F, in which Tyr-527 was changed to Phe. These results strongly suggest that Csk acts on Tyr-527 of c-Src and suppresses c-Src kinase activity in vivo. Because Csk can suppress transformation by cooverexpression of v-Crk and c-Src, we suggest that v-Crk causes activation of c-Src in vivo by altering the phosphorylation state of Tyr-527.

Abstract 12231: PECAM1 is Essential for Flow-mediated Gab1 Tyrosine Phosphorylation and Signaling in vitro and in vivo

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Suowen Xu ◽  
Marina Koroleva ◽  
Keigi Fujiwara ◽  
Zheng Gen Jin
Keyword(s):  
Tyrosine Phosphorylation ◽  
Wheel Running ◽  
Tyrosine Phosphatase ◽  
Common Mechanism ◽  
Membrane Localization ◽  
No Production ◽  
Voluntary Wheel Running ◽  
Voluntary Wheel

Introduction: Impaired activation of endothelial nitric oxide (NO) synthase (eNOS) and ensued NO production is a common mechanism of various cardiovascular pathologies, including hypertension and atherosclerosis. Specific signaling cascades, generated by vascular endothelial cells (ECs) in response to laminar flow, modulate EC structure and functions, NO production in particular. We have previously shown that flow-stimulated Gab1 (Grb2-associated binder-1) tyrosine phosphorylation mediates eNOS activation. However, the upstream mechanism that regulates Gab1 tyrosine phosphorylation remains unclear. Hypothesis: We hypothesized that platelet endothelial cell adhesion molecule-1 (PECAM1), a key molecule in an endothelial mechanosensing complex, specifically mediates Gab1 tyrosine phosphorylation and its downstream Akt and eNOS activation in ECs upon flow rather than hepatocyte growth factor (HGF) stimulation. Methods: Western blot, en face staining and voluntary wheel running. Results: Small interfering RNA (siRNA) targeting PECAM1 abolished flow- but not HGF-induced Gab1 tyrosine phosphorylation and Akt, eNOS activation as well as Gab1 membrane translocation. Protein-tyrosine phosphatase SHP2, which has been shown to interact with Gab1, was involved in a flow signaling pathway as well as HGF-induced signaling, as SHP2 siRNA diminished the flow- and HGF-induced Gab1 tyrosine phosphorylation, membrane localization and downstream signaling. Pharmacological inhibition of PI3K by LY294002 decreased flow, but not HGF-mediated Gab1 phosphorylation and membrane localization as well as eNOS activation. Finally, we observed that flow-mediated Gab1 and eNOS phosphorylation in vivo induced by voluntary wheel running was reduced in PECAM1 knockout mice. Conclusions: These results demonstrate a specific role of PECAM1 in flow-mediated Gab1 tyrosine phosphorylation and eNOS signaling in ECs

Role for tyrosine kinases in carbachol-regulated Ca entry into colonic epithelial cells

1995 ◽  
Vol 268 (1) ◽  
pp. C154-C161 ◽  
Author(s):  
G. Bischof ◽  
B. Illek ◽  
W. W. Reenstra ◽  
T. E. Machen
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Divalent Cations ◽  
Tyrosine Phosphatase ◽  
Inhibitory Effect ◽  
Colonic Epithelial Cells

We studied a possible role of tyrosine kinases in the regulation of Ca entry into colonic epithelial cells HT-29/B6 using digital image processing of fura 2 fluorescence. Both carbachol and thapsigargin increased Ca entry to a similar extent and Ca influx was reduced by the tyrosine kinase inhibitor genistein (50 microM). Further experiments were performed in solutions containing 95 mM K to depolarize the membrane potential, and the effects of different inhibitors on influx of Ca, Mn, and Ba were compared. Genistein, but not the inactive analogue daidzein nor the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2- methylpiperazine, decreased entry of all three divalent cations by 47-59%. In high-K solutions, carbachol or thapsigargin both caused intracellular Ca to increase to a plateau of 223 +/- 19 nM. This plateau was reduced by the tyrosine kinase inhibitors genistein (to 95 +/- 8 nM), lavendustin A (to 155 +/- 17 nM), and methyl-2,5-dihydroxycinnamate (to 39 +/- 3 nM). Orthovanadate, a protein tyrosine phosphatase inhibitor, prevented the inhibitory effect of genistein. Ca pumping was unaffected by genistein. Carbachol increased tyrosine phosphorylation (immunoblots with anti-phosphotyrosine antibodies) of 110-, 75-, and 70-kDa proteins, and this phosphorylation was inhibited by genistein. We conclude that carbachol and thapsigargin increase Ca entry, and tyrosine phosphorylation of some key proteins may be important for regulating this pathway.

scholarly journals Tyrosine 425 within the activated erythropoietin receptor binds Syp, reduces the erythropoietin required for Syp tyrosine phosphorylation, and promotes mitogenesis

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4495-4501 ◽  
Author(s):  
T Tauchi ◽  
JE Damen ◽  
K Toyama ◽  
GS Feng ◽  
HE Broxmeyer ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Intracellular Signaling ◽  
Tyrosine Phosphatase ◽  
Erythropoietin Receptor ◽  
Sh2 Domains ◽  
Proliferation And Differentiation ◽  
Stimulation Of

Erythropoietin (Epo), the primary in vivo stimulator of erythroid proliferation and differentiation, acts, in part, by altering the tyrosine phosphorylation levels of various intracellular signaling molecules. These phosphorylation levels are tightly regulated by both tyrosine kinases and tyrosine phosphatases. We have recently shown that the SH2 containing tyrosine phosphatase, Syp, binds directly to both the tyrosine phosphorylated form of the Epo receptor (EpoR) and to Grb2 after Epo stimulation of M07e cells engineered to express high levels of human EpoRs (T. Tauchi, et al: J Biol Chem 270:5631, 1995). To determine which tyrosine within the EpoR is responsible for binding Syp, we examined DA-3 cell lines expressing full-length mutant EpoRs bearing tyrosine to phenylalanine substitutions for each of the eight tyrosines within the intracellular domain of the EpoR. We found that: (1) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, coimmunoprecipitated with Syp; (2) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, bound to a GST-fusion protein containing both SH2 domains of Syp; (3) Jak2 could phosphorylate GST-Syp in vitro after Epo stimulation of wild-type (wt) EpoR expressing DA-3 cells; (4) Epo-stimulated tyrosine phosphorylation of Syp in vivo was markedly reduced in Y425F EpoR expressing DA-3 calls; and (5) DA-3 cells expressing the Y425F EpoR grow less well in response to Epo than wt EpoR expressing cells. These results suggest that Syp binds via its SH2 domains to phosphorylated Y425 within the EpoR and is then phosphorylated on tyrosine residues by Jak2. Moreover, Y425 in the EpoR reduces the Epo requirement for Syp tyrosine phosphorylation and promotes proliferation.

Anaplastic Lymphoma Kinase Is Activated through the Pleiotrophin/Receptor Protein Tyrosine Phosphatase (RPTP)β/ζ Signaling Pathway: A Unique Mechanism of Activation of Receptor Protein Tyrosine Kinases.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4355-4355
Author(s):  
Pablo Perez-Pinera ◽  
Wei Zhang ◽  
Zhaoyi Wang ◽  
James R. Berenson ◽  
Thomas F. Deuel
Keyword(s):  
Tyrosine Kinase ◽  
Signaling Pathway ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Kinases ◽  
Tyrosine Phosphatase ◽  
Receptor Protein ◽  
Protein Tyrosine ◽  
Anaplastic Lymphoma ◽  
Unique Mechanism

Abstract Anaplastic Lymphoma Kinase (ALK) is a receptor-type transmembrane tyrosine kinase (RTK) of the insulin receptor superfamily that structurally is most closely related to leukocyte tyrosine kinase. It was first discovered as a chimeric protein (NPM-ALK) of nucleophosmin and the C-terminal (kinase) domain of ALK in anaplastic large cell lymphomas (ALCL). NPM-ALK is constitutively active and generates the oncogenic signals that are the pathogenic mechanisms of these highly malignant cancers. The full-length ALK also is believed to have an important role in the pathogenesis of other human malignancies, since its expression is found in rhabdomyosarcomas, neuroblastomas, neuroectodermal tumors, glioblastomas, breast carcinomas, and melanomas. Recently it was proposed that pleiotrophin (PTN the protein, Ptn the gene) is the ligand that stimulates ALK to transduce signals to activate downstream targets. However, this proposal contrasted with earlier studies that demonstrated Receptor Protein Tyrosine Phosphatase (RPTP)β/ζ is the functional receptor for PTN. PTN was shown to inactivate RPTPβ/ζ and thereby permit the activity of different tyrosine kinases to increase tyrosine phosphorylation of the substrates of RPTPβ/ζ at the sites that are dephosphorylated by RPTPβ/ζ in cells not stimulated by PTN. Subsequent studies identified β-catenin, β-adducin, Fyn, GIT1/Cat-1, P190RhoGAP, and histone deacetylase 2 (HDAC-2) as downstream targets of the PTN/RPTPβ/ζ signaling pathway and demonstrated that their levels of tyrosine phosphorylation increase in PTN-stimulated cells. This diversity of PTN-regulated targets is one basis for the pleiotrophic activities of PTN. We now demonstrate that tyrosine phosphorylation of ALK is increased in PTN-stimulated cells through the PTN/RPTPβ/ζ signaling pathway. It is furthermore shown that ALK is activated in PTN-stimulated cells when it is expressed in cells without its extracellular domain, that β-catenin is a substrate of ALK, that the tyrosine phosphorylation site in β-catenin phosphorylated by ALK is the same site dephosphorylated by RPTPβ/ζ, and that PTN-stimulated tyrosine phosphorylation of β-catenin requires expression of ALK. The data suggest a unique mechanism to activate ALK; the data support a mechanism in which β-catenin is phosphorylated in tyrosine through the coordinated inactivation of RPTPβ/ζ, the activation of the tyrosine kinase activity of ALK, and the phosphorylation of β-catenin by ALK at the same site regulated by RPTPβ/ζ in PTN-stimulated cells. Since PTN often is inappropriately expressed in the same malignancies that express ALK, the data suggest a mechanism through which ALK signaling may contribute to those malignancies that express full length ALK through the activity of PTN to signal constitutively the same pathways as NPM-ALK in ALCL.

scholarly journals Tyrosine phosphorylation of BCR by FPS/FES protein-tyrosine kinases induces association of BCR with GRB-2/SOS.

1995 ◽  
Vol 15 (2) ◽  
pp. 835-842 ◽  
Author(s):  
Y Maru ◽  
K L Peters ◽  
D E Afar ◽  
M Shibuya ◽  
O N Witte ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Sh2 Domain ◽  
Binding Domain ◽  
Ras Signaling ◽  
Cytoplasmic Protein ◽  
Nucleotide Exchange ◽  
Protein Tyrosine

The human bcr gene encodes a protein with serine/threonine kinase activity, CDC24/dbl homology, a GAP domain, and an SH2-binding region. However, the precise physiological functions of BCR are unknown. Coexpression of BCR with the cytoplasmic protein-tyrosine kinase encoded by the c-fes proto-oncogene in Sf-9 cells resulted in stable BCR-FES protein complex formation and tyrosine phosphorylation of BCR. Association involves the SH2 domain of FES and a novel binding domain localized to the first 347 amino acids of the FES N-terminal region. Deletion of the homologous N-terminal BCR-binding domain from v-fps, a fes-related transforming oncogene, abolished transforming activity and tyrosine phosphorylation of BCR in vivo. Tyrosine phosphorylation of BCR in v-fps-transformed cells induced its association with GRB-2/SOS, the RAS guanine nucleotide exchange factor complex. These data provide evidence that BCR couples the cytoplasmic protein-tyrosine kinase and RAS signaling pathways.

scholarly journals Mechanism of vanadate-induced activation of tyrosine phosphorylation and of the respiratory burst in HL60 cells. Role of reduced oxygen metabolites

1991 ◽  
Vol 276 (3) ◽  
pp. 611-619 ◽  
Author(s):  
S Trudel ◽  
M R Pâquet ◽  
S Grinstein
Keyword(s):  
Tyrosine Phosphorylation ◽  
Respiratory Burst ◽  
Tyrosine Kinases ◽  
Tyrosine Phosphatase ◽  
O2 Consumption ◽  
Phosphatase Inhibitors ◽  
Hl60 Cells ◽  
Diphenylene Iodonium ◽  
Undifferentiated Cells

Vanadate induces phosphotyrosine accumulation and activates O2 consumption in permeabilized differentiated HL60 cells. NADPH, the substrate of the respiratory burst oxidase, was found to be necessary not only for the increased O2 consumption, but also for tyrosine phosphorylation. The effect of NADPH was not due to reduction of vanadate to vanadyl. Instead, NADPH was required for the synthesis of superoxide, which triggered the formation of peroxovanadyl [V(4+)-OO] and vanadyl hydroperoxide [V(4+)-OOH]. One or both of these species, rather than vanadate itself, appears to be responsible for phosphotyrosine accumulation and activation of the respiratory burst. Accordingly, the stimulatory effects of vanadate and NADPH were abrogated by superoxide dismutase. Moreover, phosphorylation was activated in the absence of NADPH by treatment with V(4+)-OO and/or V(4+)-OOH, generated by treatment of orthovanadate with KO2 or H2O2 respectively. The main source of the superoxide involved in the formation of V(4+)-OO and V(4+)-OOH is the NADPH oxidase. This was shown by the inhibitory effects of diphenylene iodonium and by the failure of undifferentiated cells, which lack oxidase activity, to undergo tyrosine phosphorylation when treated with vanadate and NADPH. By contrast, exogenously generated V(4+)-OO induced marked phosphorylation in the undifferentiated cells, demonstrating the presence of the appropriate tyrosine kinases and phosphatases. A good correlation was found to exist between induction of tyrosine phosphorylation and activation of the respiratory burst, suggesting a causal relationship. Therefore an amplification cycle appears to exist in cells treated with vanadate, whereby trace amounts of superoxide initiate the formation of V(4+)-OO and/or V(4+)-OOH. These peroxides promote phosphotyrosine formation, most likely by inhibition of tyrosine phosphatases. Accumulation of critical tyrosine-phosphorylated proteins then initiates a respiratory burst, with abundant production of superoxide. The newly formed superoxide catalyses the formation of additional V(4+)-OO and/or V(4+)-OOH, thereby magnifying the response. Since vanadium derivatives are ubiquitous in animal tissues, V(4+)-OO and/or V(4+)-OOH could be formed in vivo by reduced O2 metabolites, becoming potential endogenous tyrosine phosphatase inhibitors. Because of their potency, peroxides of vanadate may be useful as probes for the study of protein phosphotyrosine turnover.

Signalling by the W/Kit receptor tyrosine kinase is negatively regulated in vivo by the protein tyrosine phosphatase Shp1

1996 ◽  
Vol 13 (3) ◽  
pp. 309-315 ◽  
Author(s):  
Robert F. Paulson ◽  
Shirly Vesely ◽  
Katharine A. Siminovitch ◽  
Alan Bernstein
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine ◽  
Kit Receptor
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