scholarly journals GPR68 Contributes to Persistent Acidosis-Induced Activation of AGC Kinases and Tyrosine Phosphorylation in Organotypic Hippocampal Slices

2021 ◽  
Vol 15 ◽  
Author(s):  
Guokun Zhou ◽  
Xiang-ming Zha
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Neurological Diseases ◽  
Hippocampal Slices ◽  
Signaling Cascades ◽  
Dependent Manner ◽  
Cyclin Dependent Kinase ◽  
Ataxia Telangiectasia Mutated ◽  
Agc Kinases ◽  
Profiling Analysis

Persistent acidosis occurs in ischemia and multiple neurological diseases. In previous studies, acidic stimulation leads to rapid increase in intracellular calcium in neurons. However, it remains largely unclear how a prolonged acidosis alters neuronal signaling. In our previous study, we found that GPR68-mediated PKC activities are protective against acidosis-induced injury in cortical slices. Here, we first asked whether the same principle holds true in organotypic hippocampal slices. Our data showed that 1-h pH 6 induced PKC phosphorylation in a GPR68-dependent manner. Go6983, a PKC inhibitor worsened acidosis-induced neuronal injury in wild type (WT) but had no effect in GPR68−/− slices. Next, to gain greater insights into acid signaling in brain tissue, we treated organotypic hippocampal slices with pH 6 for 1-h and performed a kinome profiling analysis by Western blot. Acidosis had little effect on cyclin-dependent kinase (CDK) or casein kinase 2 activity, two members of the CMGC family, or Ataxia telangiectasia mutated (ATM)/ATM and RAD3-related (ATR) activity, but reduced the phosphorylation of MAPK/CDK substrates. In contrast, acidosis induced the activation of CaMKIIα, PKA, and Akt. Besides these serine/threonine kinases, acidosis also induced tyrosine phosphorylation. Since GPR68 is widely expressed in brain neurons, we asked whether GPR68 contributes to acidosis-induced signaling. Deleting GPR68 had no effect on acidosis-induced CaMKII phosphorylation, attenuated that of phospho-Akt and phospho-PKA substrates, while abolishing acidosis-induced tyrosine phosphorylation. These data demonstrate that prolonged acidosis activates a network of signaling cascades, mediated by AGC kinases, CaMKII, and tyrosine kinases. GPR68 is the primary mediator for acidosis-induced activation of PKC and tyrosine phosphorylation, while both GPR68-dependent and -independent mechanisms contribute to the activation of PKA and Akt.

Thrombin Mediated Tyrosine Phosphorylation of PKCδ in Human Platelets Occurs in a Calcium- and Src Family Tyrosine Kinase- Dependent Manner.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3541-3541
Author(s):  
Swaminathan Murugappan ◽  
Haripriya Shankar ◽  
Satya Kunapuli
Keyword(s):  
Tyrosine Kinase ◽  
Intracellular Calcium ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Human Platelets ◽  
Dependent Manner ◽  
Protein Signaling ◽  
Glycoprotein Vi ◽  
Pkc Isoforms

Abstract Protein kinase C (PKC)-δ is a novel PKC that has been shown to be tyrosine phosphorylated upon stimulation with agonists in platelets. Tyrosine phosphorylation of PKCδ has been shown to occur in a Fyn-dependent manner downstream of glycoprotein VI (GPVI) signaling in platelets. Although thrombin causes tyrosine phosphorylation of PKCδ in platelets, the mechanism of this event is not elucidated. In this study, we investigated whether G-protein signaling pathways utilize similar pathways as GPVI in tyrosine phosphorylation of PKCδ. Protease activated receptor (PAR) -1 selective peptide, SFLLRN and PAR - 4 selective peptide, AYPGKF caused a time- and concentration-dependent increase in tyrosine phosphorylation of PKCδ in human platelets. However, AYPGKF failed to cause tyrosine phosphorylation of PKCδ in Gq-deficient mouse platelets. Both U73122, a phospholipase C (PLC) inhibitor, and dimethyl-BAPTA, an intracellular calcium chelator, inhibited the tyrosine phosphorylation of PKCδ downstream of the PAR activation suggesting a role for Gq/PLC pathways and intracellular calcium in mediating this event. Inhibition of PKC isoforms using GF109203X potentiated the tyrosine phosphorylation of PKCδ. The Src family tyrosine kinase inhibitors, PP1 and PP2 inhibited the tyrosine phosphorylation of PKCδ suggesting a role for Src family tyrosine kinase members in this event. We also found that both Lyn and Src are physically associated with PKCδ in a constitutive manner in platelets. Finally we found that there was a time-dependent activation of Src following activation of platelets with thrombin. Thus, the precomplexed Src and Lyn tyrosine kinases get activated following PAR stimulation resulting in the tyrosine phosphorylation of PKCδ. All these data indicate that tyrosine phosphorylation of PKCδ downstream of thrombin occurs in a calcium- and Src-family kinase dependent manner in human platelets.

T-cell receptor–induced phosphorylation of the ζ chain is efficiently promoted by ZAP-70 but not Syk

Blood ◽  
2004 ◽  
Vol 104 (3) ◽  
pp. 760-767 ◽  
Author(s):  
Marcos Steinberg ◽  
Oumeya Adjali ◽  
Louise Swainson ◽  
Peggy Merida ◽  
Vincenzo Di Bartolo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Receptor ◽  
Tyrosine Kinases ◽  
Initial Step ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Signaling Cascades ◽  
Leukemia Cells

AbstractEngagement of the T-cell receptor (TCR) results in the activation of Lck/Fyn and ZAP-70/Syk tyrosine kinases. Lck-mediated tyrosine phosphorylation of signaling motifs (ITAMs) in the CD3-ζ subunits of the TCR is an initial step in the transduction of signaling cascades. However, ζ phosphorylation is also promoted by ZAP-70, as TCR-induced ζ phosphorylation is defective in ZAP-70–deficient T cells. We show that this defect is corrected by stable expression of ZAP-70, but not Syk, in primary and transformed T cells. Indeed, these proteins are differentially coupled to the TCR with a 5- to 10-fold higher association of ZAP-70 with ζ as compared to Syk. Low-level Syk-ζ binding is associated with significantly less Lck coupled to the TCR. Moreover, diminished coupling of Lck to ζ correlates with a poor phosphorylation of the positive regulatory tyr352 residue of Syk. Thus, recruitment of Lck into the TCR complex with subsequent ζ chain phosphorylation is promoted by ZAP-70 but not Syk. Importantly, the presence of ZAP-70 positively regulates the TCR-induced tyrosine phosphorylation of Syk. The interplay between Syk and ZAP-70 in thymocytes, certain T cells, and B-chronic lymphocytic leukemia cells, in which they are coexpressed, will therefore modulate the amplitude of antigen-mediated receptor signaling.

scholarly journals Reactive oxygen intermediates activate NF-kappa B in a tyrosine kinase- dependent mechanism and in combination with vanadate activate the p56lck and p59fyn tyrosine kinases in human lymphocytes

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1212-1220 ◽  
Author(s):  
GL Schieven ◽  
JM Kirihara ◽  
DE Myers ◽  
JA Ledbetter ◽  
FM Uckun
Keyword(s):  
Ionizing Radiation ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Reactive Oxygen ◽  
Lymphoid Cells ◽  
Dependent Manner ◽  
Reactive Oxygen Intermediates ◽  
Nf Kappa B ◽  
Oxygen Intermediates

We have previously observed that ionizing radiation induces tyrosine phosphorylation in human B-lymphocyte precursors by stimulation of unidentified tyrosine kinases and this phosphorylation is substantially augmented by vanadate. Ionizing radiation generates reactive oxygen intermediates (ROI). Because H2O2 is a potent ROI generator that readily crosses the plasma membrane, we used H2O2 to examine the effects of ROI on signal transduction. We now provide evidence that the tyrosine kinase inhibitor herbimycin A and the free radical scavenger N- acetyl-cysteine inhibit both radiation-induced and H2O2-induced activation of NF-kappa B, indicating that activation triggered by ROI is dependent on tyrosine kinase activity. H2O2 was found to stimulate Ins-1,4,5-P3 production in a tyrosine kinase-dependent manner and to induce calcium signals that were greatly augmented by vanadate. The synergistic induction of tyrosine phosphorylation by H2O2 plus vanadate included physiologically relevant proteins such as PLC gamma 1. Although treatment of cells with H2O2 alone did not affect the activity of src family kinases, treatment with H2O2 plus vanadate led to activation of the p56lck and p59fyn tyrosine kinases. The combined inhibition of phosphatases and activation of kinases provides a potent mechanism for the synergistic effects of H2O2 plus vanadate. Induction of tyrosine phosphorylation by ROI may thus lead to many of the pleiotropic effects of ROI in lymphoid cells, including downstream activation of PLC gamma 1 and NF-kappa B.

scholarly journals Staurosporine inhibits agrin-induced acetylcholine receptor phosphorylation and aggregation.

1994 ◽  
Vol 125 (3) ◽  
pp. 661-668 ◽  
Author(s):  
B G Wallace
Keyword(s):  
Tyrosine Phosphorylation ◽  
Acetylcholine Receptor ◽  
Tyrosine Kinases ◽  
Neuromuscular Junctions ◽  
Beta Subunit ◽  
Dependent Manner ◽  
Tyrosine Residues ◽  
Serine Kinases ◽  
Cultured Myotubes ◽  
Dose Dependent

Agrin, a protein that mediates nerve-induced acetylcholine receptor (AChR) aggregation at developing neuromuscular junctions, has been shown to cause an increase in phosphorylation of the beta, gamma, and delta subunits of AChRs in cultured myotubes. As a step toward understanding the mechanism of agrin-induced AChR aggregation, we examined the effects of inhibitors of protein kinases on AChR aggregation and phosphorylation in chick myotubes in culture. Staurosporine, an antagonist of both protein serine and tyrosine kinases, blocked agrin-induced AChR aggregation in a dose-dependent manner; 50% inhibition occurred at approximately 2 nM. The extent of inhibition was independent of agrin concentration, suggesting an effect downstream of the interaction of agrin with its receptor. Staurosporine blocked agrin-induced phosphorylation of the AChR beta subunit, which occurs at least in part on tyrosine residues, but did not reduce phosphorylation of the gamma and delta subunits, which occurs on serine/threonine residues. Staurosporine also prevented the agrin-induced decrease in the rate at which AChRs are extracted from intact myotubes by mild detergents. H-7, an antagonist of protein serine kinases, inhibited agrin-induced phosphorylation of the gamma and delta subunits but did not block agrin-induced phosphorylation of the AChR beta subunit, AChR aggregation, or the decrease in AChR extractability. The results provide support for the hypothesis that tyrosine phosphorylation of the beta subunit plays a role in agrin-induced AChR aggregation.

PECAM-1 (CD31) functions as a reservoir for and a modulator of tyrosine-phosphorylated beta-catenin

1999 ◽  
Vol 112 (18) ◽  
pp. 3005-3014 ◽  
Author(s):  
N. Ilan ◽  
S. Mahooti ◽  
D.L. Rimm ◽  
J.A. Madri
Keyword(s):  
Endothelial Cell ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Nuclear Translocation ◽  
Adherens Junction ◽  
Cell Contact ◽  
Beta Catenin ◽  
Dependent Manner

Catenins function as regulators of cellular signaling events in addition to their previously documented roles in adherens junction formation and function. Evidence to date suggests that beta and gamma catenins can act as signaling molecules, bind transcriptional factors and translocate to the nucleus. Beta- and gamma-catenin are also major substrates for protein tyrosine kinases, and tyrosine phosphorylation of junctional proteins is correlated with decreased adhesiveness. One way in which catenin functions are modulated is by dynamic incorporation into junctional complexes which controls, in part, the cytoplasmic levels of catenins. Here we show that: (1) vascular endothelial growth factor (VEGF) induces beta-catenin tyrosine phosphorylation in a time-, and dose-dependent manner and that VEGF receptors co-localize to areas of endothelial cell-cell contact in vitro and in vivo. (2) Platelet-endothelial cell adhesion molecule (PECAM)-1 can function as a reservoir for, and modulator of, tyrosine phosphorylated beta-catenin. (3) PECAM-1 can prevent beta-catenin nuclear translocation in transfected SW480 colon carcinoma cells. We suggest that PECAM-1 may play a role in modulating beta-catenin tyrosine phosphorylation levels, localization and signaling and by doing so, functions as an important modulator of the endothelium.

[3H]taurine andd-[3H]aspartate release from astrocyte cultures are differently regulated by tyrosine kinases

1999 ◽  
Vol 276 (5) ◽  
pp. C1226-C1230 ◽  
Author(s):  
Alexander A. Mongin ◽  
Jyoti M. Reddi ◽  
Carol Charniga ◽  
Harold K. Kimelberg
Keyword(s):  
Amino Acids ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Mammalian Brain ◽  
Dependent Manner ◽  
Anion Channels ◽  
Primary Astrocyte ◽  
Tyrphostin 23

Volume-dependent anion channels permeable for Cl− and amino acids are thought to play an important role in the homeostasis of cell volume. Astrocytes are the main cell type in the mammalian brain showing volume perturbations under physiological and pathophysiological conditions. We investigated the involvement of tyrosine phosphorylation in hyposmotic medium-induced [3H]taurine andd-[3H]aspartate release from primary astrocyte cultures. The tyrosine kinase inhibitors tyrphostin 23 and tyrphostin A51 partially suppressed the volume-dependent release of [3H]taurine in a dose-dependent manner with half-maximal effects at ∼40 and 1 μM, respectively. In contrast, the release ofd-[3H]aspartate was not significantly affected by these agents in the same concentration range. The inactive analog tyrphostin 1 had no significant effect on the release of both amino acids. The data obtained suggest the existence of at least two volume-dependent anion channels permeable to amino acids in astrocyte cultures. One of these channels is permeable to taurine and is under the control of tyrosine kinase(s). The other is permeable to both taurine and aspartate, but its volume-dependent regulation does not require tyrosine phosphorylation.

scholarly journals Adhesive ligand binding to integrin alpha IIb beta 3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK

1993 ◽  
Vol 122 (2) ◽  
pp. 473-483 ◽  
Author(s):  
MM Huang ◽  
L Lipfert ◽  
M Cunningham ◽  
JS Brugge ◽  
MH Ginsberg ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Actin Polymerization ◽  
Protein Tyrosine Kinase ◽  
Free Calcium ◽  
Dependent Manner ◽  
Integrin Receptors ◽  
Fibrinogen Binding ◽  
Granule Secretion

Tyrosine phosphorylation of multiple platelet proteins is stimulated by thrombin and other agonists that cause platelet aggregation and secretion. The phosphorylation of a subset of these proteins, including a protein tyrosine kinase, pp125FAK, is dependent on the platelet aggregation that follows fibrinogen binding to integrin alpha IIb beta 3. In this report, we examined whether fibrinogen binding, per se, triggers a process of tyrosine phosphorylation in the absence of exogenous agonists. Binding of soluble fibrinogen was induced with Fab fragments of an anti-beta 3 antibody (anti-LIBS6) that directly exposes the fibrinogen binding site in alpha IIb beta3. Proteins of 50-68 KD and 140 kD became phosphorylated on tyrosine residues in a fibrinogen-dependent manner. This response did not require prostaglandin synthesis, an increase in cytosolic free calcium, platelet aggregation or granule secretion, nor was it associated with tyrosine phosphorylation of pp125FAK. Tyrosine phosphorylation of the 50-68-kD and 140-kD proteins was also observed when (a) fibrinogen binding was stimulated by agonists such as epinephrine, ADP, or thrombin instead of by anti-LIBS6; (b) fragment X, a dimeric plasmin-derived fragment of fibrinogen was used instead of fibrinogen; or (c) alpha IIb beta 3 complexes were cross-linked by antibodies, even in the absence of fibrinogen. In contrast, no tyrosine phosphorylation was observed when the ligand consisted of monomeric cell recognition peptides derived from fibrinogen (RGDS or gamma 400-411). Fibrinogen-dependent tyrosine phosphorylation was inhibited by cytochalasin D. These studies demonstrate that fibrinogen binding to alpha IIb beta 3 initiates a process of tyrosine phosphorylation that precedes platelet aggregation and the phosphorylation of pp125FAK. This reaction may depend on the oligomerization of integrin receptors and on the state of actin polymerization, organizational processes that may juxtapose tyrosine kinases with their substrates.

scholarly journals Reactive oxygen intermediates activate NF-kappa B in a tyrosine kinase- dependent mechanism and in combination with vanadate activate the p56lck and p59fyn tyrosine kinases in human lymphocytes

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1212-1220 ◽  
Author(s):  
GL Schieven ◽  
JM Kirihara ◽  
DE Myers ◽  
JA Ledbetter ◽  
FM Uckun
Keyword(s):  
Ionizing Radiation ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Reactive Oxygen ◽  
Lymphoid Cells ◽  
Dependent Manner ◽  
Reactive Oxygen Intermediates ◽  
Nf Kappa B ◽  
Oxygen Intermediates

Abstract We have previously observed that ionizing radiation induces tyrosine phosphorylation in human B-lymphocyte precursors by stimulation of unidentified tyrosine kinases and this phosphorylation is substantially augmented by vanadate. Ionizing radiation generates reactive oxygen intermediates (ROI). Because H2O2 is a potent ROI generator that readily crosses the plasma membrane, we used H2O2 to examine the effects of ROI on signal transduction. We now provide evidence that the tyrosine kinase inhibitor herbimycin A and the free radical scavenger N- acetyl-cysteine inhibit both radiation-induced and H2O2-induced activation of NF-kappa B, indicating that activation triggered by ROI is dependent on tyrosine kinase activity. H2O2 was found to stimulate Ins-1,4,5-P3 production in a tyrosine kinase-dependent manner and to induce calcium signals that were greatly augmented by vanadate. The synergistic induction of tyrosine phosphorylation by H2O2 plus vanadate included physiologically relevant proteins such as PLC gamma 1. Although treatment of cells with H2O2 alone did not affect the activity of src family kinases, treatment with H2O2 plus vanadate led to activation of the p56lck and p59fyn tyrosine kinases. The combined inhibition of phosphatases and activation of kinases provides a potent mechanism for the synergistic effects of H2O2 plus vanadate. Induction of tyrosine phosphorylation by ROI may thus lead to many of the pleiotropic effects of ROI in lymphoid cells, including downstream activation of PLC gamma 1 and NF-kappa B.

scholarly journals Rho Family GTPases Are Required for Activation of Jak/STAT Signaling by G Protein-Coupled Receptors

2003 ◽  
Vol 23 (4) ◽  
pp. 1316-1333 ◽  
Author(s):  
Stéphane Pelletier ◽  
François Duhamel ◽  
Philippe Coulombe ◽  
Michel R. Popoff ◽  
Sylvain Meloche
Keyword(s):  
Tyrosine Phosphorylation ◽  
G Protein ◽  
Tyrosine Kinases ◽  
Rho Gtpases ◽  
G Protein Coupled Receptors ◽  
Dependent Manner ◽  
Signal Transducers ◽  
Regulatory Effects ◽  
Rapid Activation ◽  
G Protein Coupled

ABSTRACT As do cytokine receptors and receptor tyrosine kinases, G protein-coupled receptors (GPCRs) signal to Janus kinases (Jaks) and signal transducers and activators of transcription (STATs). However, the early biochemical events linking GPCRs to this signaling pathway have been unclear. Here we show that GPCR-stimulated Rac activity and the subsequent generation of reactive oxygen species are necessary for activating tyrosine phosphorylation of Jaks and STAT-dependent transcription. The requirement for Rac activity can be overcome by addition of hydrogen peroxide. Expression of activated mutants of Rac1 is sufficient to activate Jak2 and STAT-dependent transcription, and the activation of Jak2 correlates with the ability of Rac1 to bind to NADPH oxidase subunit p67phox. We further show that GPCR agonists stimulate tyrosine phosphorylation of STAT1 and STAT3 proteins in a Rac-dependent manner. The tyrosine phosphorylation of STAT3 is biphasic; the first peak of phosphorylation is weak and correlates with rapid activation of Jaks by GPCRs, whereas the second peak is stronger and requires the synthesis of an autocrine factor. Rho also plays an essential role in the induction of STAT transcriptional activity. Our results highlight a novel role for Rho GTPases in mediating the regulatory effects of GPCRs on STAT-dependent gene expression.

scholarly journals Src-mediated tyrosine phosphorylation of PRC1 and kinastrin/SKAP on the mitotic spindle

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mariko Morii ◽  
Sho Kubota ◽  
Chizu Hasegawa ◽  
Yumi Takeda ◽  
Shiori Kometani ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Mitotic Spindle ◽  
Tyrosine Kinases ◽  
Mass Spectrometry Analysis ◽  
Dependent Manner ◽  
Spectrometry Analysis ◽  
Microtubule Polymerization ◽  
Underlying Mechanisms ◽  
Src Family Tyrosine Kinases ◽  
Impaired Binding

AbstractSrc-family tyrosine kinases (SFKs) play important roles in a number of signal transduction events during mitosis, such as spindle formation. A relationship has been reported between SFKs and the mitotic spindle; however, the underlying mechanisms remain unclear. We herein demonstrated that SFKs accumulated in the centrosome region at the onset of mitosis. Centrosomal Fyn increased in the G2 phase in a microtubule polymerization-dependent manner. A mass spectrometry analysis using mitotic spindle preparations was performed to identify tyrosine-phosphorylated substrates. Protein regulator of cytokinesis 1 (PRC1) and kinastrin/small kinetochore-associated protein (kinastrin/SKAP) were identified as SFK substrates. SFKs mainly phosphorylated PRC1 at Tyr-464 and kinastrin at Tyr-87. Although wild-type PRC1 is associated with microtubules, phosphomimetic PRC1 impaired the ability to bind microtubules. Phosphomimetic kinastrin at Tyr-87 also impaired binding with microtubules. Collectively, these results suggest that tyrosine phosphorylation of PRC1 and kinastrin plays a role in their delocalization from microtubules during mitosis.

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