Activation Of Proline-rich Tyrosine Kinase Research Articles

Galangin Inhibits LPS-Induced MMP-9 Expression via Suppressing Protein Kinase-Dependent AP-1 and FoxO1 Activation in Rat Brain Astrocytes.

PurposeNeuroinflammation, characterized by the increased expression of inflammatory proteins such as matrix metalloproteinases (MMPs), plays a critical role in neurodegenerative disorders. Lipopolysaccharide (LPS) has been shown to upregulate MMP-9 expression through the activation of various transcription factors, including activator protein 1 (AP-1) and forkhead box protein O1 (FoxO1). The flavonoid 3,5,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one (galangin) has been demonstrated to possess antioxidant and anti-inflammatory properties in various types of cells. Here, we investigated the mechanisms underlying the inhibitory effect of galangin on LPS-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells).MethodsPharmacological inhibitors and siRNAs were employed to explore the effects of galangin on LPS-challenged RBA-1 cells. Gelatin zymography, Western blotting, real-time PCR, and a luciferase reporter assay were used to detect MMP-9 activity, protein expression, mRNA levels, and promoter activity, respectively. The protein kinases involved in the LPS-induced MMP-9 expression were determined by Western blot. A chromatin immunoprecipitation (ChIP) assay was employed to evaluate the activity of c-Jun at the MMP-9 promoter.ResultsGalangin treatment attenuated the LPS-mediated induction of MMP-9 protein and mRNA expression, as well as the activity at the MMP-9 promoter. In addition, galangin exerted its inhibitory effects on MMP-9 expression through suppressing the LPS-stimulated activation of proline-rich tyrosine kinase (Pyk2), platelet-derived growth factor receptor beta (PDGFRβ), phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), and mitogen-activated protein kinases (MAPKs). Pretreatment with galangin attenuated the LPS-induced phosphorylation of c-Jun and FoxO1. LPS-induced cell migration was also suppressed by galangin pretreatment.ConclusionGalangin attenuates the LPS-induced inflammatory responses, including the induction of MMP-9 expression and cell migration, via inhibiting Pyk2/PDGFRβ/PI3K/Akt/mTOR/JNK1/JNK2 and p44/p42 MAPK cascade-dependent AP-1 and FoxO1 activities. These results provide new insights into the mechanisms through which galangin mitigates LPS-induced inflammatory responses, and suggest novel strategies for the management of LPS-related brain diseases.

Fyn-mediated phosphorylation of Pyk2 promotes its activation and dissociation downstream of gonadotropin-releasing hormone receptor.

The receptor for gonadotropin-releasing hormone (GnRH) is highly expressed in hypothalamic GnRH neurons, as well as in anterior pituitary gonadotrophs. In our previous study, we found that stimulation of the GnRH receptor activated protein kinase D1 (PKD1), and PKD1 was involved in the Fyn-mediated activation of proline-rich tyrosine kinase 2 (Pyk2) in cultured GnRH neurons (GT1-7 cells). In the present study, we examined the molecular mechanisms of Pyk2 activation and the interaction of Pyk2 and Fyn in GT1-7 cells. Experiments with site-directed mutants of Pyk2 indicated that tyrosine 402 (Tyr402) was phosphorylated both by autophosphorylation and by Fyn, whereas Tyr579 was phosphorylated mainly by Fyn. We found that dasatinib, a Src family inhibitor, enhanced the interaction of Pyk2 and Fyn. Experiments with site-directed mutants of Pyk2 and Fyn indicated that dasatinib enhanced the binding of Pyk2 autophosphorylated at Tyr402 and the Src homology 2 domain in Fyn. Our present data may suggest that fully activated Pyk2 dissociates from Fyn after Fyn-mediated phosphorylation of Pyk2 at sites other than Tyr402 and Tyr579.

Glycolipid iGb3 feedback amplifies innate immune responses via CD1d reverse signaling.

The cross-talk between cellular lipid metabolism and the innate immune responses remains obscure. In addition to presenting lipid antigens to Natural Killer T-cells (NKT cells), the Cluster of Differentiation 1D Glycoprotein (CD1d) might mediate reverse signaling in antigen-presenting cells (APCs). Here we found CD1d deficiency attenuated Toll-like receptor (TLR)-triggered inflammatory innate responses in macrophages and dendritic cells, protecting mice from endotoxin shock. TLR activation in macrophages induced metabolic changes of glycosphingolipids (GSLs), among which glycolipid isoglobotrihexosylceramide (iGb3) was rapidly produced. The endogenously generated iGb3 bound CD1d in endosomal compartments and then synergized with the initially activated TLR signal to induce Tyr332 phosphorylation of CD1d intracellular domain. This led to the recruitment and activation of proline-rich tyrosine kinase 2 (Pyk2). Pyk2 interacted with IκB kinase β (IKKβ) and TANK-binding kinase 1 (TBK1), and enhanced tyrosine phosphorylation of Tyr188/199 of IKKβ and Tyr179 of TBK1 and thus, their activation to promote full activation of TLR signaling. Thus, intracellular CD1d reverse signaling, triggered by endogenous iGb3, amplifies inflammatory innate responses in APCs. Our findings identify a non-canonical function of CD1d reverse signaling activated by lipid metabolite in the innate immune response.

S-Nitrosylation of proline-rich tyrosine kinase 2 involves its activation induced by oxygen–glucose deprivation

Previous studies have demonstrated that activation of proline-rich tyrosine kinase 2 (PYK2) in cerebral ischemia is involved in the modulation of N-methyl-d-aspartate-type (NMDA) glutamate receptor activity and Ca2+ dynamics, resulting in ischemic neuron death ultimately. A number of reports indicate that PYK2 is a redox sensitive kinase that must be activated by an estrogen-induced reactive oxygen species (ROS). However, the mechanism of PYK2 activation remains incompletely illustrated. Accumulating attention is focused on nitric oxide (NO, a free radical) which plays a critical role in cellular signal transduction through stimulus-coupled S-nitrosylation of cysteine residues. Here we reported that PYK2 over-expressed in human embryonic kidney (HEK293) cells was S-nitrosylated (forming SNO–PYK2) by reacting with GSNO, an exogenous NO donor, at one critical cysteine residue (Cys534) with a biotin switch assay. Moreover, our results showed that S-nitrosylation and phosphorylation of PYK2 over-expressed in SH-SY5Y cells was significantly increased after oxygen–glucose deprivation (OGD). We further investigated whether the activation (phosphorylation) of PYK2 was associated with S-nitrosylation following SH-SY5Y cells OGD. Our results showed that the cysteine534 residue (site of S-nitrosylation) mutant PYK2 over-expressed in SH-SY5Y cells diminished S-nitrosylation of PYK2 and inhibited its phosphorylation induced by OGD. In addition, overexpression of the mutant PYK2 protein could prevent nuclear accumulation and abrogate neuronal cell death compared to wild type PYK2 in SH-SY5Y cells induced by OGD. These data suggest that the activation of PYK2 following OGD may be modulated by S-nitrosylation, which provides a new avenue for stroke therapy by targeting the post-translational modification machinery.

Tyrosine Phosphorylation of Mitochondrial Ca2+ Uniporter Regulates Mitochondrial Ca2+ Uptake

Mitochondrial Ca2+ has a critical role for balancing cell survival and death. Ca2+ influx into mitochondrial matrix is mediated primarily by the mitochondrial calcium uniporter (MCU). However, the signaling pathways that regulate MCU channel functions via post-translational modifications of MCU are completely unknown. Here we show that adrenergic signaling induces MCU tyrosine phosphorylation and accelerates mitochondrial Ca2+ uptake in cardiac cells. Adrenergic signaling induces activation of proline-rich tyrosine kinase 2 (Pyk2) and translocation into the mitochondrial matrix; enhancing the interaction between Pyk2 and MCU, which subsequently accelerates mitochondrial Ca2+ uptake via Pyk2-dependent MCU tyrosine phosphorylation. MCU contains 15 tyrosine residues (5 in the N-terminus, 0 in the pore-forming region, 4 in transmembrane domains and 6 in the C-terminus), which are conserved across all eukaryotic species. Among them, only 3 of these tyrosine residues (Y157 at N-terminus, Y288, and Y316 at C-terminus in mouse MCU) remained as potential phosphorylation candidate sites for protein tyrosine kinases using phosphorylation prediction programs. We mutated these tyrosine residues to phenylalanine and generated non-phosphorylation mimetic MCU mutants (MCU-YFs). We confirmed that only two tyrosine sites were phosphorylated in response to adrenergic stimulation in situ using cell lines stably expressing MCU-YFs. In addition, overexpression of these MCU-YFs failed to increase mitochondrial Ca2+ uptake in response to cytosolic Ca2+ elevation by thapsigargin, whereas wild-type MCU transfection dramatically accelerates mitochondrial Ca2+ uptake compared to non-transfected cells. In summary, MCU contains Pyk2-specific phosphorylation site(s) and Pyk2-dependent tyrosine phosphorylation of MCU can modulate its channel functions and regulate mitochondrial Ca2+ uptake.

Expression and Activation of Proline-Rich Tyrosine Kinase 2 (PYK2) in Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus

This study analysed the expression and activation of proline-rich tyrosine kinase 2 (PYK2) in peripheral blood mononuclear cells (PBMCs) from 36 systemic lupus erythematosus (SLE) patients and explored whether activation of PYK2 correlates with disease activity or organ damage in SLE. Samples from 19 patients with rheumatoid arthritis (RA) and 15 healthy individuals were included as controls. There was a significant increase in both total PYK2 protein and its activated/phosphorylated form in PBMCs from patients with SLE, particularly in those with the complication of World Health Organization class IV lupus nephritis. There was a clear correlation between the activation of PYK2 and the level of serum complement, but no relationship was found between the activation of PYK2 and SLE Disease Activity Index (SLEDAI). As previous studies have shown that PYK2 provides important signals during the activation of lymphocytes, PYK2 could be a major contributor to the complex autoimmune pathogenesis of SLE.

Dependence of Peroxisome Proliferator-activated Receptor Ligand-induced Mitogen-activated Protein Kinase Signaling on Epidermal Growth Factor Receptor Transactivation

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that function as ligand-activated transcription factors regulating lipid metabolism and homeostasis. In addition to their ability to regulate PPAR-mediated gene transcription, PPARalpha and gamma ligands have recently been shown to induce activation of mitogen-activated protein kinases (MAPKs), which in turn phosphorylate PPARs, thereby affecting transcriptional activity. However, the mechanism for PPAR ligand-dependent MAPK activation is unclear. In the current study, we demonstrate that various PPARalpha (nafenopin) and gamma (ciglitazone and troglitazone) agonists rapidly induced extracellular signal-regulated kinase (Erk) and/or p38 phosphorylation in rat liver epithelial cells (GN4). The selective epidermal growth factor receptor (EGFR) kinase inhibitors, PD153035 and ZD1839 (Iressa), abolished PPARalpha and gamma agonist-dependent Erk activation. Consistent with this, PPAR agonists increased tyrosine autophosphorylation of the EGFR as well as phosphorylation at a putative Src-specific site, Tyr845. Experiments with the Src inhibitor, PP2, and the antioxidant N-acetyl-L-cysteine revealed critical roles for Src and reactive oxygen species as upstream mediators of EGFR transactivation in response to PPAR ligands. Moreover, PPARalpha and gamma ligands increased Src autophosphorylation as well as kinase activity. EGFR phosphorylation, in turn, led to Ras-dependent Erk activation. In contrast, p38 activation by PPARalpha and gamma ligands occurred independently of Src, oxidative stress, the EGFR, and Ras. Interestingly, PPARalpha and gamma agonists caused rapid activation of proline-rich tyrosine kinase or Pyk2; Pyk2 as well as p38 phosphorylation was reduced by intracellular Ca2+ chelation without an observable effect on EGFR and Erk activation, suggesting a possible role for Pyk2 as an upstream activator of p38. In summary, PPARalpha and gamma ligands activate two distinct signaling cascades in GN4 cells leading to MAPK activation.

Two types of calcium channels regulating activation of proline-rich tyrosine kinase 2 induced by transient brain ischemia in rat hippocampus

Tyrosine phosphorylation is an important means for regulating post-ischemic signal transduction. In this article, brain ischemia was induced by four-vessel occlusion, and the effect of ischemia/reperfusion on proline-rich tyrosine kinase 2 (Pyk2) was studied. Tyrosine phosphorylation of Pyk2 in Sprague–Dawley rat hippocampus after transient (15 min) brain ischemia and reperfusion was examined by immunoprecipitation and immunoblot. Kinase activity of Pyk2 was examined by the method of 32P-incorporation into poly(Glu-Tyr). Tyrosine phosphorylation and kinase activity of Pyk2 decreased slightly after ischemia, then increased after reperfusion and reached the peak levels (5.1 and 1.8 times the levels of the sham-operated group, respectively) at 1 h of reperfusion. Both the increases were partly inhibited by NMDA receptor antagonist ketamine and L-type voltage-gated calcium channel antagonist nifedipine administered 20 min before ischemia. The results suggested that Pyk2 was activated after transient brain ischemia and reperfusion, and it might play an important role in mediating post-ischemic signal transduction events.

Chronic ethanol feeding impairs endothelin-1-stimulated glucose uptake via decreased G alpha 11 expression in rat adipocytes.

Chronic ethanol feeding decreases insulin-stimulated glucose uptake in rat adipocytes. Here, we show that chronic ethanol also decreases endothelin-stimulated glucose uptake. Endothelin-1 increased uptake of 2-deoxyglucose 2.4-fold in adipocytes isolated from pair-fed rats. However, in adipocytes isolated from rats that had consumed a diet containing 35% ethanol for 4 wk, endothelin-1 did not increase glucose uptake. Although endothelin-1 increased GLUT4 quantity at the plasma membrane in adipocytes from pair-fed rats, there was no increase in GLUT4 after chronic ethanol feeding. Loss of endothelin-1-stimulated glucose uptake after ethanol feeding was associated with a specific decrease in the quantity of Galpha11 in plasma membranes, with no change in Galphaq quantity. Activation of proline-rich tyrosine kinase 2 (PYK2), a downstream target of Galphaq/11 that is required for endothelin-1-stimulated GLUT4 translocation in 3T3-L1 adipocytes, was also suppressed after chronic ethanol feeding. In contrast, activation of p38 MAPK by endothelin-1 was not affected by chronic ethanol exposure. These data demonstrate that chronic ethanol feeding suppresses endothelin-1-stimulated glucose uptake and suggest that decreased expression of Galpha11 coupled to impaired endothelin-1-dependent activation of PYK2 contributes to this response.