Tyrosine Phosphorylation Signaling Pathways Research Articles

Diffusion-weighted magnetic resonance imaging reflects activation of signal transducer and activator of transcription 3 during focal cerebral ischemia/reperfusion.

Signal transducer and activator of transcription (STAT) is a unique protein family that binds to DNA, coupled with tyrosine phosphorylation signaling pathways, acting as a transcriptional regulator to mediate a variety of biological effects. Cerebral ischemia and reperfusion can activate STATs signaling pathway, but no studies have confirmed whether STAT activation can be verified by diffusion-weighted magnetic resonance imaging (DWI) in rats after cerebral ischemia/reperfusion. Here, we established a rat model of focal cerebral ischemia injury using the modified Longa method. DWI revealed hyperintensity in parts of the left hemisphere before reperfusion and a low apparent diffusion coefficient. STAT3 protein expression showed no significant change after reperfusion, but phosphorylated STAT3 expression began to increase after 30 minutes of reperfusion and peaked at 24 hours. Pearson correlation analysis showed that STAT3 activation was correlated positively with the relative apparent diffusion coefficient and negatively with the DWI abnormal signal area. These results indicate that DWI is a reliable representation of the infarct area and reflects STAT phosphorylation in rat brain following focal cerebral ischemia/reperfusion.

Proteomic analysis of SP600125‐controlled TrkA‐dependent targets in SK‐N‐MC neuroblastoma cells: Inhibition of TrkA activity by SP600125

The c-Jun N-terminal kinase (JNK) is well known to play an important role in cell death signaling of the p75 neurotrophin receptor. However, little has been studied about a role of JNK in the signaling pathways of the tropomyosin-related kinase A (TrkA) neurotrophin receptor. In this study, we investigated JNK inhibitor SP600125-controlled TrkA-dependent targets by proteomic analysis to better understand an involvement of JNK in TrkA-mediated signaling pathways. PDQuest image analysis and protein identification results showed that hnRNP C1/C2, α-tubulin, β-tubulin homolog, actin homolog, and eIF-5A-1 protein spots were upregulated by ectopic expression of TrkA, whereas α-enolase, peroxiredoxin-6, PROS-27, HSP70, PP1-gamma, and PDH E1-alpha were downregulated by TrkA, and these TrkA-dependent upregulation and downregulation were significantly suppressed by SP600125. Notably, TrkA largely affected certain PTM(s) but not total protein amounts of the SP600125-controlled TrkA-dependent targets. Moreover, SP600125 strongly suppressed TrkA-mediated tyrosine phosphorylation signaling pathways as well as JNK signaling, indicating that SP600125 could function as a TrkA inhibitor. Taken together, our results suggest that TrkA could play an important role in the cytoskeleton, cell death, cellular processing, and glucose metabolism through activation or inactivation of the SP600125-controlled TrkA-dependent targets.

Proteomic analysis of novel targets associated with TrkA‐mediated tyrosine phosphorylation signaling pathways inSK‐N‐MCneuroblastoma cells

Tropomyosin-related kinase A (TrkA) is a receptor-type protein tyrosine kinase and exploits pleiotypic roles via nerve growth factor (NGF)-dependent or NGF-independent mechanisms in various cell types. Here, we showed that the inhibition of TrkA activity by GW441756 resulted in the suppression of tyrosine phosphorylation of cellular proteins including extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK). To find novel targets associated with TrkA-mediated tyrosine phosphorylation signaling pathways, we investigated GW441756 effects on TrkA-dependent targets in SK-N-MC neuroblastoma cells by proteomic analysis. The major TrkA-dependent protein spots controlled by GW441756 were determined by PDQuest image analysis, identified by MALDI-TOF MS and MALDI-TOF/TOF MS/MS, and verified by 2DE/Western blot analysis. Thus, we found that most of the identified protein spots were modified forms in a normal condition, and their modifications were regulated by TrkA activity. Especially, our results demonstrated that the modifications of α-tubulin and heterogeneous nuclear ribonucleoproteins C1/C2 (hnRNP C1/C2) were significantly upregulated by TrkA, whereas α-enolase modification was downregulated by TrkA, and it was suppressed by GW441756, indicating that TrkA activity is required for their modifications. Taken together, we suggest here that the major novel TrkA-dependent targets such as α-tubulin, hnRNP C1/C2, and α-enolase could play an essential role in TrkA-mediated tyrosine phosphorylation signaling pathways via regulation of their posttranslational modifications.

Regulation of airway goblet cell mucin secretion by tyrosine phosphorylation signaling pathways

Mucus hyperproduction in pulmonary obstructive diseases results from increased goblet cell numbers and possibly increased cellular mucin synthesis, occurring in response to inflammatory mediators acting via receptor tyrosine kinases (RYK) and tyrosine phosphorylation (Y-Pi) signaling pathways. Yet, increased mucin synthesis does not lead necessarily to increased secretion, as mucins are stored in secretory granules and secreted in response to extracellular signals, commonly assumed to be mediated by G protein-coupled receptors (GPCRs). We asked whether activation 1) of Y-Pi signaling pathways, in principal, and 2) of the novel PKC isoform, nPKCdelta, by Y-Pi, specifically, might lead to regulated mucin secretion. nPKCdelta in SPOC1 cells was tyrosine phosphorylated by exposure to purinergic agonist (ATPgammaS) or PMA, actions that were blocked by the Src kinase inhibitor, PP1. Mucin secretion, however, was not affected by PP1. Hence, activation of nPKCdelta by Y-Pi is unlikely to participate in GPCR-related mucin secretion. Mucin secretion from both SPOC1 and normal human bronchial epithelial (NHBE) cells was stimulated by generalized protein Y-Pi induced by the tyrosine phosphatase inhibitor, pervanadate (PV). PV-induced SPOC1 cell mucin secretion was not affected by inhibition of Src kinases (genistein or PP1), or of PI3 kinase (LY-294002). MAP kinase pathway inhibitors, RAF1 kinase inhibitor-I and U0126 (MEK), inhibited SPOC1 cell PV-induced secretion by approximately 50%. Significantly, the phospholipase C (PLC) inhibitor, U-73122, essentially abolished PV- and ATPgammaS-induced mucin secretion from both SPOC1 and NHBE cells. Hence, PLC signaling may play a key role in regulated mucin secretion, whether the event is initiated by mediators interacting with GPCRs or RYKs.

The Docking Protein Cas Links Tyrosine Phosphorylation Signaling to Elongation of Cerebellar Granule Cell Axons

Crk-associated substrate (Cas) is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of the actin cytoskeletal organization and cell migration in fibroblasts. The function of Cas in neurons, however, is poorly understood. Here we report that Cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. During cerebellar development, Cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. Cas coimmunoprecipitates with Src family protein tyrosine kinases, Crk, and cell adhesion molecules and colocalizes with these proteins in granule cells. The axon extension of granule cells is inhibited by either RNA interference knockdown of Cas or overexpression of the Cas mutant lacking the YDxP motifs, which are tyrosine phosphorylated and thereby interact with Crk. These findings demonstrate that Cas acts as a key scaffold that links the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation.

Prolactin stimulates integrin-mediated adhesion of circulating mononuclear cells to endothelial cells

Attachment of leukocytes to endothelial cells is an essential step for the extravasation and recruitment of cells at sites of inflammation. The pituitary hormone prolactin (PRL) is involved in the inflammatory process. Here, we show that treatment with PRL of human peripheral blood mononuclear cells (PBMC) stimulates their adhesion to human umbilical vein endothelial cells (HUVEC) activated by interleukin-1β. Stimulation of adhesion by PRL is mediated via integrins leukocyte functional antigen-1 (LFA-1) and very late antigen-4 (VLA-4), because immunoneutralization of both integrins prevents PRL action. Also, PRL promotes the adhesion of PBMC to immobilized intercellular adhesion molecule-1 and fibronectin, ligands for LFA-1 and VLA-4, respectively. Stimulation of integrin-mediated cell adhesion by PRL may involve the activation of chemokine receptors, because PRL upregulates the expression of the G-protein-coupled chemokine receptor CXCR3 in PBMC, and pertussis toxin, a specific G-protein inhibitor, blocks PRL stimulation of PBMC adhesion to HUVEC. In addition, PRL stimulates tyrosine phosphorylation pathways leading to leukocyte adhesion. PRL triggered the tyrosine phosphorylation of Janus kinase-2, of signal transducer and activator of transcription-3 and 5, and of the focal adhesion protein paxillin. Furthermore, genistein, a tyrosine kinase inhibitor, blocked PRL-stimulated adhesion of PBMC and Jurkat T-cells to HUVEC. These results suggest that PRL promotes integrin-mediated leukocyte adhesion to endothelial cells via chemokine receptors and tyrosine phosphorylation signaling pathways.

Tyrosine-317 of p52(Shc) mediates androgen-stimulated proliferation signals in human prostate cancer cells.

The involvement of tyrosine phosphorylation signaling pathways in steroid-induced cell proliferation has received much attention. However, the adaptor molecule that mediates this interaction remains to be identified. In this communication, we identify p52(Shc) as the mediator between tyrosine phosphorylation signaling and steroid signaling in steroid-responsive cell proliferation. Although the different LNCaP prostate cancer cells, C-33, C-51 and C-81, express similar levels of functional androgen receptor (AR), they exhibit different levels of androgen sensitivity. C-33 cell proliferation is highly responsive to the presence of androgens, whereas C-51 cell proliferation is comparatively less responsive to androgens. In contrast, C-81 cell proliferation is independent of androgens. In these cells, tyrosine phosphorylation levels of both p52(Shc) and ErbB-2 were greatest in C-81 cells, comparatively less in C-51 cells and weaker in C-33 cells. The levels and activity of protein tyrosine phosphatase, cellular prostatic acid phosphatase, decreased correspondingly in those cells. In both androgen-independent, rapidly growing C-81 and ErbB-2 cDNA-transfected C-33 cells, p52(Shc) was hyperphosphorylated at Tyr317 (Y317). Conversely, p52(Shc) tyrophosphorylation was decreased in prostatic acid phosphatase cDNA-transfected stable subclones of C-81 cells, which restore androgen-sensitive proliferation and leads to slow growth rates. In C-33 cells, androgen-stimulated cell proliferation correlated with tyrophosphorylation of ErbB-2 and increased phosphorylation of p52(Shc) at Y317, but not at Y239, differing from phosphorylation patterns associated with epidermal growth factor (EGF) stimulation. Furthermore, overexpression of a mutant of p52(Shc), that is Y317F, blocks Y317 phosphorylation of endogenous p52(Shc) and abolishes androgen-stimulated proliferation, but not EGF-stimulated proliferation. Thus, Y317 of p52(Shc) serves as an important regulatory site that allows tyrosine phosphorylation pathways to moderate androgen sensitivity in human prostate cancer cells.

Expression of Voltage-Gated Potassium Channels Decreases Cellular Protein Tyrosine Phosphorylation

Protein tyrosine phosphorylation by endogenous and expressed tyrosine kinases is reduced markedly by the expression of functional voltage-gated potassium (Kv) channels. The levels of tyrosine kinase protein and cellular protein substrates are unaffected, consistent with a reduction in tyrosine phosphorylation that results from inhibition of protein tyrosine kinase activity. The attenuation of protein tyrosine phosphorylation is correlated with the gating properties of expressed wild-type and mutant Kv channels. Furthermore, cellular protein tyrosine phosphorylation is reduced within minutes by acute treatment with the electrogenic potassium ionophore valinomycin. Because tyrosine phosphorylation in turn influences Kv channel activity, these results suggest that reciprocal modulatory interactions occur between Kv channel and protein tyrosine phosphorylation signaling pathways.

Antigen- and ionophore-induced signal transduction in rat basophilic leukemia cells involves protein tyrosine phosphorylation.

Treatment of rat basophilic leukemia cells (RBL-2H3) with antigen or ionophore leads to an increase in cellular protein tyrosine phosphorylation. Three major proteins of molecular mass of 72, 92, and 110 kDa are targeted by antigen and a 110-kDa species by ionophore, A23187. The antigen- and ionophore-induced tyrosine phosphorylation responses are dose-dependent and correlate with increases in serotonin release from activated cells. The presence of extracellular Ca2+ is required to sustain the antigen- and ionophore-stimulated tyrosine phosphorylation as well as mediator release. A protein tyrosine kinase inhibitor, RG 50864, differentially inhibits the antigen-stimulated tyrosine phosphorylation in the decreasing order of 72, 91, and 110-kDa proteins. The compound inhibition of the 72-kDa protein tyrosine phosphorylation correlates with that of serotonin release. In ionophore-stimulated cells, the inhibition of the 110-kDa protein tyrosine phosphorylation and serotonin release by RG 50864 occurs in parallel. These results suggest that the 72- and 110-kDa phosphoproteins may represent the respective regulators of serotonin release in antigen- and ionophore-activated cells. The 110-kDa tyrosine phosphorylated proteins from antigen- and ionophore-stimulated cells exhibit identical electrophoretic mobility and V8 protease-generated phosphopeptide maps, suggesting that these two proteins may be the same. These results provide new evidence that both the stimulatory actions of antigen and ionophore on mediator release are mediated through enhanced protein tyrosine phosphorylation in RBL-2H3 cells. Significantly, the present study suggests the presence of multiple tyrosine phosphorylation signaling pathways in RBL cells and that their selective utility may be determined by the nature of the stimulus.