Oncostatin M induces tyrosine phosphorylation in endothelial cells and activation of p62yes tyrosine kinase.

Oncostatin M is a polypeptide cytokine produced by activated and transformed T lymphocytes that has diverse biologic effects, including growth inhibition of tumor cells and induction of IL-6 expression in cultured human endothelial cells (HEC). HEC are highly responsive to oncostatin M and express high levels of oncostatin M receptors relative to other cell types. Oncostatin M has previously been found to bind a specific receptor of 150 to 160 kDa. We have found through the use of anti-phosphotyrosine immunoblotting that oncostatin M induces tyrosine phosphorylation in HEC. Anti-phosphotyrosine antibodies specifically immunoprecipitated labeled oncostatin M cross-linked to its receptor, demonstrating that the oncostatin M receptor is either directly phosphorylated on tyrosine after ligand binding or is tightly associated with a phosphotyrosyl protein in these cells. The tyrosine kinase inhibitor herbimycin A blocked the induction of IL-6 by oncostatin M in HEC. In addition, immune complex kinase assays showed that oncostatin M markedly increased the activity of the p62yes tyrosine kinase with a small increase in p59fyn but no increase in p56lyn tyrosine kinase activity in HEC. We conclude that oncostatin M utilizes a tyrosine phosphorylation signal transduction pathway in HEC involving the activation of the p62yes tyrosine kinase, and that this tyrosine phosphorylation pathway leads to the induction of IL-6 expression.     

Reduced tyrosine phosphorylation and nonresponsiveness to EGF-mediated cytotoxicity in EGF receptor-hyperproducing UCVA-1 cells

UCVA-1 cells, derived from human pancreas adenocarcinoma, have a high number of epidermal growth factor (EGF) receptors (1.0 x 10(6) per cell) but their growth is not inhibited by EGF, unlike other EGF receptor-hyperproducing tumour cells. In UCVA-1 cells EGF activates neither the phosphatidylinositol turnover nor protein kinase C. EGF, however, enhances the phosphorylation of EGF receptors at specific tyrosine residues, indicating that the EGF receptor kinase is active and subject to autophosphorylation. Downmodulation of EGF receptors by 12-O-tetradecanoylphorbol 13-acetate (TPA) is also observed. Using an anti-phosphotyrosine antibody several phosphoproteins, including EGF receptors, were immunoprecipitated from UCVA-1 cell lysates, whereas more than 20 phosphoproteins were detected in other EGF receptor-hyperproducing tumour cells (NA), indicating that tyrosine-phosphorylation of endogenous substrates by EGF receptor kinase is significantly reduced in UCVA-1 cells. Thus, non-responsiveness of UCVA-1 cells to EGF is correlated with the reduced tyrosine phosphorylation.     

Growth inhibition and protein tyrosine phosphorylation in MCF 7 breast cancer cells by a novel K vitamin

We have now found that the most potent, Cpd 5 [2-(2-mercaptoethanol)-3-methyl-1, 4-napthoquinone], inhibits growth of doxorubicin-resistant and doxorubicin-sensitive breast cancer cells (MCF 7r and MCF 7w) in culture. Growth inhibition by Cpd 5 was antagonized by the thiol antioxidants glutathione and cysteine, but not by catalase or superoxide dismutase, suggesting that growth inhibition is probably via conjugation of cellular thiols. In support of this, we found that Cpd 5 inhibited the activity of thiol containing cellular protein tyrosine phosphatase (PTP) enzyme, with consequent induction of various tyrosine phosphoproteins, but not serine or tyrosine phosphoproteins. The tyrosine phosphorylation was also inhibited by exogenous glutathione or cysteine and could be enhanced by depletion of cellular glutathione by BSO. This effect of Cpd 5 on protein tyrosine phosphorylation was highly selective, however. Tyrosine phosphorylation of EGF-R, Erb-B2, and ERK1/2 was increased, but not that of Insulin-R or JNK. ERK1/2 tyrosine phosphorylation and growth inhibition increased with increasing concentrations of Cpd 5. Furthermore, suppression of Cpd 5-mediated ERK1/2 phosphorylation by an ERK-kinase inhibitor antagonized growth inhibition. These results suggest a strong correlation between ERK1/2 phosphorylation by Cpd 5 and growth inhibition. This novel K-vitamin analog thus inhibits MCF 7 cell growth and induces selective protein tyrosine phosphorylation.     

EGF-mediated phosphorylation of extracellular signal-regulated kinases in osteoblastic cells

Epidermal growth factor (EGF) induces a rapid increase in the phosphorylation of extracellular signal-regulated kinases (ERKs) in the human osteosarcoma osteoblastic cell line G292 and in primary cultures of rat osteoblastic cells. This phosphorylation is transient and time-dependent. Maximal stimulation is attained within 1 min in G292 and within 5 min in rat osteoblastic cells. Enzymatic activity in G292 cells is also induced rapidly after EGF stimulation. Western blot analysis revealed that enhancement of the phosphorylation of ERKs in the EGF-stimulated cells is not due to an increase in ERK protein, since EGF-treatment does not lead to an increase in the absolute amount of ERKs present even after 2 days of stimulation. The pattern of expression of the ERKs observed in the two cell types differs in the apparent molecular weights observed. The most slowly migrating immunoreactive protein (～45 kDa) in normal rat osteoblastic cells is ERK1, identified by an ERK1-selective antiserum. The same antiserum reacts only weakly with one of the ERK proteins (44 kDa) blotted from the human osteosarcoma cell line G292. Phorbol 12-myristate 13-acetate (PMA) is also capable of inducing ERK phosphorylation, albeit to a lesser degree. The combination of PMA and EGF does not produce a greater response than EGF alone. The role of protein kinase C (PKC) in the EGF-stimulated ERK signaling pathway was further examined by inhibition of PKC with the staurosporine analog, CGP41251, and by down-regulation of PKC via chronic treatment with PMA. Chronic PMA treatment results in a partial inhibition of the EGF-mediated phosphorylation. CGP41251 completely abolishes the increased ERK activity produced by PMA, but the effect of EGF in this regard is potentiated. We conclude that PKC and EGF act through parallel pathways to stimulate ERK phosphorylation and activity. The inhibitor studies, in addition, indicate that activation of PKC may moderate the actions of the EGF pathway via a tonic inhibitory feedback. © 1995 Wiley-Liss, Inc.     

Immunoaffinity profiling of tyrosine phosphorylation in cancer cells

Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.     

Oncostatin M promotes osteogenesis and suppresses adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells

Oncostatin M (OSM) is a multifunctional cytokine of the interleukin-6 family and has been implicated in embryonic development, differentiation, inflammation, and regeneration of liver and bone. In the present study, we demonstrated that treatment of human adipose mesenchymal stem cells (hADSCs) with OSM-attenuated adipogenic differentiation, as indicated by decreased accumulation of intracellular lipid droplets and down-regulated expression of adipocytic markers, such as lipoprotein lipase and PPARgamma. However, OSM treatment stimulated osteogenic differentiation, as demonstrated by the increase in matrix mineralization and expression levels of osteogenic differentiation markers, including alkaline phosphatase, Runx2, and osteocalcin. OSM treatment induced activation of JAK2, JAK3, and ERK in hADSCs, and pre-treatment of hADSCs with the JAK2 inhibitor, AG490, significantly restored the OSM-induced inhibition of adipogenic differentiation. Whereas, the JAK3 inhibitor, WHI-P131, and the MEK inhibitor, U0126, had no effects on the anti-adipogenic activity of OSM. On the other hand, the pro-osteogenic activity of OSM was prevented by treatment of the cells with WHI-P131 or U0126, but not with AG490. These results indicate that distinct signaling pathways, including JAK2, JAK3, and MEK-ERK, play specific roles in the OSM-induced anti-adipogenic and pro-osteogenic differentiation of hADSCs.     

Heat shock induces protein tyrosine phosphorylation in cultured cells

We examined the effect of heat shock on protein tyrosine phosphorylation in cultured animal cells using antiphosphotyrosine antibodies in immunoblotting and immunofluorescence microscopy experiments. Heat shock significantly elevated the level of phosphotyrosine in proteins in most of the cultured cells examined, including fibroblasts, epithelial cells, nerve cells, and muscle cells, but not in Rous sarcoma virus-transformed fibroblasts. The increase in protein tyrosine phosphorylation induced by heat shock occurred in proteins with a wide range of molecular masses and was dependent on the temperature and duration of the heat shock.     

Effects of peroxynitrite-induced protein tyrosine nitration on insulin-stimulated tyrosine phosphorylation in HepG2 cells

Tyrosine nitration by peroxynitrite can affect signal transduction pathways involving tyrosine phosphorylation. The present study was undertaken to investigate the effects of peroxynitrite-induced protein tyrosine nitration on insulin-stimulated tyrosine phosphorylation in HepG2 cells. We show here that exposure of HepG2 cells to peroxynitrite led to a dose-dependent increase in tyrosine nitration of cellular proteins, mainly membrane and nuclear proteins. Furthermore, peroxynitrite induced differential responses in tyrosine phosphorylation of membrane proteins as well as cytosolic proteins according to peroxynitrite concentrations used. Our findings indicate at low concentrations peroxynitrite upregulates the insulin signaling and may operate as a signaling molecule, but at higher concentrations peroxynitrite downregulates the insulin signaling and may be involved in insulin resistance, suggesting peroxynitrite plays a dual role in regulation of the insulin signaling.     

Enhanced phosphorylation of progesterone receptor by protein kinase C in human breast cancer cells

Affinity-isolated progesterone receptor (PR) from human breast cancer cells incubated with [32P]orthophosphate was shown to exist as a phosphoprotein. Exposure of the cells to 10 nM phorbol-12-myristate-13-acetate (PMA) for 10 min increased by 30-40% the amount of label incorporated into the 116-kDa receptor protein. A two-fold increase in the total number of steroid binding sites was also observed in cells receiving PMA treatment. This apparent unmasking of PR binding sites by phosphorylation probably involved conformational changes to existing receptor complexes and affected the eventual state of receptor dissociation or transformation. An increase primarily in the 8 S sedimenting molecular species was observed but PMA treatment also led to the appearance of a smaller, 2-3 S form of receptor (10% of total) that was not present in control samples. When cytosols were partially transformed in vitro by ATP and salt, all molecular species of receptor (8, 4, and 2-3 S) from the PMA-treated samples consistently migrated faster in sucrose gradients. The larger amount of 2-3 S receptor in PMA-treated samples disappeared when ATP, but not salt, was the transforming agent. These results suggest a major role for phosphorylating reactions in the receptor-mediated action of steroids by regulating hormone-binding and influencing receptor transformation. Tumor promoters such as the phorbol esters may act by artificially increasing the level of processing of steroid receptor.     

Taurine suppresses platelet-derived growth factor (PDGF) BB-induced PDGF-beta receptor phosphorylation by protein tyrosine phosphatase-mediated dephosphorylation in vascular smooth muscle cells

In atherosclerosis, abnormal vascular smooth muscle cell (VSMC) proliferation plays an important role to form fibroproliferative lesions and platelet-derived growth factor (PDGF)-BB is one of the most potent chemoattractants and proliferative factors for VSMCs. Taurine, sulfur-containing beta-amino acid, has been considered to prevent the development of atherosclerosis, although the molecular mechanism remains obscure. Previously, we demonstrated that taurine significantly suppressed PDGF-BB-induced cell proliferation, DNA synthesis, immediate-early gene expressions and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in VSMCs. The present study was aimed at elucidating the precise molecular mechanism of taurine in PDGF-BB signaling pathway. We showed that taurine significantly suppressed PDGF-BB-induced phosphorylation of PDGF-beta receptor and activation of its downstream signaling molecules such as Ras, MAPK/ERK kinase (MEK)1/2 and Akt. Because taurine did not attenuate phorbol 12-myristate 13-acetate (PMA)-induced PDGF-beta receptor-independent ERK1/2 phosphorylation, we further investigated the suppressive mechanism of taurine in PDGF-beta receptor level. Although taurine did not directly affect PDGF receptor autophosphorylation in vitro, taurine promoted PDGF-beta receptor dephosphorylation and restored PDGF-BB-induced suppression of protein tyrosine phosphatase (PTPase) activity. Taken together, we propose that taurine could prevent or delay the progression of atherosclerosis by PTPase-mediated suppression of PDGF-beta receptor phosphorylation, and by decreasing the activation of its downstream signaling molecules in VSMCs.     

EGF-mediated migration signaling activated by N-acetylglucosaminyltransferase-V via receptor protein tyrosine phosphatase kappa

N-acetylglucosaminyltransferase-V (GnT-V) has been reported to be closely associated with tumor migration, but the mechanism has been not currently clear. In this study we found that GnT-V activated EGFR signaling and promoted cell migration through receptor protein tyrosine phosphatase kappa (RPTPκ). The overexpression of GnT-V gene in human hepatoma SMMC-7721 cell line not only induced the addition of β1,6 GlcNAc branch to N-glycan of RPTPκ but also decreased the protein level of RPTPκ, which both contributed to the decreased phosphatase activity of RPTPκ activating subsequently EGFR signaling. Moreover, we found that the knockdown of RPTPκ and its addition of β1,6 GlcNAc branch both promoted cell migration, which could be ascribed to the activation of EGFR signaling regulated by RPTPκ. Therefore, our findings could provide an insight into the molecular mechanism of how GnT-V promoted cell migration, suggesting that RPTPκ could be one of factors regulating the EGF-mediated migration signals.     

Reduced tyrosine phosphorylation in polyamine-starved cells.

Onset of cell proliferation is associated with enhanced turnover of the polyamines putrescine, spermidine, and spermine, particularly evident in the massive increase in the activity of the rate-limiting enzyme in their production, ornithine decarboxylase (ODC). The physiological functions of these polyamines, however, have remained unclear. Here we report that treatment of LSTRA cells for 2-18 h with alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, decreased the amount of phosphotyrosine in several cellular substrates including the T cell protein tyrosine kinase p56lck. No reductions in the amount of p56lck, overall synthesis of protein and DNA, or cell viability were observed until much later. DFMO did not affect the catalytic activity of p56lck in vitro and the activity of p56lck immunoprecipitated from DFMO-treated cells was unaltered. Addition of putrescine, the reaction product of ODC, completely reversed the effect of DFMO on tyrosine phosphorylation. Finally, we provide evidence that polyamines reduce the activity of cellular protein tyrosine phosphatases toward endogenous substrates. Our results suggest that polyamines may influence the extent of tyrosine phosphorylation during cell proliferation and malignant transformation, perhaps by modulating the rate of dephosphorylation of specific target proteins.     

Insulin-stimulated protein tyrosine phosphorylation in intact cells evaluated by giant two-dimensional gel electrophoresis

We have studied the insulin-stimulated phosphorylation of proteins in NIH 3T3 cells expressing high numbers of human insulin receptors (HIR 3.5 cells) using the technique of giant two-dimensional gel electrophoresis. In serum-deprived cells, insulin stimulated the phosphorylation of more than 25 proteins; all but two of these were also phosphorylated in response to 15% (v/v) fetal bovine serum, which also stimulated the phosphorylation of additional proteins thought to be direct substrates for protein kinase C. In cells pretreated insulin specifically stimulated the phosphorylation insulin specifically stimulated the phosphorylation of at least 26 predominantly cytosolic proteins, only one of which was observed in insulin-treated cells not exposed to phenylarsine oxide. Serum was without effect in cells pretreated with phenylarsine oxide. In phenylarsine oxide-pretreated cells, phosphoamino acid analysis of 10 of the most highly labeled insulin-stimulated phosphoproteins showed that all 10 were labeled predominantly or exclusively on tyrosine residues. The phosphorylation of several of these could be stimulated in vitro by the addition of insulin to a detergent extract of cells in the presence of Mn2+ and ATP. In general, the insulin-stimulated phosphorylations observed in the presence of phenylarsine oxide were more rapid than those observed in its absence. Finally, a variety of other growth factors and mitogens did not stimulate any of the insulin-stimulated phosphorylations in the presence of phenylarsine oxide. Thus, the use of this inhibitor apparently unmasked a number of novel insulin-specific protein phosphorylations that were ordinarily undetectable. We suggest that at least some of these proteins may be direct substrates for the insulin receptor protein tyrosine kinase and may play significant roles in insulin action.     

Heparin rapidly and selectively regulates protein tyrosine phosphorylation in vascular smooth muscle cells

Aberrant vascular smooth muscle cell (VSMC) hyperplasia is the hallmark of atherosclerosis and restenosis seen after vascular surgery. Heparin inhibits VSMC proliferation in animal models and in cell culture. To test our hypothesis that heparin mediates its antiproliferative effect by altering phosphorylation of key mitogenic signaling proteins in VSMC, we examined tyrosine phosphorylation of cellular proteins in quiescent VSMC stimulated with serum in the presence or absence of heparin. Western blot analysis with anti-phosphotyrosine antibodies shows that heparin specifically alters the tyrosine phosphorylation of only two proteins (42 kDa and 200 kDa). The 200 kDa protein (p200) is dephosphorylated within 2.5 min after heparin treatment with an IC50 that closely parallels the IC50 for growth inhibition. Studies using the tyrosine phosphatase inhibitor, sodium orthovanadate, indicate that heparin blocks p200 phosphorylation by inhibiting a kinase. Phosphorylation of p200 is not altered in heparin-resistant cells, supporting a role for p200 in mediating the antiproliferative effect of heparin. Purification and sequence analysis indicate that p200 exhibits very high homology to the heavy chain of nonmuscle myosin IIA. The 42 kDa protein, identified as mitogen activated protein kinase (MAPK), undergoes dephosphorylation within 15 min after heparin treatment, and this effect is also not seen in heparin-resistant cells. The identification of only two heparin-regulated tyrosine phosphoproteins suggests that they may be key mediators of the antiproliferative effect of heparin.     

C-reactive protein suppresses insulin signaling in endothelial cells: role of spleen tyrosine kinase

Although few epidemiological studies have demonstrated that C-reactive protein (CRP) is related to insulin resistance, no study to date has examined the molecular mechanism. Here, we show that recombinant CRP attenuates insulin signaling through the regulation of spleen tyrosine kinase (Syk) on small G-protein RhoA, jun N-terminal kinase (JNK) MAPK, insulin receptor substrate-1 (IRS-1), and endothelial nitric oxide synthase in vascular endothelial cells. Recombinant CRP suppressed insulin-induced NO production, inhibited the phosphorylation of Akt and endothelial nitric oxide synthase, and stimulated the phosphorylation of IRS-1 at the Ser307 site in a dose-dependent manner. These events were blocked by treatment with an inhibitor of RhoA-dependent kinase Y27632, or an inhibitor of JNK SP600125, or the transfection of dominant negative RhoA cDNA. Next, anti-CD64 Fcgamma phagocytic receptor I (FcgammaRI), but not anti-CD16 (FcgammaRIIIa) or anti-CD32 (FcgammaRII) antibody, partially blocked the recombinant CRP-induced phosphorylation of JNK and IRS-1 and restored, to a certain extent, the insulin-stimulated phosphorylation of Akt. Furthermore, we identified that recombinant CRP modulates the phosphorylation of Syk tyrosine kinase in endothelial cells. Piceatannol, an inhibitor of Syk tyrosine kinase, or infection of Syk small interference RNA blocked the recombinant CRP-induced RhoA activity and the phosphorylation of JNK and IRS-1. In addition, piceatannol also restrained CRP-induced endothelin-1 production. We conclude that recombinant CRP induces endothelial insulin resistance and dysfunction, and propose a new mechanism by which recombinant CRP induces the phosphorylation of JNK and IRS-1 at the Ser307 site through a Syk tyrosine kinase and RhoA-activation signaling pathway.     

Signal processing by protein tyrosine phosphorylation in plants

Protein phosphorylation is a reversible post-translational modification controlling many biological processes. Most phosphorylation occurs on serine and threonine, and to a less extend on tyrosine (Tyr). In animals, Tyr phosphorylation is crucial for the regulation of many responses such as growth or differentiation. Only recently with the development of mass spectrometry, it has been reported that Tyr phosphorylation is as important in plants as in animals. The genes encoding protein Tyr kinases and protein Tyr phosphatases have been identified in the Arabidopsis thaliana genome. Putative substrates of these enzymes, and thus Tyr-phosphorylated proteins have been reported by proteomic studies based on accurate mass spectrometry analysis of the phosphopeptides and phosphoproteins. Biochemical approaches, pharmacology and genetic manipulations have indicated that responses to stress and developmental processes involve changes in protein Tyr phosphorylation. The aim of this review is to present an update on Tyr phosphorylation in plants in order to better assess the role of this post-translational modification in plant physiology.Key words: protein tyrosine phosphorylation, kinases, phosphatases, proteomics, mass spectrometry, signaling     

Smooth muscle contractility and protein tyrosine phosphorylation

During the last 5 years several studies have documented an involvement of protein tyrosine kinases (PTKs) in smooth muscle contraction and Ca²⁺mobilization. Most of these studies have utilized highly selective inhibitors of PTKs, genistein and tyrphostin and have shown that these inhibitors attenuated smooth muscle contraction induced by growth factors - epidermal growth factor (EGF) and platelet derived growth factor (PDGF) and several vasoactive peptides. It has also been demonstrated that inhibitors of protein tyrosine phosphatases (PTPases) such as vanadate and pervanadate mimic growth factors and vasoactive peptides in causing the contraction of smooth muscle. In this brief review, we have summarized some of the recent observations suggesting a possible link between protein tyrosine phosphorylation pathway and smooth muscle contraction.