Characterization of Tyrosine-Phosphorylated Isoform of Protein Kinase C Isolated from Chinese Hamster Ovary Cells
Makoto KadotaniTeruaki NishiumaMasakazu NanahoshiYosuke TsujishitaKouji OgitaSaori NakamuraUshio KikkawaYoshinori Asaoka
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Phorbol ester treatment of Chinese hamster ovary cells stably overexpressing the δ isoform of protein kinase C induced the association of the isoform with the particulate fraction and the tyrosine phosphorylation of a small portion of the δ isoform. The δ isoform without tyrosine phosphorylation was recovered as an enzyme dependent on phospholipid and diacylglycerol, whereas the tyrosine-phosphorylated δ isoform was recovered in two fractions, one dependent on, and the other independent of, phospholipid and diacylglycerol. The tyrosine-phosphorylated δ isoform independent of lipid activators might be associated with phorbol ester and phospholipids. Immunoblot analysis revealed that the δ isoform is a doublet protein of 76 and 78 kDa, and that the δ isoform fraction without tyrosine phosphorylation contained 76- and 78-kDa proteins, whereas the tyrosine-phosphorylated δ isoform contained the 78-kDa protein but not the 76-kDa protein. In vitro analysis showed that the 78-kDa protein of the δ isoform without tyrosine phosphorylation is an efficient substrate of tyrosine kinase only when phosphatidylserine and either diacylglycerol or phorbol ester are present; however, the 76-kDa protein can not be tyrosine-phosphorylated even in the presence of these lipid activators. The phospholipid and diacylglycerol-dependent form of the tyrosine-phosphorylated enzyme isolated from the cell line required lower concentrations of phosphatidylserine and phorbol ester for its activity in vitro as compared with the enzyme without tyrosine phosphorylation. These results suggest that the tyrosine-phosphorylated enzyme generated upon stimulation of the cells may associate with membranes and exert its full activity even with the lower concentrations of the lipid activators.Sodium orthovanadate
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Abstract Tyrosine kinases play a prominent role in various intracellular signal transduction pathways. In this study, β2 integrin (CD11/CD18)-mediated adhesive interactions of human neutrophils (PMN) were mimicked by antibody cross-linking of GD11/CD18. Cross-linking of CD18 induced tyrosine phosphorylation of several proteins with apparent molecular masses of ~120, 78, 72, 65, and 56 kDa, respectively, as shown by anti-phosphotyrosine immunoblotting of whole cell lysates. Cross-linking of the α-subunits of the β2 integrins demonstrated that only cross-linking of Mac-1 but not LFA-1 or gp150/95 triggered tyrosine signaling. The tyrosine phosphorylations showed a rapid and transient time course. Comparison of the CD18-mediated tyrosine phosphorylation patterns with those induced by chemoattractants gave similar results. The observed tyrosine phosphorylation was specific, since binding and non-binding irrelevant primary antibodies had no effect. Furthermore, F(ab’)2 fragments of the anti-CD18 antibody IB4 and addition of F(ab’)2 fragments of secondary antibody were sufficient to induce tyrosine phosphorylation. Inhibition of tyrosine kinases by herbimycin A resulted in partial inhibition of the CD18-mediated tyrosine phosphorylation of the 120- and 65-kDa proteins and in complete inhibition of tyrosine phosphorylation of the 78- and 56-kDa proteins. In unstimulated PMN, the tyrosine phosphatase inhibitor sodium orthovanadate had no effect on tyrosine phosphorylation of the 120-kDa protein, but induced phosphorylation of the 78-, 72-, 65-, and 56-kDa proteins. These results indicate that the β2 integrin-mediated intracellular signaling cascade involves different tyrosine phosphorylation (and dephosphorylation) events, which may play a role in the regulation of PMN functions during inflammation.
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Abstract Activation signals of lymphocytes are negatively regulated by the membrane molecules carrying the immunoreceptor tyrosine-based inhibition motif (ITIM). Upon tyrosine phosphorylation, ITIMs recruit SH2-containing phosphatases such as SHP-1, resulting in down-modulation of cell activation. We showed that the cytoplasmic domain of the CD72 molecule carries an ITIM and is associated in vitro with SHP-1 upon tyrosine phosphorylation. Moreover, cross-linking of B cell Ag receptor (BCR) enhances both tyrosine phosphorylation of CD72 and association of CD72 with SHP-1 in B cell line WEHI-231. These results indicate that CD72 recruits SHP-1 upon tyrosine phosphorylation induced by BCR signaling, suggesting that CD72 is a negative regulator of BCR signaling.
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Ligation of the CD95 receptor resulted in a transient increase of cellular tyrosine phosphorylation. The inhibition of protein tyrosine phosphatases by pervanadate, a potent activator of B cells and T cells through the induction of tyrosine phosphorylation and downstream signaling events in the activation cascade, antagonized CD95-triggered apoptosis. Pervanadate exerted its inhibitory effect only during the early phase of apoptosis prior to the CD95-induced decrease of the mitochondrial transmembrane potential. Inhibition of tyrosine phosphatases delayed the cleavage and activation of caspase-8 and caspase-3 and antagonized the tyrosine dephosphorylation of the CD95 receptor-associated phosphoproteins p61 and p89/92. In contrast, ligation of the tumor necrosis factor (TNF) receptor resulted in a continuous tyrosine dephosphorylation of cellular proteins. Pervanadate-induced tyrosine phosphorylation increased the TNF-alpha-induced cytotoxicity and NF-kappaB activation, suggesting that it stimulates early signaling events prior to the separation of the two signaling pathways.
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In this study we compare the effect of CD3 and CD2 ligation on tyrosine kinase activation in human peripheral blood T cells. Using antiphosphotyrosine antibody to detect tyrosine phosphorylation of cellular substrates, we demonstrate that mAb stimulation of either CD3 or CD2 results in tyrosine phosphorylation of the TCR-zeta chain and 135- and 100-kDa proteins. However, differences are observed between CD3 and CD2 ligation; only the former results in rapid tyrosine phosphorylation of 72-, 65-, and 40-kDa substrates. Co-aggregation of CD2 and CD45, a tyrosine phosphatase, results in inhibition of intracellular calcium elevation and T cell proliferation. We demonstrate in this study that this manipulation also inhibits polyphosphoinositide hydrolysis and tyrosine phosphorylation of the 100-kDa substrate. The failure of tyrosine phosphorylation of the 100-kDa substrate is specific in that phosphorylation of the 135-kDa protein is not inhibited. Similar results are observed when CD2 and CD45 are independently cross-linked rather than co-aggregated. The observation that CD45 cross-linking alters tyrosine phosphorylation of T cell substrates and effects polyphosphoinositide hydrolysis is further evidence that tyrosine phosphorylation regulates early events in T cell activation including, perhaps, phospholipase C activity.
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The low molecular weight protein tyrosine phosphatase (LMW−PTP) is a ubiquitously expressed enzyme with several proposed roles in cell signaling. Previously, two tyrosine phosphorylation modifications of LMW−PTP at sites Tyr-131 and Tyr-132 in response to growth factor stimulation have been mapped and suggested to stimulate LMW−PTP phosphatase activity. Biochemical analysis of tyrosine phosphorylation of a tyrosine phosphatase is challenging because of the intrinsic instability of these modifications. Here we used expressed protein ligation to site-specifically incorporate a phosphotyrosine mimic (phosphonomethylenephenylalanine, Pmp) at the Tyr-131 and Tyr-132 positions and measured the catalytic activity of these semisynthetic LMW−PTPs. The phosphonate-modified LMW−PTPs were 10- to 23-fold less active in dephosphorylating phosphotyrosine peptides derived from the PDGF receptor and p190RhoGap, two putative cellular substrates. These findings suggest the first example of a tyrosine phosphatase that is inhibited by tyrosine phosphorylation and provide a new model for the regulation of LMW−PTP and its role in cell adhesion.
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This study examined the temporal relationships of endothelin-1-stimulated rabbit platelets tyrosine phosphorylated proteins. The effect of endothelin-1 on tyrosine phosphorylation was dose- and time-dependent and caused a rapid tyrosine phosphorylation of three groups of proteins in the molecular mass range 70-100 kDa, 100-150 kDa and 150-200 kDa. Significant protein tyrosine phosphatase activity and amount were found to be associated with the cytoskeleton of endothelin-1-stimulated rabbit platelets. Under our experimental conditions, translocation from the cytosolic fraction to the cytoskeleton reached its highest levels within 10-20 sec of endothelin-1 stimulation. Endothelin-1-induced translocation of protein tyrosine phosphatase, associated with the increase in its activity was demonstrated by immunoblotting and immunoelectron microscopy.
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Early signaling events that control the process of programmed cell death are largely unknown. Tyrosine phosphorylation plays a major role in transmembrane signal transduction through most cell surface receptors. Granulocyte/macrophage colony-stimulating factor (GM-CSF), a cytokine released by activated T cells, has been shown to increase tyrosine phosphorylation in several cells and to inhibit granulocyte cell death in vitro. In this study, we demonstrate that the effect of GM-CSF on granulocyte cell death can be blocked by the tyrosine kinase inhibitor genistein, suggesting that increases in tyrosine phosphorylation are essential to inhibit cell death. To analyze the role of tyrosine phosphorylation for the regulation of granulocyte cell death more precisely, we increased levels of tyrosine phosphorylation using the protein-tyrosine phosphatase inhibitor phenylarsine oxide (PAO). Similar to GM-CSF, treatment of the cells with PAO was followed by high increases in tyrosine phosphorylation and inhibition of programmed cell death in human eosinophils and neutrophils. Strikingly, at low concentrations of the inhibitor and low induction of tyrosine phosphorylation, acceleration of apoptosis was observed. Genistein and herbimycin A reversed the effects of PAO on tyrosine phosphorylation and granulocyte apoptosis. These results suggest that programmed eosinophil and neutrophil death is regulated by early events of signal transduction pathways such as tyrosine phosphorylation.
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Protein tyrosine phosphorylation, modulated by the rate of both protein tyrosine kinase and protein tyrosine phosphatase activities, is critical for cellular signal transduction cascades. We report that endothelin‐1 stimulation of rabbit platelets resulted in a dose‐ and time‐dependent tyrosine phosphorylation of four groups of proteins in the molecular mass ranges of 50, 60, 70–100 and 100–200 kDa and that one of these corresponds to focal adhesion kinase. This effect is also related to the approximately 60% decrease in protein tyrosine phosphatase activity. Moreover, this inhibited activity was less sensitive to orthovanadate. In the presence of forskolin that increases the cAMP level a dose‐dependent inhibition of the endothelin‐stimulated tyrosine phosphorylation of different protein substrates and a correlation with an increase in the protein tyrosine phosphatase activity (11.6‐fold compared to control) have been found. Further studies by immunoblotting of immunoprecipitated soluble fraction with anti‐protein tyrosine phosphatase‐1C from endothelin‐stimulated platelets have demonstrated that the tyrosine phosphorylation of platelet protein tyrosine phosphatase‐1C is correlated with the decrease in its phosphatase activity. As a consequence, modulation and regulation by endothelin‐1 in rabbit platelets can be proposed through a cAMP‐dependent pathway and a tyrosine phosphorylation process that may affect some relevant proteins such as focal adhesion kinase.
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The importance of tyrosine phosphorylation in diverse cellular processes such as cell growth, gene expression, metabolism, differentiation, cytoskeletal dynamics, and cell motility was discovered primarily through studies of the protein tyrosine kinases (PTKs). The discovery that defective PTKs are encoded by oncogenes that induce cell transformation sustained the early focus on these enzymes. This work established the concept that defects in signaling pathways dependent on tyrosine phosphorylation can lead to diseases including cancer. For many years, research on tyrosine phosphorylation centered around the kinases and very little was known about the phosphatases that remove phosphate from tyrosine residues. Because it is a dynamic and reversible process, protein tyrosine phosphorylation is necessarily governed by the combined action of protein tyrosine phosphatases and PTKs. Consequently, an understanding of protein tyrosine phosphatases and their action in opposing PTKs is essential for understanding any signaling pathway involving tyrosine phosphorylation.KeywordsCatalytic DomainTyrosine PhosphorylationTyrosine PhosphataseProtein Tyrosine PhosphataseNeural Cell Adhesion MoleculeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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