pervanadate and herbimycin

In the capping of cell-surface receptors two stages can be distinguished: 1) clustering of the receptors (patching) induced by cross-linking with specific antibodies and 2) subsequent assembly of patches into a cap which is driven by the actin-based cytoskeleton. We found that patching of Fcgamma receptor II in U937 cells was correlated with tyrosine phosphorylation of certain proteins, most prominently those of 130, 110, 75 and 28 kDa. The phosphotyrosine-bearing proteins were accumulated at the receptor patches. Formation of the receptor caps was coincident with dephosphorylation of these proteins. Inhibition of protein tyrosine kinases with herbimycin A and genistein attenuated the protein tyrosine hyperphosphorylation and blocked capping in a dose-dependent manner. Phenylarsine oxide and pervanadate, inhibitors of protein tyrosine phosphatases, also suppressed capping of Fcgamma receptor II in a concentration-dependent fashion. Simultaneously, tyrosine hyperphosphorylation of proteins occurred. In the presence of the tyrosine kinase and phosphatase inhibitors the receptors were arrested at the patching stage. In contrast, okadaic acid, a serine/threonine phosphatase blocker, did not affect assembly of the receptor caps. The inhibitory effect of phenylarsine oxide was rapidly reversed by dithiols, 2,3-dimercapto-1-propanoldithiol and dithiotreitol, and was coincident with dephosphorylation of protein tyrosine residues. Extensive washing of pervanadate-exposed cells also resulted in progressive restoration of the cap assembly. Using streptolysin O-permeabilized cells we confirmed regulatory function played by dephosphorylation of tyrosine residues in capping of Fcgamma receptor II. Exogenous phosphatases, applied to permeabilized cells in which activity of endogenous tyrosine phosphatases was blocked, evoked dephosphorylation of protein tyrosine residues that was accompanied by recovery of capping ability in the cells.

Topics: Arsenicals; Bacterial Proteins; Benzoquinones; Cell Membrane Permeability; Dose-Response Relationship, Drug; Enzyme Inhibitors; Genistein; Humans; Immunoblotting; Lactams, Macrocyclic; Microscopy, Fluorescence; Okadaic Acid; Phosphorylation; Quinones; Receptor Aggregation; Receptors, IgG; Rifabutin; Streptolysins; Temperature; Time Factors; Tyrosine; U937 Cells; Vanadates

The chemokine eotaxin is a potent and relatively eosinophil-specific chemoattractant implicated in the cell migration to inflammatory sites in allergic diseases. Eotaxin exerts its activity solely through the CCR3 receptor, but the signaling pathways are poorly defined. In this study, we show that eotaxin induces an increase in tyrosine phosphorylation of multiple cellular proteins in normal human eosinophils. Eotaxin-dependent tyrosine phosphorylation was detected 1 min after stimulation and increased for at least 15 min with kinetics similar to those of eotaxin-induced cell shape changes. Herbimycin A, a tyrosine kinase inhibitor, blocked both eotaxin-induced tyrosine phosphorylation and cell shape changes as well as chemotaxis. Immunofluorescence microscopy analyses showed that eotaxin-induced cell shape changes were accompanied by redistribution of tyrosine-phosphorylated proteins and F-actin reorganization that were sensitive to herbimycin A. Coimmunoprecipitation studies revealed that binding of eotaxin to CCR3 greatly enhanced association of the Src family kinases, Hck and c-Fgr, with CCR3 after internalization of CCR3. These results may indicate that recruitment of Hck and c-Fgr to CCR3 in a compartment triggers tyrosine phosphorylation, leading to rapid cell shape changes required for cell migration.

Topics: Benzoquinones; Chemokine CCL11; Chemokines, CC; Chemotaxis, Leukocyte; Cytokines; Drug Synergism; Enzyme Inhibitors; Eosinophils; Humans; Lactams, Macrocyclic; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-hck; Quinones; Receptors, CCR3; Receptors, Chemokine; Rifabutin; src-Family Kinases; Tyrosine; Vanadates

The mediators involved in eosinophil accumulation in diseases such as allergy continue to be an area of interest, even though little is known regarding the signaling involved in the human cell type recruitment. In the present study, we demonstrate a novel modulatory role of tyrosine kinase and tyrosine phosphatase activities on normal human eosinophil chemotaxis induced by different groups of chemoattractant.. Purified eosinophils were obtained from normal healthy volunteers with the CD16-negative procedure. Chemotactic activities against platelet-activating factor (PAF), vasoactive intestinal peptide (VIP) and eotaxin were assessed using a 48-well microchemotaxis chamber assay. Purified eosinophils were pretreated with herbimycin A, erbastatin or pervanadate to examine the role of tyrosine kinase in chemoattractant signaling.. Pretreatment of eosinophils with the tyrosine kinase inhibitors herbimycin A and erbstatin significantly blocked chemotaxis induced by eotaxin whilst both inhibitors augmented chemotaxis induced by VIP; however, they had no effect on PAF-induced chemotaxis. On the other hand, pretreatment of eosinophils with the phosphotyrosine phosphatase inhibitor pervanadate resulted in augmentation of eotaxin-induced chemotaxis and inhibition of VIP-induced chemotaxis, but it had no effect on PAF-induced chemotaxis.. These results suggest that protein kinase plays a modulatory role in eosinophil chemotaxis induced by various chemoattractants.

Topics: Benzoquinones; Chemokine CCL11; Chemokines, CC; Chemotactic Factors, Eosinophil; Chemotaxis, Leukocyte; Cytokines; Enzyme Inhibitors; Eosinophils; Humans; Hydroquinones; In Vitro Techniques; Lactams, Macrocyclic; Platelet Activating Factor; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Quinones; Rifabutin; Vanadates; Vasoactive Intestinal Peptide

Endothelin (ET-1), a contractor and mitogen in the vasculature, enhanced cAMP production (t1/2, 2.2 min; EC50, 89 +/- 6.3 nM) and stimulated activity of the cAMP-dependent protein kinase (PKA) in pig coronary arteries. These responses were blunted by the protein tyrosine kinase (PTK) inhibitors genistein and herbimycin-A, but not by inhibitors of protein kinase C or cyclooxygenase. In contrast, forskolin-stimulated cAMP production was unaffected by PTK inhibition. Immunoblot analysis revealed that ET-1 induced a concentration-dependent protein tyrosine (PT) phosphorylation. Sarafotoxin-c, a selective ETB receptor agonist, had no effect on either cAMP levels or PT phosphorylation. Moreover, pervanadate (PV), a potent inhibitor of PT phosphatases, enhanced both cAMP formation and PT phosphorylation, both of which were blocked by PTK inhibitors. The effects of ET-1 and PV were not additive, suggesting a similar mode of activation, whereas responses to ET-1 and forskolin were synergistic. These findings indicate that AC and PKA are activatable via a nonreceptor PTK-dependent pathway downstream from the G-protein-linked ETA receptor. Because cAMP is a dilator and antimitogen in smooth muscle, stimulation of AC activity may be a negative feedback mechanism regulating ET-1-induced vasoconstriction and/or mitogenesis.

Topics: Adenylyl Cyclases; Animals; Benzoquinones; Colforsin; Coronary Vessels; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Genistein; GTP-Binding Proteins; In Vitro Techniques; Kinetics; Lactams, Macrocyclic; Muscle, Smooth, Vascular; Peptides; Protein-Tyrosine Kinases; Quinones; Receptor, Endothelin A; Receptors, Endothelin; Rifabutin; Signal Transduction; Swine; Vanadates; Viper Venoms

Although transmembrane signaling defect has been recognized as one of the major functional alterations involved in immune senescence, its biochemical nature as well as its precise molecular localization are still unknown. The available data indicate that an early step in the signaling cascade may be affected during the aging process. Because protein tyrosine kinases (PTK) are ubiquitously implicated in the initiation of physiological signals, they appear as prime candidates for age-related changes. The present investigation examined the effect of age on the activity of PTK associated with CD3, CD4, CD8 or the IL-2 receptor (IL-2R) in human T lymphocytes. By comparison with cells derived from young individuals, anti-CD3-activated T lymphocytes from elderly donors were more susceptible to herbimycin A, a PTK inhibitor known to prevent signal transduction by the T cell antigen receptor. This increased sensitivity of cells from senescent organisms to PTK inhibitors is most likely related to a lesser PTK activity since a significant decrease in the tyrosine phosphorylation of particular endogenous substrates was observed as a consequence of either CD3, CD4, CD8 or IL-2R activation. However, no age-related difference in tyrosine phosphorylation could be demonstrated when T cells were activated by pervanadate, a pharmacological activator of PTK. These results suggest that the intrinsic activity of the enzymes is preserved and that the age-associated defect in PTK activation occurs as a consequence of an upstream biochemical alteration. The defect in PTK activation could be the primary cause for the dysfunction of various components of the signaling cascade observed during the course of aging.

Topics: Adult; Aged; Aged, 80 and over; Aging; Antigens, CD; Benzoquinones; Blood Donors; CD3 Complex; CD4 Antigens; Enzyme Inhibitors; Humans; Lactams, Macrocyclic; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Receptors, Interleukin-2; Rifabutin; T-Lymphocytes; Vanadates