Inhibitors of tyrosine phosphorylation induce apoptosis in human leukemic cell lines

Experimental evidence suggests that hematopoietic growth factors promote cell survival by suppressing apoptosis or programmed cell death. Since interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) induce tyrosine phosphorylation of a common set of proteins in the factor-dependent cell line M07e, we have investigated whether growth-factor-induced tyrosine phosphorylation is involved in the promotion of cell survival and suppression of apoptosis. Experiments were carried out with the leukemic cell lines HL-60 and M07e and the tyrosine kinase inhibitors genistein and tyrphostin AG82. Both the tyrosine kinase inhibitors induced apoptosis of HL-60 and M07e cells. This was indicated by the appearance of DNA degradation and morphologic evidence of nuclear condensation and fragmentation. It was also confirmed by flow cytometry of DNA, which showed apoptotic cells as a fraction of cells characterized by a diminished DNA stainability, represented on the DNA frequency histograms as a distinct peak below the G0/G1 population. Kinase inhibitors also reduced the fraction of cells in the S phase of the cell cycle. That tyrphostin specifically inhibited tyrosine kinases was further suggested by the prevention of its effects by the tyrosine phosphatase inhibitor sodium orthovanadate (vanadate), at least during the first 18-24 h of treatment. The incomplete prevention of genistein effects by vanadate suggests that genistein is a less specific inhibitor of tyrosine kinases than tyrphostin, and may also act as an inhibitor of topoisomerase II. Vanadate also prevented apoptosis and reduction of the S phase in M07e cells cultured for 24 h in the absence of growth factors. These results suggest that tyrosine phosphorylation is an essential step in IL-3 and GM-CSF signal transduction. Since in our experimental model the effects of tyrosine kinase inhibition and growth factor deprivation could be reversed by concomitant inhibition of tyrosine phosphatases, it is suggested that a balance between tyrosine kinases and tyrosine phosphatases establishes whether a cell will survive or undergo apoptosis.     

Proteasome inhibitors induce mitochondria-independent apoptosis in human glioma cells

6-Chloro-5,10-dihydro-5-[( 1-methyl-4-piperidinyl)acetyl]-11H-dibenzo[b,e][1,4]diazepine-11one++ + hydrochloride (UH-AH 37) is an analog of pirenzepine that has previously been reported to interact with classical muscarinic antagonists in a competitive manner, yet its binding has also been found to be sensitive to the same epitope as is that of the allosteric ligand gallamine. The present study was carried out with wild-type and chimeric muscarinic receptors to determine whether UH-AH 37 might also have an allosteric mode of action. In assays that detect only allosteric interactions, UH-AH 37 slowed the rate of dissociation of [3H]N-methylscopolamine (NMS) from all five muscarinic receptor subtypes, with the highest apparent affinity at m2. By contrast, studies carried out under equilibrium conditions have found UH-AH 37 to have the lowest affinity for the m2 subtype. Studies with m2/m5 chimeric receptors found the allosteric potency of UH-AH 37 to be sensitive to an epitope in the seventh transmembrane domain (TM). Again, this contrasts with equilibrium studies, wherein an epitope in the sixth TM has been implicated. Simultaneous analysis of the interactions between UH-AH 37 and [3H]NMS at the m2 receptor under equilibrium and non-equilibrium conditions found that a simple allosteric model could not accommodate both sets of data. On the other hand, the model did accommodate such data for gallamine; gallamine also displays concordance in order-of-potency and epitope sensitivity between equilibrium and non-equilibrium assays. Based on these results, we conclude that UH-AH 37 interacts at the classical muscarinic binding site with high affinity and at a second (allosteric) site with lower affinity.     

Shrinkage-induced protein tyrosine phosphorylation in Chinese hamster ovary cells

To investigate the signal transduction of osmotic stress, we examined hypertonicity-induced tyrosine phosphorylations in Chinese hamster ovary cells. Hyperosmosis elicited characteristic phosphotyrosine accumulation in at least 3 proteins (approximately 42, approximately 85, and approximately 120 kDa). The most prominent response occurred in the 85-kDa band (p85) whose phosphorylation was rapid, sustained, apparent already at mild hypertonicity (350 mosM), proportional to the extracellular osmotic concentration, and reversible. Hyperosmotic environment could not induce tyrosine phosphorylation if cell shrinkage was prevented by nystatin and appropriately composed media. Conversely, isotonic shrinkage caused strong tyrosine phosphorylation. Thus, the initial signal is a decrease in cell volume and not an increase in the intra- or extracellular osmotic concentration, or a rise in cytosolic K+ and Cl- levels. Tyrosine phosphorylation of p85 was not due to the hypertonicity-induced protein kinase C-dependent stimulation of the extracellular signal-regulated protein kinase, nor to the activation of stress-activated protein kinases. Tonicity-responsive proteins interacted with Grb2-glutathione S-transferase fusion proteins: the 120-kDa protein complexed with the SH2 and both SH3 domains, whereas p85 associated with the SH2 and the N-terminal SH3 domains of the adapter. Tyrosine phosphorylation of p85 is a sensitive indicator of reduced intracellular hydration and might signify a hitherto unrecognized, early volume-dependent signaling event.     

Farnesyltransferase inhibitors induce dramatic morphological changes of KNRK cells that are blocked by microtubule interfering agents

Farnesyltransferase inhibitors (FTIs) exhibit the remarkable ability to inhibit transformed phenotypes of a variety of human cancer cell lines and to block the growth of cancer cells in a number of animal model systems. In this paper, we report that the addition of FTI to v-K-ras- transformed NRK cells (KNRK) results in dramatic morphological changes. Within 24 h after the addition of FTI, the round morphology of KNRK cells was changed to an elongated (flattened and spread out) morphology resembling those of untransformed NRK cells. No morphological effects were seen when similar concentrations of FTI were added to NRK cells. Phalloidin staining showed that FTI treatment did not restore the disrupted actin cytoskeleton in KNRK cells. In contrast, FTI addition resulted in the appearance of extensive microtubule networks in KNRK cells. The addition of a low concentration (1.2 nM) of vincristine or vinblastine, agents that interfere with microtubule dynamics, blocked the FTI-induced morphological changes in KNRK cells. In contrast, cytochalasin B, which interferes with actin polymerization, did not block the morphological changes. The FTI-induced morphological changes were associated with a decrease in the percentage of cells in S-phase, and the addition of 1.2 nM vincristine did not have additional effects on cell cycle progression. A higher concentration (12 nM) of vincristine caused synergistic effect with FTI to enrich dramatically KNRK cells in G2/M phase. These results suggest that FTI affects cell morphology and that microtubule dynamics are involved in these processes.     

Mechanical strain increases protein tyrosine phosphorylation in airway smooth muscle cells

The 'comet' assay is being increasingly employed for evaluating DNA damage in biological systems. Using this technique, we examined DNA damage in whole in density-separated trout erythrocytes. Results clearly show that all the three considered parameters (tail length, tail intensity and tail moment) increased with the density of the fractions, possibly reflecting different degrees of DNA damage. Probably, this behaviour is due to different periods of exposure of the density fractions to the hazard of active oxygen radicals; older cells have been exposed to oxidative stress for a longer time.     

Normal Syk protein level but abnormal tyrosine phosphorylation in B-CLL cells

One characteristic of B cells that accumulate during chronic lymphocytic leukemia (CLL) is their highly heterogeneous functional responses to B cell receptor (BCR) stimulation. Leukemic B cells with very poor responses have defective rapid tyrosine phosphorylation of numerous substrates, especially phospholipase C (PLC)gamma, as well as a defective calcium elevation on BCR stimulation. This points to a defect in BCR-associated protein tyrosine kinase (PTK). We investigated whether a defect in Syk, a PTK that is pivotal in coupling BCR to downstream signaling events, could account for these alterations. Syk tyrosine phosphorylation triggered by BCR ligation was severely impaired in B-CLL cells with low calcium responses to anti-mu stimulation. Syk associations were also defective, as concomitant tyrosine phosphorylation of a Syk-associated 145 kDa protein comigrating with PLCgamma-2 was only detected in responding B-CLL cells. By contrast, we found similar expression of the kinase regardless of B-CLL cell responsiveness. These results are consistent with the possibility that very proximal BCR signaling elements in some B-CLL cells are unable to connect with downstream biochemical events dominated by tyrosine phosphorylation and the potential docking function of Syk PTK.     

Inhibition of neuropeptide-stimulated tyrosine phosphorylation and tyrosine kinase activity stimulates apoptosis in small cell lung cancer cells

Small cell lung cancer (SCLC) cell growth is sustained by multiple autocrine and paracrine growth loops involving neuropeptides. The bombesin family of peptides are autocrine growth factors in H345 SCLC cells and provide a paradigm for the study of growth factors and mitogenic signaling in SCLC cells. We show that bombesin (and other neuropeptides) stimulates protein tyrosine phosphorylation (particularly focal adhesion kinase) and protein tyrosine kinase (PTK) activity in intact SCLC cells. Furthermore, the broad spectrum neuropeptide receptor antagonist [D-Arg, D = Phe, D-Trp, Leu11]substance P inhibits all neuropeptide-mediated signals (including PTK activation), SCLC cell growth in vivo and in vitro, and also increases the natural rate of apoptosis seen in growing SCLC cell lines. Hence the effect of selective PTK inhibition on SCLC cell growth and apoptosis was examined. We show that selective inhibition of PTK activity, with genistein and (3,4,5-tri-hydroxyphenyl)-methylene(-propanedinitrile) tyrphostin-25 inhibits basal and neuropeptide-stimulated SCLC cell growth. Genistein and tyrphostin-25 also stimulate apoptosis in SCLC cells. Inhibition of proliferation in these cells is intimately linke to apoptosis, because these changes occurred without any effect on SCLC cell cycle kinetics, suggesting that apoptosis occurs independently of the cell cycle and that failure to progress through the cell cycle results in apoptosis. Because tyrphostin-25 fails to influence p53 or Bcl-2 expression in these cells, this mode of programmed cell death appears to be via a p53- and Bcl-2-independent mechanism. These results provide evidence that tyrosine phosphorylation is a mitogenic signal in SCLC cells and suggest that regulation of the level of protein tyrosine phosphorylation represents a critical determinant of whether SCLC cells survive and proliferate or die by apoptosis. Thus PTK inhibition may provide a novel therapeutic option in SCLC that has become resistant to conventional chemotherapeutic agents.     

Calcium-regulated protein tyrosine phosphorylation is required for endothelin-1 to induce prostaglandin endoperoxide synthase-2 mRNA expression and protein synthesis in mesangial cells

The role of endothelin (ET)-1-mediated cytosolic calcium ([Ca2+]i) elevation in regulating ET-1-induced prostaglandin endoperoxide synthase, prostaglandin G/H synthase (PGHS)-2 mRNA expression and protein synthesis was investigated in mesangial cells (MC). Ionomycin, a calcium ionophore, and thapsigargin, an inhibitor of calcium ATPase, mimicked the ET-1-stimulated PGHS-2 mRNA and protein induction. Inhibition of [Ca2+]i increases with (2-?C2-bis-(carboxymethyl)-amino-5 methylphenoxy]methyl?-6-methoxy-8-bis-(carboxymethyl)-aminoquinoline tetra-(acetoxymethyl)ester (Quin/AM), a calcium chelator, or with the combined presence of [8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, HCl] (TMB), an inhibitor of intracellular calcium stores release, and ethyleneglycol-bis-(beta-aminoethyl)- N,N,N',N'-tetra-acetic acid (EGTA) suppressed ET-1, as well as ionomycin and thapsigargin-mediated PGHS-2 mRNA and protein formation. Also, the ET-1-, ionomycin-, and thapsigargin-induced PGHS-2 mRNA expression and protein formation was inhibited in MC pretreated with inhibitors of calcium calmodulin kinase. In contrast, these conditions did not inhibit interleukin (IL)-1-induced PGHS-2 mRNA expression and protein synthesis. Pretreatment with tyrosine kinase inhibitors abolished the ET-1-, ionomycin-, thapsigargin-, and IL-1-mediated PGHS-2 mRNA and protein induction. ET-1-, ionomycin-, and thapsigargin- induced protein tyrosine phosphorylation, but not IL-1-induced protein tyrosine phosphorylation, was suppressed by inhibiting either [Ca2+]i elevation or calcium calmodulin kinase activation. It was concluded that elevation of [Ca2+]i and activation of calcium calmodulin kinases are upstream mediators of ET-1-induced PGHS-2 gene expression through activation of non-receptor-linked protein tyrosine kinase in MC.     

Cell growth inhibition by a novel vitamin K is associated with induction of protein tyrosine phosphorylation

We have shown that a synthetic vitamin K analog, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or compound 5 (Cpd 5), potently inhibits cell growth and suggested that the analog exerts its effects mainly via sulfhydryl arylation rather than redox cycling. Since protein-tyrosine phosphatases (PTPases), which have pivotal roles in many cellular functions, have a critical cysteine in their active site, we have proposed PTPases as likely targets for Cpd 5. To test this hypothesis, we examined the effects of Cpd 5 on protein tyrosine phosphorylation of cellular proteins and on the activity of PTPases. We found that Cpd 5 rapidly induced protein tyrosine phosphorylation in a human hepatocellular carcinoma cell line (Hep3B) at growth inhibitory doses, and the effect was blocked by thiols but not by non-thiol antioxidants or tyrosine kinase inhibitors. Cpd 5 inhibited PTPase activity, which was also significantly antagonized by reduced glutathione. Furthermore, the well studied PTPase inhibitor orthovanadate also induced protein tyrosine phosphorylation and growth inhibition in Hep3B cells. These results suggest that inhibition of cellular PTPases by sulfhydryl arylation and subsequent perturbation of protein tyrosine phosphorylation may be involved in the mechanisms of Cpd 5-induced cell growth inhibition.     

HMG-CoA reductase inhibitors induce apoptosis in mouse proximal tubular cells in primary culture

Renal cyst formation in polycystic diseases or after nephron reduction is attributed to enhanced tubular cell proliferation with unbalanced cell death. The induction of tubular cell death could be effective to reduce renal cyst formation. In this study, we examined the effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors on apoptosis in mouse proximal tubular (MPT) cells in primary culture. After treatment with HMG-CoA reductase inhibitors, the extracted DNA was analyzed by gel-electrophoresis under ultraviolet light. Apoptosis was evaluated quantitatively by estimating the ratio of fragmented DNA over intact DNA. For morphologic studies, cells were stained with Hoechst 33,258. DNA ladder pattern of 200 kDa typical of apoptosis and significant increase in DNA fragmentation were seen after 24 hours of treatment with lovastatin, a HMG-CoA reductase inhibitor. Staining with the Hoechst dye revealed cleavage of nucleus into pieces under the same condition. Geranylgeranylpyrophosphate (20 microM) and mevalonate (500 microM) completely reversed the effect of lovastatin, while farnesylpyrophosphate (20 microM) partially reversed it. Other products of HMG-CoA pathway such as cholesterol, ubiquinone, dolichol, and isopentenyladenine had no effect. Perillic acid and alpha-hydoxyfarnesylphosphonic acid, isoprenylation inhibitors, induced apoptosis of the cells. A treatment with lovastatin caused actin filament disruption. Cytochalasin D, an inhibitor of actin polymerization, induced apoptosis. Interleukin-1 beta-converting enzyme inhibitor II, a protease inhibitor, had no effect on the apoptosis induced by either HRI or cytochalasin D. The present study suggests that in mouse proximal tubules, HMG-CoA reductase inhibitors induce apoptosis via inhibition of isoprenoid production, and disruption of actin filaments may play a role in the apoptosis induction.     

Influence of tyrosine phosphorylation on protein interaction with FcgammaRIIa

The cytoplasmic tail of Fc(gamma)RIIa present on human neutrophils shares with other antigen receptors a common amino acid sequence called ITAM (Immunoreceptor Tyrosine-based Activation Motif). After receptor ligation, the tyrosine residues within this motif become phosphorylated. We prepared a recombinant fusion protein of the cytoplasmic tail of Fc(gamma)RIIa (containing the ITAM) with glutathione-S-Transferase (GST-CT) to characterize the phosphorylation of Fc(gamma)RIIa and its ability to interact with other proteins involved in signal transduction. The GST-CT became phosphorylated in the presence of Lyn, Hck and Syk (immunoprecipitated from human neutrophils), but not in the presence of Fgr. Of the active kinases, only Lyn (mainly present in the membrane fraction) was found to associate with the GST-CT in the absence of ATP. This association was also observed in immunoprecipitates of Fc(gamma)RIIa from resting neutrophils, suggesting that Lyn might be the kinase responsible for the initial Fc(gamma)RIIa phosphorylation. Moreover, we observed specific association of Syk and the p85 subunit of PI 3-kinase after incubation of the GST-CT with neutrophil cytosol. This interaction was dependent on tyrosine phosphorylation of the GST-CT. Substitution of 269Tyr by Phe almost completely abolished tyrosine phosphorylation of the fusion protein. Substitution of either 253Tyr or 269Tyr eliminated Syk binding, but only 253Tyr appeared to be essential for p85 binding. We hypothesize that, upon activation, the membrane-associated Lyn is responsible for the initial tyrosine phosphorylation of Fc(gamma)RIIa, thus creating a docking site for Syk and PI 3-kinase.     

Activation of protein phosphorylation by oxidants in vascular endothelial cells: identification of tyrosine phosphorylation of caveolin

The colonial protochordate Botryllus schlosseri possesses a historecognition system which has long invited comparison to the vertebrate MHC. Upon contact, colonies either fuse or reject one another in a manner resembling graft acceptance or rejection in vertebrates. This response is controlled by a single highly polymorphic genetic region, the FuHC locus. Colonial protochordates such as B. schlosseri are among the closest relatives of the vertebrate lineage, and therefore may possess a recognizable MHC homologue. Since linkage between heat shock protein 70 (HSP70) genes and MHC appears to be conserved within the vertebrate lineage, we have analyzed HSP70 genes from B. schlosseri as a first step toward isolating the historecognition locus. Two HSP70 genes (HSP70.1 and HSP70.2) have been cloned and sequenced, and exhibit 93.6% sequence identity within the predicted coding regions. The B. schlosseri genes share a number of characteristics with vertebrate MHC-linked HSP70 genes: Northern blotting and sequence analysis suggest that the protochordate genes are cytoplasmically-expressed heat-inducible members of the HSP70 gene family (FAGAN and WEISSMAN 1996). However, unlike vertebrate MHC-linked HSP70 genes, HSP70.1 and HSP70.2 are not closely linked (FAGAN and WEISSMAN 1997). Furthermore, neither is closely linked to the locus determining historecognition (FAGAN and WEISSMAN 1997). These results do not support the hypothesis that the B. schlosseri FuHC locus is an MHC homolog. A discussion of the implications of these results for evolution of the vertebrate MHC is included.     

Effect of genistein on amylase release and protein tyrosine phosphorylation in parotid acinar cells

We evaluated the role of protein tyrosine phosphorylation in amylase exocytosis from parotid acinar cells by using genistein, a tyrosine kinase inhibitor. Amylase release stimulated by isoproterenol was dose-dependently inhibited by genistein. Genistein also inhibited the exocytosis evoked by dibutyryl- or 8-chlorophenylthio-cAMP. Daidzein, a negative control agent of genistein, elicited no inhibitory effect. Isoproterenol had dual effects on protein tyrosine phosphorylation; it increased that phosphorylation of 190- and 210-kDa proteins and decreased that of a 90-kDa one. The phosphorylation was dose-dependently inhibited by genistein but not by daidzein. These results suggest that protein tyrosine phosphorylation plays a role in the process of amylase exocytosis from parotid acinar cells.     

Adhesion-dependent protein tyrosine phosphorylation in neutrophils treated with tumor necrosis factor

Human neutrophils (PMN) respond to tumor necrosis factor (TNF) by releasing their granules, reorganizing their cytoskeleton, and massively secreting hydrogen peroxide. This response is dependent on adhesion to extracellular matrix proteins and expression of CD11b/CD18 integrins (Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, and S. D. Wright. 1989. J. Cell Biol. 109:1341-1349). We investigated the role of tyrosine phosphorylation in the response of PMN to TNF. PMN adherent to protein-coated surfaces but not suspended PMN showed tyrosine phosphorylation of several proteins (approximately 150, approximately 115, approximately 75, and approximately 65 kD) in response to TNF. Tyrosine phosphorylation was evident 5 min after addition of TNF and lasted at least 2 h. The tyrosine kinase inhibitors K252a, genistein and ST638 suppressed tyrosine phosphorylation and blocked hydrogen peroxide production in a reversible manner at low concentrations. Tyrosine kinase inhibitors also blocked the spreading of PMN in response to TNF. Dihydrocytochalasin B did not inhibit tyrosine phosphorylation, but in its presence phosphorylation was rapidly reversed. By immunocytochemistry, the majority of tyrosine phosphoproteins were localized to focal adhesions. Thus TNF-induced tyrosine phosphorylation depends on adhesion of PMN to extracellular matrix proteins, and participates in the transduction of the signals that direct the cells to spread on a biological surface and undergo a respiratory burst.     

Differential regulation of insulin receptor substrate-2 and mitogen-activated protein kinase tyrosine phosphorylation by phosphatidylinositol 3-kinase inhibitors in SH-SY5Y human neuroblastoma cells

Insulin-like growth factor I (IGF-I) is a potent neurotropic factor promoting the differentiation and survival of neuronal cells. SH-SY5Y human neuroblastoma cells are a well characterized in vitro model of nervous system growth. We report here that IGF-I stimulated the tyrosine phosphorylation of the type I IGF receptor (IGF-IR) and insulin receptor substrate-2 (IRS-2) in a time- and concentration-dependent manner. These cells lacked IRS-1. After being tyrosine phosphorylated, IRS-2 associated transiently with downstream signaling molecules, including phosphatidylinositol 3-kinase (PI 3-K) and Grb2. Treatment of the cells with PI 3-K inhibitors (wortmannin and LY294002) increased IGF-I-induced tyrosine phosphorylation of IRS-2. We also observed a concomitant increase in the mobility of IRS-2, suggesting that PI 3-K mediates or is required for IRS-2 serine/threonine phosphorylation, and that this phosphorylation inhibits IRS-2 tyrosine phosphorylation. Treatment with PI 3-K inhibitors induced an increased association of IRS-2 with Grb2, probably as a result of the increased IRS-2 tyrosine phosphorylation. However, even though the PI 3-K inhibitors enhanced the association of Grb2 with IRS-2, these compounds suppressed IGF-I-induced mitogen-activated protein kinase activation and neurite outgrowth. Together, these results indicate that although PI 3-K participates in a negative regulation of IRS-2 tyrosine phosphorylation, its activity is required for IGF-IR-mediated mitogen-activated protein kinase activation and neurite outgrowth.     

Depolarization-induced tyrosine phosphorylation of paxillin in PC12h cells

Treatment of PC12h cells with a high concentration of KC1 induces depolarization of the plasma membrane and Ca2+ influx into the cells. We have previously shown that KC1 induced tyrosine phosphorylation of cellular proteins of 120, 110, 68, 44 and 42 kDa. In the present study, we found that the 68-kDa protein is paxillin, a tyrosine kinase substrate associated with the actin cytoskeleton. A calcium ionophore, A23187, also induced tyrosine phosphorylation of the 68-kDa protein, while KC1 did not in the presence of EGTA or nifedipine, indicating that the effect of KC1 was due to the Ca2+ influx into the cells. Tyrosine phosphorylation of paxillin was also induced by nerve growth factor and epidermal growth factor, but its migration patterns on an SDS/polyacrylamide gel were different, that is, nerve growth factor and epidermal growth factor caused upward shifts of the bands, while KC1 did not. However, both forms could associate with Csk and Crk. The effect of KC1 was blocked by cytochalasin D, indicating that tyrosine phosphorylation required the integrity of actin filaments. These results suggest that tyrosine phosphorylation of paxillin may be involved in Ca2+ -dependent events in neuronal and neuroendocrine cells.     

Low levels of ionic mercury modulate protein tyrosine phosphorylation in lymphocytes

The ability of ionic mercury to induce protein tyrosine phosphorylation in mouse spleen cells and in the mouse WEHI-231 B-cell lymphoma was investigated. We have confirmed previous studies which showed that exposure to high levels (several hundred microM) of mercury lead to very large increases in the level of protein tyrosine phosphorylation in these cell systems. However we have also demonstrated that low levels (in the order of 0.1 to 1.0 microM) of mercury also significantly upregulate protein tyrosine phosphorylation. Mercury induced protein tyrosine phosphorylation is inhibited by the mercury chelator penicillamine and by pretreating treating target cells with the sulfhydryl blocking reagent N-hydroxymaleimide. These results suggest that exposure to low levels of mercury could potentially interfere with lymphocyte signal transduction and so offer a possible explanation as to how mercury exposure could lead to immune cellular dysfunction. On a molecular level, the results suggest that the site(s) of action with respect to mercury dependent induction of protein tyrosine phosphorylation is likely a free disulfide group or groups located on the outer leaflet of the plasma membrane.     

Reorganization of contractile systems and protein tyrosine phosphorylation in platelet aggregation

Platelet aggregation is accompanied with reorganization of contractile systems. This event involves tyrosine phosphorylation of various proteins. When tyrosine phosphorylation is induced by an intracellular trigger as vanadate, a phosphorylated cytosolic 60 Kd protein acts as a nucleating center on which new actin filaments grow. When aggregation is induced by an extracellular signal as ristocetin, actin filaments of the membrane-associated skeleton and the 60 Kd protein bound to this structure migrate toward the cytosol and only in this soluble state the 60 Kd protein undergoes tyrosine phosphorylation and consequently triggers reorganization of contractile systems.     

Early inhibition of protein phosphatases preferentially blocks phorbol ester-stimulated mitogenic signalling in melanocytes: increase in specific tyrosine phosphoproteins

Inhibition of protein phosphatases has been suggested as an alternative mechanism of tumor promotion (H. Fujiki, Mol. Carcinog. 5:91, 1992). We have now used early melanocyte passages dependent on phorbol esters and serum for growth and later passages with partial phorbol ester independence, to investigate the role of protein phosphatases on melanocyte DNA synthesis. Neither okadaic acid, an inhibitor of ser/thr protein phosphatases, nor vanadate, an inhibitor of tyrosine phosphatases, can stimulate basal or serum-stimulated mitogenesis in contrast to phorbol esters. Moreover, both phosphatase inhibitors are able to suppress serum and phorbol ester-stimulated mitogenesis, if added within 4 hours of growth activation. Inhibition of mitogenesis by either inhibitor correlated with an early increase in a common set of tyrosine phosphoproteins, which included a major 33 Kd species. Our data suggest that protein phosphatase inhibitors are growth suppressors and antagonize phorbol ester effects in cells of melanocytic origin, implying an early requirement for protein phosphatase activity during mitogenic signalling in these cells.