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Biomedical subjects

A Levitzki

Publications and source records attributed to A Levitzki.

At least 109 records · Page 6Linked to original sources
PubMed1992

Inhibition of platelet activation by tyrosine kinase inhibitors.

Protein tyrosine kinase (PTK) blockers (tyrphostins) inhibit in a dose-dependent fashion thrombin-induced aggregation and serotonin release with IC50 values in the 10-35 microM concentration range. The inhibition of thrombin-induced aggregation correlates with their potency in inhibiting phosphorylation of proteins on tyrosine residues. Using metabolically 32P-labelled human platelets, it was found that the tyrphostins have no effect on the decrease in [32P]phosphatidylinositol bisphosphate but prevent the replenishment of [32P]polyphosphoinositide. Tyrphostins decreased [32P]phosphatidic acid production induced by thrombin, although never by more than 50%, and only delayed the peak of diacylglycerol, suggesting that phospholipase C was still activated. Tyrphostins inhibited the thrombin-elicited early phosphorylation of p43 and p20, substrates for protein kinase C (PKC) and myosin light chain kinase, respectively, at short times of activation. This inhibition, however, was overcome after 1 min of stimulation with thrombin. Tyrphostin AG213 also inhibited platelet aggregation and tyrosine protein phosphorylation induced by phorbol myristate acetate (PMA), but did not inhibit pleckstrin phosphorylation. These results suggest that thrombin induces the phosphorylation of proteins on tyrosine residues which most probably results in the activation of phosphoinositide kinases. The ability of tyrphostins to inhibit phosphorylation of p43 and p20 when induced by thrombin but not when induced by PMA confirms that PTKs may be involved subsequent to PKC activation.

Catechols↗
PubMed1992

The GppNHp-activated adenylyl cyclase complex from turkey erythrocyte membranes can be isolated with its beta gamma subunits.

The adenylyl cyclase complex, derived from turkey erythrocyte membranes, was activated using guanosine 5'-[beta, gamma-imido]triphosphate (Gpp[NH]p) and separated under low-detergent and low-salt conditions using conventional molecular-sieve chromatography followed by high-pressure ion-exchange and molecular-sieve chromatography. Although the complex remains activated with Gpp[NH]p throughout the isolation, the beta gamma subunits copurify with the cyclase. The stoichiometry of the cyclase to the alpha subunit of the stimulatory guanosine-nucleotide-binding regulatory protein (alpha s) to the beta subunit is close to unity, demonstrating that the beta gamma subunits do not dissociate from the Gs.cyclase complex (Gs, guanosine-nucleotide-binding regulatory protein) upon activation of the enzyme. If the final purification step was performed at high-salt concentrations, the beta gamma subunits could be separated from the alpha s.cyclase complex. Previously reported results on bovine brain cyclase also showed that the Gs.cyclase complex remains intact subsequent to activation by hormone and Gpp[NH]p [Marbach, I., Bar-Sinai, A., Minich, M. and Levitzki, A. (1990) J. Biol. Chem. 265, 9999-10,004]. These results, using adenylyl cyclase from two different sources, support our previous kinetic experiments which first suggested that beta gamma subunits are not released from Gs upon cyclase activation. We, therefore, argue that the mode of adenylyl cyclase inhibition by the inhibitory guanosine-nucleotide-binding regulatory protein cannot be via shifting the alpha s to beta gamma equilibrium as is commonly believed, and an alternate hypothesis is proposed.

Adenylyl Cyclases↗
PubMed1992

Selective interactions of transforming and normal abl proteins with ATP, tyrosine-copolymer substrates, and tyrphostins.

The transforming abl proteins p160gag-abl, p185bcr-abl, and p210bcr-abl and the normal protein p140c-abl have identical catalytic sites, but differ in their N-terminal domains. Previous studies have indicated that the transforming abl proteins possess higher tyrosine kinase activity than the normal abl proto-oncogene product. In the present study, we demonstrate that two transforming abl proteins, p210bcr-abl and p160gag-abl, exhibit a higher affinity toward ATP and synthetic tyrosine containing substrates than p140c-abl. Furthermore, protein tyrosine kinase blockers from the tyrphostin family can discriminate between normal abl and transforming abl proteins of both human and mouse origin. These results suggest that the transforming potency of the abl proteins may result from their higher affinities toward intracellular signal transducers and demonstrate for the first time that oncogene products can differ from their homologous proto-oncogene product in substrate specificity. The ability of tyrphostins to discriminate between normal and transforming abl proteins suggests that it may be possible to design specific abl kinase inhibitors to combat abl-associated human leukemias.

Adenosine Triphosphate↗
PubMed1992

The effect of tyrosine-specific protein phosphorylation on the assembly of adherens-type junctions.

Adherens-type junctions (AJs) are major subcellular targets for tyrosine specific protein phosphorylation [Volberg et al. (1991) Cell Regul., 2, 105-120]. Here we report on the apparent effect of such phosphorylation events on the assembly and integrity of AJs. We show that incubation of MDCK cells with potent inhibitors of tyrosine-specific phosphatases (PTP), namely H2O2 and vanadate, leads to a dramatic increase in AJ-associated phosphotyrosine which was apparent already within 2-5 min of treatment and progressed upon further incubation. Examination of H2O2 vanadate treated cells at later time points indicated that intercellular AJs rapidly deteriorated, concomitantly with a marked increase in the number and size of vinculin and actin containing focal contacts. In parallel, major changes were observed in cell structure and topology, as revealed by electron microscopy. These were manifested by rapid rounding-up of the cells followed by reorganization of the cell monolayer. Other intercellular junctions, including desmosomes and tight junctions, visualized by staining with desmoplakin and ZO-I antibodies, were not significantly affected. To verify that modulation of AJs was indeed related to tyrosine phosphorylation, we have carried out reciprocal experiments in which Rovs Sarcoma virus (RSV) transformed chick lens cells, expressing high levels of pp60src kinase, were treated with inhibitors of tyrosine kinases, (tyrphostins). We show that following such treatment, intercellular AJs which were deteriorated in the transformed cells, were reformed. Based on these observations, we propose that specific tyrosine phosphorylation of AJ components is involved in the downregulation of these cellular contacts.

Actins↗
PubMed1992

Inhibition of platelet-derived growth factor-induced mitogenesis and tyrosine kinase activity in cultured bone marrow fibroblasts by tyrphostins.

Tyrphostins, which block protein tyrosine kinase activity, were studied for their inhibitory action on platelet-derived growth factor (PDGF)-induced proliferation of human bone marrow fibroblasts. Of the seven tryphostins examined, tyrphostin AG370 was found to be the most potent blocker against PDGF-induced mitogenesis (IC50 = 20 microM). This PTK blocker also blocks mitogenesis induced by epidermal growth factor (IC50 = 50 microM) and human serum (IC50 = 50 microM), but with lower efficacy. In digitonin-permeabilized fibroblasts as well as in intact fibroblasts, tyrphostin AG370 inhibits PDGF receptor autophosphorylation and the tyrosine phosphorylation of intracellular protein substrates (pp120, pp85, and pp75) which coprecipitate with the PDGF receptor. In comparison to AG370, AG18, a potent EGF receptor blocker, was less efficient in inhibiting PDGF-induced proliferation of fibroblasts and phosphorylation of the intracellular protein substrates. Under the conditions in which AG370 inhibits PDGF-induced mitogenesis and phosphorylation, it does not affect [125I]PDGF internalization and enhance [125I]PDGF binding. These findings suggest that AG370, which is an indole tyrphostin, may serve as a model for developing analogues with a therapeutic potential for treatment of diseases which involve abnormal cellular proliferation induced by PDGF.

Blotting, Western↗
PubMed1992

Tyrphostins: tyrosine kinase blockers as novel antiproliferative agents and dissectors of signal transduction.

Protein tyrosine kinases (PTKs) are members of a growing family of oncoproteins and protooncoproteins that play a pivotal role in normal and abnormal proliferative processes. This hallmark identifies these unique proteins as potential targets for antiproliferative therapy. This review discusses the current status of PTK inhibitors, with special emphasis on tyrphostins as antiproliferative agents and as potential drugs for cancers, leukemias, psoriasis, and restenosis as well as other proliferative conditions. The development of tyrphostins as selective signal blockers can be viewed as a first step toward the development of "smart" cocktails as antiproliferative agents. Each of these custom-made cocktails will be aimed at proliferative conditions whose transduction pathways can be characterized by molecular tools. The review also discusses the use of PTK blockers as tools to study signal transduction processes in which protein tyrosine kinases are implicated.

Animals↗
PubMed1992

Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein.

The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a beta-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, approximately 3-fold less potently, the Mn(2+)-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Val-19 and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyr1 proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of approximately 150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the beta-galactosidase-Cdc25 fusion protein.

Adenylyl Cyclases↗
PubMed1991

Kinetic analysis of the inhibition of the epidermal growth factor receptor tyrosine kinase by Lavendustin-A and its analogue.

Lavendustin-A was reported to be a potent tyrosine kinase inhibitor of the epidermal growth factor (EGF) receptor (Onoda, T., Iinuma, H., Sasaki, Y., Hamada, M., Isshibi, K., Naganawa, H., Takeuchi, T., Tatsuta, K., and Umezawa, K. (1989) J. Nat. Prod. 52, 1252-1257). Its inhibition kinetics was studied in detail using the baculovirus-expressed recombinant intracellular domain of the EGF receptor (EGFR-IC). Lavendustin-A (RG 14355) is a slow and tight binding inhibitor of the receptor tyrosine kinase. The pre-steady state kinetic analysis demonstrates that the inhibition corresponds to a two-step mechanism in which an initial enzyme-inhibitor complex (EI) is rapidly formed followed by a slow isomerization step to form a tight complex (EI*). The dissociation constant for the initial rapid forming complex is 370 nM, whereas the overall dissociation constant is estimated to be less than or equal to 1 nM. The difference between the two values is due to the tight binding nature of the inhibitor to the enzyme in EI*. The kinetic analysis using a preincubation protocol to pre-equilibrate the enzyme with the inhibitor in the presence of one substrate showed that Lavendustin-A is a hyperbolic mixed-type inhibitor with respect to both ATP and the peptide substrate, with a major effect on the binding affinities for both substrates. An analogue of Lavendustin-A (RG 14467) showed similar inhibition kinetics to that of Lavendustin-A. The results of the pre-steady state analysis are also consistent with the proposed two-step mechanism. The dissociation constant for the initial fast forming complex in this case is 3.4 microM, whereas the overall dissociation constant is estimated to be less than or equal to 30 nM. It is a partial (hyperbolic) competitive inhibitor with respect to ATP. Its inhibition is reduced to different extents by different peptide substrates, when the peptide is added to the enzyme simultaneously with the inhibitor. When studied with the least protective peptide, K1 (a peptide containing the major autophosphorylation site of the EGF receptor), RG 14467 acts as a hyperbolic noncompetitive inhibitor with respect to the peptide.

Adenosine Triphosphate↗
PubMed1991

The antiproliferative effects of tyrosine kinase inhibitors tyrphostins on a human squamous cell carcinoma in vitro and in nude mice.

Many human tumors of epithelial origin contain cells overexpressing the epidermal growth factor (EGF) receptor, and there is convincing evidence that cancer cell growth is correlated with the loss of the normal regulation of the EGF receptor signal transduction pathway. Some cancers are clearly dependent on activation of the EGF receptor for their proliferation. Recently, a class of compounds, tyrphostins, which inhibit the protein tyrosine kinase activity of the growth factor receptor, have been described. In this report, we have examined the antiproliferative effects of potent new tyrphostins on a well-characterized human squamous cell carcinoma in vitro and in vivo. We found that two of these compounds (RG-13022 and RG-14620) suppressed not only EGF-stimulated cancer cell proliferation in vitro but also tumor growth in nude mice. RG-13022 also increased the life span of these tumor-bearing nude mice. When administered to tumor-bearing nude mice together with monoclonal antibodies to the EGF receptor at a suboptimal dose which had no effect alone, inhibition of tumor growth was markedly enhanced. These data suggest that tyrphostins have potential as anticancer agents.

Animals↗
PubMed1991

Erbstatin and tyrphostins block protein-serine kinase activation and meiotic maturation of sea star oocytes.

The effects of ten putative protein-tyrosine kinase inhibitors on the activation of protein-serine kinases and germinal vesicle breakdown (GVBD) in maturing sea star oocytes were investigated. Erbstatin and tyrphostins such as AG18 and AG125 blocked 1-methyladenine-induced GVBD in sea star oocytes with IC50 values of less than 20 microM. Inhibition of the rate of GBVD was achieved even when these compounds were added up to 15 min after exposure of the oocytes to 1-methyladenine. The action of these substances on oocyte maturation was reversed by subsequent washing and culturing of the cells in natural sea water free of the inhibitors. Cell viability was maintained for at least 12 h in their presence, as assessed by Trypan blue dye exclusion. These inhibitors prevented the 1-methyladenine-induced activations of the histone H1 kinase p34cdc2, the myelin basic protein kinase p44mpk and a ribosomal S6 peptide kinase. Erbstatin, AG18 and AG125 prevented 1-methyladenine-induced tyrosine dephosphorylation of p34cdc2, and they inhibited tyrosine phosphorylation of p44mpk. These studies imply that activation of a protein-tyrosine kinase may be necessary for stimulation of p34cdc2 in maturing sea star oocytes.

Adenine↗
PubMed1991

Tyrosine phosphorylation is an essential event in the stimulation of B lymphocytes by Staphylococcus aureus Cowan I.

Staphylococcus aureus Cowan I (SAC) is a potent mitogen for purified human B cells. By using Western blotting with antiphosphotyrosine antibodies, we demonstrated that the mitogenic effect of SAC is associated with rapid tyrosine phosphorylation of proteins of 45, 68, 75, 97, and 145 kDa. This tyrosine phosphorylation was detected within 30 s of the addition of SAC; it reached a maximum within 10 min, after which it declined gradually. In contrast to SAC, most soluble anti-IgM antibodies do not induce proliferation of isolated human B cells. As indicated by Western blotting, soluble anti-IgM antibodies induced a similar pattern of tyrosine phosphorylation, with the exception of the 68-kDa protein, which was the most heavily phosphorylated protein in SAC-treated cells. A similar but less intense 68-kDa band was also induced by mitogenic anti-IgM bound to beads. This suggested that tyrosine phosphorylation, especially of p68, may play an important role in B cell mitogenesis. To test this hypothesis, we determined the effect of specific tyrosine kinase inhibitors (tyrphostins) on SAC-induced tyrosine phosphorylation, oncogene expression, and B cell proliferation. The concentration dependencies of inhibition of these processes suggested that they were linked. Nonspecific toxic effects of the tyrphostins were ruled out by the demonstration that the tyrphostins did not alter cell viability and did not inhibit B cell proliferation induced by phorbol esters, which do not induce tyrosine phosphorylation. For maximal inhibition of SAC-induced cell proliferation, the tyrophostins needed to be added before or shortly after addition of SAC. Taken together, these data indicate that tyrosine phosphorylation is an obligatory early signal in B cell proliferation.

Antibodies, Anti-Idiotypic↗
PubMed1991

The regulation of adenylyl cyclase by receptor-operated G proteins.

The receptor regulated adenylyl cyclase system is a multiprotein complex which is a member of the family of the receptor-effector systems whose signal is transduced by heterotrimeric GTP-binding proteins. The system consists of stimulatory and inhibitory receptors (Rs and Ri), stimulatory and inhibitory G proteins (Gs and Gi) and the adenylyl cyclase enzyme (C). While quite specific in situ, receptors (stimulatory or inhibitory) from one source can activate the appropriate G protein from other cell types or species which in turn can act on C from other sources. Studies with chimeric proteins have shown that the various specificities (stimulatory or inhibitory) can be mapped to defined domains in both receptors and G proteins. The mechanism by which the heterotrimeric G proteins couple to the stimulatory and inhibitory signals is discussed in detail. Specifically, the data supporting collision coupling vs the shuttle mechanism is reviewed, as well as the role of beta gamma subunits in both the stimulatory and inhibitory signals.

Adenylyl Cyclases↗
PubMed1991

Tyrphostins. 2. Heterocyclic and alpha-substituted benzylidenemalononitrile tyrphostins as potent inhibitors of EGF receptor and ErbB2/neu tyrosine kinases.

We have previously described a novel series of low molecular weight protein tyrosine kinase inhibitors which we named tyrphostins. The characteristic active pharmacophore of these compounds was the hydroxy-cis-benzylidenemalononitrile moiety. In this article we describe three novel groups of tyrphostins: (i) one group has the phenolic moiety of the cis-benzylidenemalononitrile replaced either with other substituted benzenes or with heteroaromatic rings, (ii) another is a series of conformationally constrained derivatives of hydroxy-cis-benzylidenemalononitriles in which the malononitrile moiety is fixed relative to the aromatic ring, and (iii) two groups of compounds in which the position trans to the benzenemalononitrile has been substituted by ketones and amides. Among the novel tyrphostins examined we found inhibitors which discriminate between the highly homologous EGF receptor kinase (HER1) and ErbB2/neu kinase (HER2). These findings may lead to selective tyrosine kinase blockers for the treatment of diseases in which ErbB2/neu is involved.

Benzylidene Compounds↗
PubMed1991

The inhibition of EGF-dependent proliferation of keratinocytes by tyrphostin tyrosine kinase blockers.

Protein tyrosine kinase blockers of the tyrphostin family inhibited the EGF-dependent proliferation of human and guinea pig keratinocytes grown in culture and induced their growth arrest. These blockers also significantly inhibited the growth of epidermal keratinocytes, but not of dermal cells, in whole skin organ culture from both guinea pig and human origin. The antiproliferative activity of these tyrphostins correlated quantitatively with their potency as inhibitors of EGF receptor autophosphorylation and the EGF-dependent protein phosphorylation of intracellular target proteins in the keratinocyte. Furthermore, no significant cell cytotoxicity or reduction in serine and threonine phosphorylation of many intracellular polypeptides were observed upon incubation of the cells with tyrphostins like AG213. The complete growth arrest induced by the tyrphostins is fully reversible and upon their removal the keratinocytes resumed their growth with the original growth rate. Because of the nontoxic nature of these compounds and their growth-arresting properties, we suggest their use as agents to treat hyperproliferative conditions of human skin.

Animals↗
PubMed1991

Tyrphostins inhibit PDGF-induced DNA synthesis and associated early events in smooth muscle cells.

Tyrphostins are low-molecular-weight synthetic inhibitors of protein tyrosine kinase, which block cell proliferation. Since platelet-derived growth factor (PDGF) is thought to figure prominently in disorders of vascular smooth muscle cells (VSMC), such as atherosclerosis, hypertension, and restenosis, we examined whether tyrphostins would inhibit PDGF-induced mitogenesis in VSMC. In this communication, we demonstrate that tyrphostins with the benzenemalononitrile nucleus inhibited PDGF-dependent growth of VSMC as well as PDGF-dependent DNA synthesis in these cells, with the concentrations for 50% inhibition ranging from 0.04 to 9 microM. Up to 30-fold higher tyrphostin concentrations were required to inhibit serum-stimulated DNA synthesis of VSMC. The effect of the tyrphostins is reversible, since on their removal a normal proliferative response to PDGF was resumed. Tyrphostins also inhibited PDGF-receptor autophosphorylation and PDGF-induced phosphorylation of intracellular substrates, including the phosphorylation of phospholipase C-gamma, with a potency ratio similar to their antimitogenic activity. The expression of c-fos mRNA, a mitogenic nuclear signal, was also reduced in PDGF-stimulated VSMC treated with tyrphostins at concentrations which inhibit PDGF-induced mitogenesis. It is concluded that tyrphostins are potent reversible inhibitors of PDGF-induced mitogenesis which act by inhibiting the tyrosine kinase activity of the PDGF receptor and the subsequent signaling cascade. Tyrphostins may be useful in the study and treatment of VSMC proliferation disorders.

Animals↗
PubMed1991

Activation of phospholipase C in human B cells is dependent on tyrosine phosphorylation.

Cross-linking of the surface antigen receptor on B lymphocytes has been demonstrated to lead to activation of phospholipase C (PLC) with subsequent increases in production of inositol phosphates and diacylglycerol. In turn, these second messengers increase cytosolic free calcium [( Ca2+]i) and activate the serine threonine phosphotransferase protein kinase C (PKC). These processes are thought to play a major role in B cell activation and proliferation. However, the mechanism linking the B lymphocyte antigen receptor to phospholipase C remains to be identified. We demonstrate herein that activation of the antigen receptor on human lymphocytes, in addition to activation of PLC, increases tyrosine phosphorylation of specific substrates. Tyrphostins, a new class of tyrosine kinase inhibitors which compete for substrate binding site of specific tyrosine kinases have recently been synthesized. Preincubation of B lymphocytes with two different tyrphostins blocked anti-IgM-induced proliferation, oncogene expression, tyrosine phosphorylation, increases in [Ca2+]i, and production of inositol phosphates. The same inhibitors were without effect on B cell proliferation induced by phorbol esters and cation ionophores which directly activate PKC and increase [Ca2+]i thus bypassing PLC. These findings strongly indicate that tyrphostins do not exhibit significant nonspecific toxicity and suggest that they act proximal to PLC. The ability of the tyrphostins to block increases in [Ca2+]i and inositol phosphate production, after activation of the B cell antigen receptor, indicates that a tyrosine kinase acts as an essential link between the B cell antigen receptor and PLC.

B-Lymphocytes↗