2,3,7,8-Tetrachlorodibenzo-p-dioxin causes an increase in protein kinases growth hepatic associated with epidermal factor receptor in the plasma membrane

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), administered to male rats at a single intraperitoneal (IP) injection dose of 25 μg/kg causes down-regulation of epidermal growth factor (EGF) receptor in the plasma membrane of rat liver which starts after two days and continues throughout the experimental period (20 days). Using monoclonal antibody to EGF receptor, it was determined that TCDD-caused EFG receptor down-regulation in the rat liver was accompanied by increased protein kinase activity. Such an increase in the protein kinase activity involves, at least in part, an activation of protein tyrosine kinase. Examination of serum samples from control and treated rats revealed no detectable difference in the level of EGF itself or EGF receptor-reacting substances (eg, hormones and other growth factors). In vivo TCDD caused early eye opening and tooth eruption and poor body weight gain and hair growth in mouse neonates similar to those observed with exogenously administered EGE The results indicate that such EGF receptor–mediated effect of TCDD has some toxocilogical significance in vivo. Although TCDD causes significant reduction in [¹²⁵I]-EGF binding in the hepatic plasma membrane in susceptible strains of mice, it has only modest effects in tolerant strains. The results are consistent with the idea that the action of TCDD on the EGF receptor is mediated through the cytosoliclnuclear TCDD receptor, which is known to be regulated by the Ah locus.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin causes elevation of the levels of the protein tyrosine kinase pp60c-src

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been found to cause increases in cellular levels of pp60^src, a protein tyrosine kinase in hepatocytes from the rat and guinea pig, in the thymus of the mouse in vivo and in NIH-3T3 mouse fibroblast cell lines in vitro. Such cellular changes take place in vivo at early stages of TCDD poisoning (as early as one day after treatment in the case of mouse thymus) and at very low doses (single intraperitoneal injections of 1 μg/kg for guinea pigs, 25 μg/ kg for rats, and 30 μg/kg for mice). In addition such an effect of TCDD was observed only in a TCDD-responsive mouse strain but not in a nonresponsive strain. This effect of TCDD is a long-lasting one (eg, even 25 days after single dosing, the levels of pp60^src in the hepatic membrane remained high). In vitro this effect was observed in a wild-type 3T3 cell line but was more pronounced in one of the transfected lines with a v-src gene, a virus-derived oncogene known to code for pp60^src protein.     

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters pancreatic membrane tyrosine phosphorylation following acute treatment

To understand the basic mechanisms of TCDD's action to cause hypoinsulinemia in several experimental animals, we have studied TCDD-induced changes in various protein kinase activities in membrane preparations of guinea pig pancreas. For this purpose, young male guinea pigs were treated through a single intraperitoneal in jection with 1 or 3 μg/kg of TCDD in vivo, and, after given time periods, pancreas samples were obtained and membranes were isolated through homogenization and centrifugation procedures. Several sets of incubation conditions were selected for protein kinase activity assay, each favoring a specific type of protein kinase. It was found that overall protein phosphorylation activities were higher in the preparation from TCDD-treated an imals as compared to those found in pair-fed controls and that this trend was more pronounced when the assay medium contained Mn²⁺ in place of Mg²⁺ and EGTA. These are the conditions that are known to favor protein tyrosine kinases. Other types of protein kinases from the treated animals did not show any significant differences from the pair-fed control animals, though that of protein kinase C in the treated preparation showed a modest increase. To establish that the type of protein kin ases stimulated by TCDD are protein tyrosine kin ases, we have carried out phosphoamino acid analyses, KOH digestion, and western blot analyses using an antibody to phosphotyrosine. All the results were consistent in supporting the idea that TCDD causes a rise in protein-tyrosine kinases in pancreas at early stages of poisoning.     

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) causes increases in protein kinases particularly protein kinase C in the hepatic plasma membrane of the rat and the guinea pig

To study the cause of TCDD-evoked changes in the functions of plasma membrane constituents TCDD's effects on protein kinase activities in the liver of rats and guinea pigs were investigated. TCDD was found to cause a sharp increase in both c-AMP independent and dependent protein kinase activities in plasma membrane preparations from rat liver within 48 hours from the time of administration. Such effects reached maxima around day 20, and were quite noticeable even 40 days after a single administration of TCDD. As a result of SDS-polyacrylamide gel-electrophoresis (SDS-PAGE) analysis several substrate proteins for these increased protein kinases were observed. Among them are 170 K - 150 K bands, representing EGF receptor protein. TCDD was found to particularly stimulate protein kinase C which is known to influence many enzyme and receptor functions through protein phosphorylation. The possible significance of such an action of TCDD is discussed.     

2,3,7,8-tetrachlorodibenzo-P-dioxin induced alterations in protein phosphorylation in guinea pig adipose tissue

An in situ (explant tissue culture) model has been developed to study the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), hormones, and growth factors either alone or in combination. In our model system, the effect of TCDD on protein phosphorylation was greatly affected by the presence or the absence of externally added D-glucose. In the presence of a physiologically relevant level of glucose (13.3 mM), TCDD clearly stimulated protein phosphorylation as in the case of in vivo data. However, in the absence of Dglucose TCDD clearly inhibited protein phosphorylation. On the other hand, TCDD reduced the glucose uptake activity in isolated adipose tissue either in the presence or absence of D-glucose (13.3 mM). Therefore, the TCDD-induced reduction of glucose transport does not appear to be related directly to the simultaneous rise in protein phosphorylation. For comparison, several agents which are known to affect protein phosphorylation were tested. These hormonal agents generally affected the TCDD-untreated adipose tissues in the manner expected from their known actions, indicating that this in situ model is an adequate system to study their independent actions. The TCDD-treated adipose tissue samples showed only mild or insignificant response to these hormonal stimuli. In terms of the changes in the pattern of protein phosphorylation activities, the action of TCDD appeared to resemble that of EGF and T3. Since under in situ conditions no agents such as EGF or T3 can be expected to be present, the observed TCDD-induced changes are suggestive of the basic intracellular changes in cellular activities. The types of TCDD-induced protein kinases appear to be protein tyrosine kinases and protein kinase C.     

Effects of TCDD on the EGF receptor of XB mouse keratinizing epithelial cells

TCDD was found to cause a marked inhibition of 125I-epidermal growth factor (EGF) binding to its receptor on the cell surface of XB mouse keratinizing epithelial cells (XB cells) cultured in vitro. The EC50 concentration was estimated to be on the order of 3 x 10(-11) M 24 hours after TCDD administration. As early as 12 hours after the addition of 10(-9) M of TCDD, XB cells showed signs of a decline in 125I-EGF binding levels. The level of such EGF receptor downregulation reached a maximum at 24 hours, continued until day 2, but completely recovered by day 3. This was accompanied by a rise in protein kinase activities, particularly those of the protein tyrosine kinases during the initial period of 6-24 hours. To test the hypothesis that the EGF receptors of the cells, by showing TCDD-induced symptoms of downregulation, actually are being activated and triggering EGF-like signals, we examined the effects of both TCDD and exogenously added EGF on cell morphology, colony formation degree of keratinization, the pattern of activation of protein kinases and de novo protein synthesis, and EGF receptor phosphorylation. Based on the similarity of cell responses to these between TCDD- and EGF-treated cells, we concluded that TCDD, directly or indirectly, causes activation of the EGF receptor. In contrast, 12-O-tetradencanoylphorbol-13-acetate (TPA), which is known to downregulate EGF receptors by blocking their protein tyrosine kinase, produced dissimilar end results. The balance of evidence support the notion that the action of TCDD in this cell line is tightly coupled to the activation of the EGF receptor and that one of the key consequences of such a biochemical change is that it signals these cells to commit to terminal differentiation.     

Tubulin is phosphorylated at tyrosine by pp60c-src in nerve growth cone membranes

We show here that tubulin is the major in vivo substrate of the tyrosine-specific protein kinase pp60c-src in nerve growth cone membranes. Phosphotyrosine antibodies were used to demonstrate phosphotyrosyl residues in a subpopulation of alpha- and beta-tubulin that was highly enriched in a subcellular fraction of growth cone membranes from fetal rat brain. The presence of phosphotyrosine- modified isoforms of alpha- and beta-tubulin in vivo was confirmed by 32p labeling of rat cortical neurons in culture. Tubulin in growth cone membranes was phosphorylated at tyrosine in endogenous membrane phosphorylation reactions (0.068 mol phosphotyrosine/mol alpha-tubulin and 0.045 mol phosphotyrosine/mol beta-tubulin), and phosphorylation was specifically inhibited by antibodies directed against pp60c-src, which is localized in the growth cone membranes. pp60c-src was capable of directly phosphorylating tubulin as shown in immune complex kinase assays with purified brain tubulin. Phosphopeptide mapping revealed a limited number of sites of tyrosine phosphorylation in alpha- and beta- tubulin, with similar phosphopeptides observed in vivo and in vitro. These results reveal a novel posttranslational modification of tubulin that could regulate microtubule dynamics at the growth cone.     

Activation of p56lck through mutation of a regulatory carboxy-terminal tyrosine residue requires intact sites of autophosphorylation and myristylation.

Mutation of the major site of in vivo tyrosine phosphorylation of p56lck (tyrosine 505) to a phenylalanine constitutively enhances the p56lck-associated tyrosine-specific protein kinase activity. The mutant polypeptide is extensively phosphorylated in vivo at the site of in vitro Lck autophosphorylation (tyrosine 394) and is capable of oncogenic transformation of rodent fibroblasts. These observations have suggested that phosphorylation at Tyr-505 down regulates the tyrosine protein kinase activity of p56lck. Herein we have attempted to examine whether other posttranslational modifications may be involved in regulation of the enzymatic function of p56lck. The results indicated that activation of p56lck by mutation of Tyr-505 was prevented by a tyrosine-to-phenylalanine substitution at position 394. Furthermore, activation of p56lck by mutation of the carboxy-terminal tyrosine residue was rendered less efficient by substituting an alanine residue for the amino-terminal glycine. This second mutation prevented p56lck myristylation and stable membrane association and was associated with decreased in vivo phosphorylation at Tyr-394. Taken together, these findings imply that lack of phosphorylation at Tyr-505 may be insufficient for enhancement of the p56lck-associated tyrosine protein kinase activity. Our data suggest that activation of p56lck may be dependent on phosphorylation at Tyr-394 and that this process may be facilitated by myristylation, membrane association, or both.     

The role of protein tyrosine kinases in CYP1A1 induction by omeprazole and thiabendazole in rat hepatocytes

Benzimidazoles compounds like omeprazole (OME) and thiabendazole (TBZ) mediate CYP1A1 induction differently from classical aryl hydrocarbon receptor (AhR) ligands, 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To clarify the involvement of an intracellular signal pathway in CYP1A1 induction by OME and TBZ, the TBZ, OME and 3-MC signal-transducing pathways were compared by using specific protein tyrosine kinase inhibitors in primary culture of rat hepatocytes. The effect of OME and TBZ (75-250 microM) on cytochrome P450 1A1 (CYP1A1) expression was therefore studied in primary cultures of rat hepatocytes after 24 h, 48 h and 72 h of exposure. Both compounds provoked a dose- and time-dependent increase in CYP1A1 (EROD activity, protein and mRNA levels), but OME was less effective at all the concentrations and times tested. The mechanism of benzimidazole-mediated induction of CYP1A1 was investigated by comparison with 3-MC, a prototypical AhR ligand. As expected, OME and TBZ were unable to displace [(3)H]-TCDD from its binding sites to the AhR in competitive binding studies. Moreover, classic tyrosine kinase inhibitor herbimycin A (HA) inhibited the two benzimidazoles-mediated CYP1A1 inductions, but only partially inhibited the 3-MC-mediated one. Another two tyrosine kinase inhibitors, Lavendustin A (LA) and genistein (GEN), had no effect on CYP1A1 induction by benzimidazoles and 3-MC. These results are consistent with the implication of a tyrosine kinase, most probably the Src tyrosine kinase, in the mechanism of CYP1A1 induction in rat hepatocytes.     

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced reduction of adenosine deaminase activity in vivo and in vitro

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent immunosuppressant in several animal species. The purpose of this study was to determine if TCDD affected the activity of adenosine deaminase (ADA), a purine metabolizing enzyme that is vital to the proper functioning of the immune system. The effect of TCDD on ADA ctivity was studied in various tissues of male Balb/c mice (a TCDD-responsive strain) and DBA/2 mice (a less-responsive strain). Of the tissues examined after administration of TCDD in vivo (115 μ/kg, i.p.), ADA activity was found to be significantly reduced in thymic and splenic tissues of Balb/c mice at 24 hours postadministration. The enzyme activity in these affected tissues remained consistently low through 10 days postadministration. Such an effect of TCDD was both dose and time related in the thymic tissue of Balb/c mice. In contrast, no appreciable alterations in ADA activity were evident in any of the tissues of DBA/2 mice at any of the sampling intervals, indicating that such an effect of TCDD is likely to be mediated through the Ah receptor. This in vivo effect of TCDD on thymic ADA activity was also reproducible in situ where isolated whole thymuses were directly incubated with 10 nM TCDD. In this model, TCDD's effects on ADA activity were antagonized by known protein kinase or phosphorylation inhibitors such as quercetin, genistein, tyrphostin, and neomycin. These results indicate that the effect of TCDD on ADA activity in the thymus may be related to its property to elevate protein kinase activities in this tissue. ADA activity was also reduced in 3T3 cells that were treated with 10 nM TCDD in a low (1%) serum media. In contrast, 25 ng/mL epidermal growth factor (EGF) under such conditions consistently stimulated ADA activity. Interestingly, EGF at a similar concentration failed to elicit a stimulatory effect on ADA activity when cells were pretreated with TCDD. The property of TCDD to lower ADA activity under in vivo, in situ, as well as in vitro conditions appears to be largely related to its action to modulate protein phosphorylation activities.     

Confocal microscopic study of GABA(A) receptors in Xenopus oocytes after rat brain mRNA injection: modulation by tyrosine kinase activity

The expression of GABA(A) receptors in Xenopus oocytes injected with rat brain mRNA was studied by immunocytochemistry and evaluation of the distribution of fluorescent probes at the confocal microscope. The beta(2/3) subunit distributed exclusively on the membrane at the animal pole of the oocytes. Treatment of oocytes for 20 min with the protein tyrosine kinase inhibitor genistein, 200 microM, resulted in a lower presence of GABA(A) receptors on the membrane. The inactive genistein analogue daidzein, 200 microM, had no effect even with a 30 min treatment. Alkaline phosphatase but not a protein tyrosine phosphatase, when injected into oocytes, reduced GABA(A) receptor membrane expression. The data indicate that protein tyrosine phosphorylation modulates the expression on the plasma membrane of presynthesized GABA(A) receptors.     

TCDD causes stimulation of c-ras expression in the hepatic plasma membranes in vivo and in vitro

A series of in vivo and in vitro experiments were conducted to determine the effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) administered on the expression of c-ras. Differences in c-ras expression between control and TCDD treated groups were determined by immunoassay of p21^ras protein, or indirectly measured by the specific binding of ³H-GTP to hepatic plasma membrane preparations. Intraperitoneal injection of sublethal doses of TCDD significantly elevated (P < 0.05, Student t test) levels of hepatic p21^ras protein in Sprague-Dawley rats and TCDD sensitive C57BL/6J mice. Such an increase occurred at an early stage of poisoning in the C57BL/6J mice. The earliest increase was detectable 6 hr after dosing, and the differencebecame statistically significant by 12 and 24 hr after dosing. In contrast, TCDD tolerant DBA/2J mice had only a marginal increase in hepatic p21^ras protein which did not become statistically significant even at 24 hr post-dosing. TCDD evoked increases in hepatic p21^ras protein of C57BL/6J mice were accompanied by the increase in the specific binding of GTP to hepatic plasma membranes. Column chromatography of solubilized rat hepatic membrane proteins on sephadex G-50 showed TCDD administration increased levels of a ³H-GTP binding protein with MW of approximately 21 Kd. ³H-GTP binding in total hepatic membranes was also elevated (P<0.05, Fisher PLSD multiple comparison test) 6 hr and 24 hr after dosing of C57BL/6J mice, but as expected the effect of TCDD was not as conspicuous as that found in the plasma membrane. TCDD treatment increased levels of a 21 Kd protein found in the in vitro translation products of RNA purified from guinea pig liver. This protein was identified as a c-ras protein based upon its ability to bind GTP, precipitation by a polyclonal antibody against the rasHa and Ki proteins and subsequent SDS-PAGE which showed a single protein band of ≈ 21 Kd.     

Activation of c-Src in cells bearing v-Crk and its suppression by Csk.

The protein product of the CT10 virus, p47gag-crk (v-Crk), which contains Src homology region 2 (SH2) and 3 (SH3) domains but lacks a kinase domain, is believed to cause an increase in cellular protein tyrosine phosphorylation. A candidate tyrosine kinase, Csk (C-terminal Src kinase), has been implicated in c-Src Tyr-527 phosphorylation, which negatively regulates the protein tyrosine kinase of pp60c-src (c-Src). To investigate how c-Src kinase activity is regulated in vivo, we first looked at whether v-Crk can activate c-Src kinase. We found that cooverexpression of v-Crk and c-Src caused elevation of c-Src kinase activity, resulting in an increase of tyrosine phosphorylation of cellular proteins and morphological transformation of rat 3Y1 fibroblasts. v-Crk and c-Src complexes were not detected, although v-Crk bound to a variety of tyrosine-phosphorylated proteins in cells overexpressing v-Crk and c-Src. Overexpression of Csk in these transformed cells caused reversion to normal phenotypes and also reduced the level of c-Src kinase activity. However, Csk did not cause reversion of cells transformed by v-Src or c-Src527F, in which Tyr-527 was changed to Phe. These results strongly suggest that Csk acts on Tyr-527 of c-Src and suppresses c-Src kinase activity in vivo. Because Csk can suppress transformation by cooverexpression of v-Crk and c-Src, we suggest that v-Crk causes activation of c-Src in vivo by altering the phosphorylation state of Tyr-527.     

Activated Src tyrosine kinase phosphorylates Tyr-457 of bovine GTPase-activating protein (GAP) in vitro and the corresponding residue of rat GAP in vivo.

GTPase-activating protein (GAP) is a key regulator of the cellular Ras protein, which is implicated in oncogenic signal transduction pathways downstream of the viral Src (v-Src) kinase. Previous studies demonstrated that v-Src induces tyrosine phosphorylation of GAP, suggesting that GAP may provide a biochemical link between v-Src and Ras signaling pathways. To determine the precise residues in GAP phosphorylated by Src kinases, we used a baculovirus/insect cell expression system for investigating in vitro phosphorylation of GAP. Phosphopeptide mapping analysis revealed that v-Src and normal cellular Src (c-Src) phosphorylate tyrosine residues in bovine GAP at one major site and one minor site in vitro. Significantly, the major site of GAP phosphorylation in vitro is also the major site of in vivo tyrosine phosphorylation of GAP in rat fibroblasts transformed by v-Src. Analyses of GAP deletion mutants and TrpE-GAP fusion proteins established that Tyr-457 of bovine GAP (and the corresponding residue of rat and human GAP) is the major site of tyrosine phosphorylation. Our results demonstrate that the v-Src kinase induces phosphorylation of the same tyrosine residue of GAP in vitro and in vivo, suggesting that GAP is a direct substrate of activated Src kinases in vivo. Because epidermal growth factor receptor phosphorylates the equivalent tyrosine residue in human GAP (Tyr-460), these findings are consistent with the hypothesis that specific phosphorylation of GAP at this site may have a physiologically important role in regulating mitogenic Ras signaling pathways.     

Localization of phospho-beta-dystroglycan (pY892) to an intracellular vesicular compartment in cultured cells and skeletal muscle fibers in vivo

beta-Dystroglycan is a ubiquitously expressed integral membrane protein that undergoes tyrosine phosphorylation in an adhesion-dependent manner. Tyrosine 892 is now thought to be the principal site for recognition by the c-Src tyrosine kinase; however, little is known about the regulation of this phosphorylation event in vivo. Here, we generated a novel monoclonal antibody probe that recognizes only tyrosine 892 phosphorylated beta-dystroglycan (pY892). We show that upon tyrosine phosphorylation, beta-dystroglycan undergoes a profound change in its sub-cellular localization (e.g., from the plasma membrane to an internal membrane compartment). One possibility is that the net negative charge at position 892 causes the redistribution of beta-dystroglycan to this intracellular vesicular location. In support of this notion, mutation of tyrosine 892 to glutamate (Y892E) is sufficient to drive this intracellular localization, while other point mutants (Y892F and Y892A) remain at the plasma membrane. Interestingly, our colocalization studies with endosomal markers (EEA1, transferrin, and transferrin receptor) suggest that these phospho-beta-dystroglycan containing internal vesicles represent a subset of recycling endosomes. At the level of these internal vesicular structures, we find that tyrosine phosphorylated beta-dystroglycan is colocalized with c-Src. In addition, we demonstrate that known ligands for alpha-dystroglycan, namely, agrin and laminin, are able to induce the tyrosine phosphorylation of beta-dystroglycan. Finally, we show that tyrosine phosphorylated beta-dystroglycan is also detectable in skeletal muscle tissue lysates and is localized to an internal vesicular membrane compartment in skeletal muscle fibers in vivo. The generation of a phospho-specific beta-dystroglycan (pY892) mAb probe provides a new powerful tool for dissecting the role of dystroglycan phosphorylation in normal cellular functioning and in the pathogenesis of muscular dystrophies.     

Phosphorylation of tyrosine hydroxylase by calmodulin-dependent multiprotein kinase

Tyrosine hydroxylase purified from rat pheochromocytoma was phosphorylated stoichiometrically by either cyclic AMP-dependent protein kinase or calmodulin-dependent multiprotein kinase from skeletal muscle, but not by five other protein kinases tested. The activity of tyrosine hydroxylase was elevated 3-fold by cyclic AMP-dependent protein kinase, but no activation was observed after phosphorylation by calmodulin-dependent multiprotein kinase. Phosphorylation produced by cyclic AMP-dependent protein kinase and calmodulin-dependent multiprotein kinase was additive, suggesting different sites of phosphorylation. This was confirmed by high-performance liquid chromatography analysis of tryptic phosphopeptides which demonstrated that the major sites phosphorylated by each protein kinase were distinct. A calmodulin-dependent multiprotein kinase that had identical properties and substrate specificity to the skeletal muscle enzyme was partially purified from rat pheochromocytoma. The possibility that this protein kinase is involved in the regulation of tyrosine hydroxylase activity in adrenergic tissue in vivo is discussed.     

Activation of the oncogenic potential of the avian cellular src protein by specific structural alteration of the carboxy terminus.

The role of tyrosine phosphorylation in the regulation of tyrosine protein kinase activity was investigated using site-directed mutagenesis to alter the structure and environment of the three tyrosine residues present in the C terminus of avian pp60c-src. Mutations that change Tyr 527 to Phe or Ser activate in vivo tyrosine protein kinase activity and induce cellular transformation of chicken cells in culture. In contrast, alterations of tyrosine residues present at positions 511 or 519 in c-src do not induce transformation or in vivo tyrosine protein kinase activity. Amber mutations, which alter the structure of the pp60c-src C terminus by inducing premature termination of the c-src protein at either residue 518 or 523 also induce morphological transformation and increase in vivo tyrosine phosphorylation, whereas removal of the last four residues of c-src by chain termination at residue 530 does not alter the kinase activity or the biological activity of the resultant c-src protein. We conclude from these studies that C-terminal alterations which either remove or replace Tyr 527 serve to activate the c-src protein resulting in cellular transformation and increased in vivo tyrosine protein kinase activity.     

The role of tyrosine phosphorylation in signal transduction through surface Ig in human B cells. Inhibition of tyrosine phosphorylation prevents intracellular calcium release.

Cross-linking surface Ig on human B cells, or the TCR complex on T cells leads to the rapid appearance of newly tyrosine phosphorylated proteins. This is associated with inositol phospholipid turnover and a rise in intracellular calcium. Incubation of human B or T lymphocytes with the tyrosine kinase inhibitors, herbimycin and genistein, inhibits new tyrosine phosphorylation after receptor-linked activation. This is associated with complete abrogation of the increase in intracellular calcium in these lymphocytes and inhibition of inositol phospholipid turnover. Herbimycin- and genistein-treated lymphocytes are nevertheless still capable of responding to aluminum fluoride with a rise in intracellular calcium. These data support the contention that a B cell-associated protein tyrosine kinase regulates signal transduction via phospholipase C. CD45, the membrane associated protein tyrosine phosphatase, and PMA that activates protein kinase C, both inhibit the calcium response in B lymphocytes induced by receptor cross-linking. PMA and cross-linking CD45 both induced the appearance of tyrosine phosphorylated proteins in human B cells, although the pattern is quite distinct from that seen when surface lg is cross-linked. However, the induction of new tyrosine phosphorylation by anti-mu does not appear to be affected by these reagents. Although this may reflect an insensitivity of the tyrosine phosphorylation assay, it could indicate that regulation of the calcium response and regulation of the tyrosine kinase can be independent processes.