Involvement of the tyrosine phosphorylation on GSH transport in NIH3T3 fibroblasts

This study demonstrates the involvement of phosphotyrosine phosphatases on the activity and regulation of GSH ATP-dependent transport system that we have previously identified in NIH3T3 fibroblasts. This is shown by the fact that increases of the initial rate of GSH uptake were measured in NIH3T3 overexpressing a synthetic gene coding for a low-Mr-phosphotyrosine protein phosphatase (LMW-PTP), while decreases were obtained in NIH3T3 overexpressing the phosphatase inactive mutant (LMW-C12SPTP), with respect to NIH3T3neo. Moreover, these results have been confirmed by experiments performed in the same cells by vanadate, and H2O2 treatment on both GSH transport and mediated passive transport of glucose. A possible regulation of this transport system by platelet-derived growth factor receptor (PDGFr) with tyrosine kinase activity is also demonstrated. Moreover, these data show a relationship among GSH, PDGFr and phosphotyrosine phosphatase activity, and suggest a role of GSH transport systems on the cell proliferation process.     

The insulinomimetic agents H2O2 and vanadate stimulate protein tyrosine phosphorylation in intact cells

H2O2 and vanadate are known insulinomimetic agents. Together they induce insulin's bioeffects with a potency which exceeds that seen with insulin, vanadate, or H2O2 alone. Employing Western blotting with anti-P-Tyr antibodies, we have identified in Fao cells at least four proteins (pp180, 150, 114, and 100) whose P-Tyr content is rapidly increased upon treatment of the cells with 3 mM H2O2. Tyrosine phosphorylation of these and additional proteins was markedly potentiated (6-10-fold) when 100 microM sodium orthovanadate was added together with H2O2. The effects of H2O2 and vanadate on protein tyrosine phosphorylation were rapid and specific. The enhanced tyrosine phosphorylation was accompanied by a concomitant inhibition of a cytosolic protein tyrosine phosphatase activity. The latter was inhibited by 50% in 3 mM H2O2-treated cells. The inhibitory effect was augmented in the combined presence of H2O2 and vanadate. Half- and maximal effects of vanadate were obtained at 15 microM and 1 mM, respectively. Vanadate (1 mM) alone, added to the cells, had only a trivial effect on protein tyrosine phosphatase activity. A 45-s challenge with insulin (10(-7) M) of cells pretreated with H2O2 largely mimicked the potentiating effects of vanadate on protein tyrosine phosphorylation but not on protein tyrosine phosphatase activity. Our results suggest the involvement of multiple tyrosine-phosphorylation proteins in mediating the biological effects of H2O2/vanadate. Their enhanced phosphorylation can be attributed at least in part, to the inhibitory effects exerted by H2O2 alone, or in combination with vanadate, on protein tyrosine phosphatase activity. The similarity between proteins phosphorylated in Fao cells in response to H2O2/vanadate or H2O2/insulin, suggests that either treatment stimulates protein tyrosine kinases having similar substrate specificities. The insulin receptor kinase is a likely candidate as its activity is markedly enhanced either by insulin (plus H2O2) or by H2O2/vanadate.     

Interleukin 3 stimulation of tyrosine kinase activity in FDC-P1 cells

Interleukin 3 stimulates the proliferation of FDC-P1, a murine myeloid cell line, however the biochemical events subsequent to binding of IL3 have only recently begun to be investigated. We have previously described the activation of protein kinase C (PK-C) and serine/threonine phosphorylation of a 68 kd protein following IL3 treatment of FDC-P1 cells. Here we have used an anti-phosphotyrosine antibody to purify proteins containing phosphotyrosine following IL3 administration to FDC-P1 cells. We find that tyrosine phosphorylation of two proteins of 50 (pp50) and 70 (pp70) kilodaltons occurs rapidly following IL3 treatment. In addition to phosphotyrosine both proteins also contained phosphoserine. Together with previous evidence these results suggest that coactivation of serine/threonine and tyrosine kinase activities which target unique proteins may be an important element in IL3 signal transduction.     

The carboxyl-terminal tyrosine residue of protein-tyrosine phosphatase alpha mediates association with focal adhesion plaques

The receptor protein-tyrosine phosphatase alpha (PTPalpha) is involved in the activation of c-Src kinase as well as in down-regulation of the insulin signal. To investigate the role of PTPalpha in activation of the Src kinase in more detail we tried to overexpress this phosphatase in NIH3T3 fibroblasts. Although PTPalpha has been overexpressed in rat embryonic fibroblasts and in embryonic carcinoma cells and should increase mitogenic responses we were not able to achieve a detectable overexpression. In contrast, expression of partially (C442S) or completely inactive (C442S,C732S) PTPalpha or of phosphatase active PTPalpha containing mutation Y781F or Y798F was possible. The level of expression, however, was reduced to background after several passages of lines expressing PTPalphaC442S,C732S and PTPalphaY781F. When employed in a focus formation assay, only infection with virus encoding PTPalphaY798F induced Src-dependent formation of foci. In immunofluorescence studies, PTPalphaC442S and PTPalphaY781F but not PTPalphaY798F colocalized with proteins found in focal adhesion plaques. Treatment of PTPalphaC442S-overexpressing cells with vanadate abolished this colocalization and led to proteolytic processing of the phosphatase. We conclude that tyrosine 798 in PTPalpha is important for localization at focal adhesion plaques. Inhibition of phosphatases by vanadate treatment releases PTPalpha from focal adhesions.     

Laminin stimulates protein tyrosine dephosphorylation in PC12 cells.

Laminin stimulates neurite outgrowth in rat pheochromocytoma cells (PC12 cells). Here, we investigated laminin signal transduction mechanisms by adding the tyrosine kinase/phosphatase modulators, genistein, quercetin, aurin tricarboxylic acid (ATA), and vanadate to PC12 cells. At 10 microM both genistein and quercetin enhanced laminin-mediated neurite outgrowth by 1.7- and 2.3-fold, respectively, while at 10 microM, ATA inhibited laminin-mediated neurite outgrowth by 92%. Vanadate inhibited neurite outgrowth by 63% at 10 microM. Immunoblot analysis revealed four proteins of approximately 240, 22, 110, and 35 kDa, which were dephosphorylated on tyrosine residues in laminin-treated PC12 cells, but not in NIH 3T3 cells. These results demonstrate that laminin-mediated neurite outgrowth involves protein tyrosine dephosphorylation and suggests that this mechanism may have specificity to neuronal cells.     

Activation of human T cells is associated with tyrosine phosphorylation of several cellular proteins

Human T lymphocytes are activated to proliferate after triggering the T Cell Antigen Receptor Complex. CD3-Ti, with either antigen, mitogenic lectins or monoclonal antibodies against its different subunits. Stimulation of Jurkat leukemic human T cells with anti-CD3 or anti-Ti monoclonal antibodies was found to induce, within 1 min, an increase in the phosphorylation of a set of cellular proteins that can be precipitated with anti-phosphotyrosine antibodies. Seven phosphotyrosine-containing proteins were separated with respective mol. wt of 21, 25, 38, 55, 70, 80 and 110 kDa, among which the 38 kDa species is predominant. Moreover, incubation of Jurkat T cells with sodium orthovanadate, a potent inhibitor of phosphotyrosine protein-phosphatases, was found to potentiate the effects of anti-CD3 mAb on tyrosine phosphorylation. In addition vanadate also induced IL-2 secretion in Jurkat cells when associated with the phorbol ester TPA, further demonstrating the importance of these phosphorylation reactions in the process of T cell activation. Our results therefore allow us to identify several protein substrates of a tyrosine kinase activity, whose stimulation appears to be an early event in human T cell activation through the antigen receptor pathway.     

Ras-induced activation of Raf-1 is dependent on tyrosine phosphorylation.

Although Rafs play a central role in signal transduction, the mechanism(s) by which they become activated is poorly understood. Raf-1 activation is dependent on the protein's ability to bind Ras, but Ras binding is insufficient to activate Raf-1 tyrosine phosphorylation to this Ras-induced activation, in the absence of an over-expressed tyrosine kinase. We demonstrate that Raf-1 purified form Sf9 cells coinfected with baculovirus Ras but not Src could be inactivated by protein tyrosine phosphatase PTP-1B. 14-3-3 and Hsp90 proteins blocked both the tyrosine dephosphorylation and inactivation of Raf-1, suggesting that Raf-1 activity is phosphotyrosine dependent. In Ras-transformed NIH 3T3 cells, a minority of Raf-1 protein was membrane associated, but essentially all Raf-1 activity and Raf-1 phosphotyrosine fractionated with plasma membranes. Thus, the tyrosine-phosphorylated and active pool of Raf-1 constitute a membrane-localized subfraction which could also be inactivated with PTP-1B. By contrast, B-Raf has aspartic acid residues at positions homologous to those of the phosphorylated tyrosines (at 340 and 341) of Raf-1 and displays a high basal level of activity. B-Raf was not detectably tyrosine phosphorylated, membrane localized, or further activated upon Ras transformation, even though B-Raf has been shown to bind to Ras in vitro. We conclude that tyrosine phosphorylation is an essential component of the mechanism by which Ras activates Raf-1 kinase activity and that steady-state activated Ras is insufficient to activate B-Raf in vivo.     

Vanadate inhibition of protein tyrosine phosphatases in Jurkat cells: modulation by redox state

 Vanadate is a potent reversible inhibitor of protein tyrosine phosphatases (PTP) in vitro. Vanadate has been shown to increase the phosphotyrosine levels in some cell types whereas in others, like the Jurkat T-lymphoma, vanadate has no effect. The reason for the apparent lack of effect of vanadate in Jurkat cells was investigated in this study. Alteration of the redox state of these cells by reducing the glutathione level with 1-chloro-2,4-dinitrobenzene (DnpCl) had no effect on phosphotyrosine levels. However, the cells became sensitive to vanadate, as measured by an increase in phosphotyrosine levels on a wide range of proteins including the MAP kinases. The increase in phosphotyrosine levels most likely results from inhibition of cellular PTP and suggests that protein tyrosine kinases are constitutively active in cells, resulting in a dynamic phosphorylation-dephosphorylation cycle. The mode of inhibition of PTP by vanadate was investigated by measuring the PTP activity of Jurkat membranes isolated after treatment of cells with vanadate and DnpCl. In contrast to the reversible inhibition of PTP in vitro, the effect of vanadate in the presence of DnpCl was irreversible, raising the possibility that it is peroxovanadate formed in situ that is responsible for the inhibition of PTP in intact cells. Received: 4 December 1998 · Accepted: 22 March 1999     

Evidence that increased tyrosine phosphorylation causes disassembly of adherens junctions but does not perturb paracellular permeability in Caco-2 cells

In this study, we report on the apparent effect of increased tyrosine phosphorylation events on the assembly and integrity of adherens junctions (AJs) and on paracellular permeability in Caco-2 cells. Cell monolayers were incubated with the phosphotyrosine phosphatase inhibitor vanadate/H2O2. Addition of this compound to monolayer resulted in disruption of the AJs, as revealed by electron microscopy and by a loss of membrane association of the AJ-associated protein uvomorulin/E-cadherin (U/E-c). However, tight junctions (TJs) were unaltered, as determined by measuring the transepithelial resistance (Rt), by ruthenium red labeling, as seen by transmission electron microscopy, and the distribution of TJ strands as seen in freeze-fracture replicas and by hyperphosphorylation of triton-insoluble occludin. Also examination of vanadate/H2O2 treated cells indicated a specific increase in AJ-associated phosphotyrosine residues as evaluated by immunofluorescence microscopy, but no modification of F-actin distribution, as revealed by confocal laser scanning microscopy analysis. To verify that modulation of AJs was indeed related to tyrosine phosphorylation, we tested a range of distinct protein kinase inhibitors. Of the three inhibitors tested (tyrphostin 25, genistein and staurosporine), tyrphostin 25 completely blocked the effects of vanadate/ H2O2 on assembly and integrity of AJs, redistribution of U/E-c and phosphotyrosine labeling. Our results indicate that, after addition of vanadate/H2O2 to Caco-2 monolayers, specific tyrosine phosphorylation of proteins cause disruption of AJs, but no modifications of the TJs' structure and functionality. These observations suggest that, in contrast to what happens with epithelial cells, TJs and AJs of Caco-2 cells are regulated by independent mechanisms.     

Reversible tyrosine phosphorylation/dephosphorylation of proline-directed protein kinase FA/glycogen synthase kinase-3α in A431 cells

Modulation of protein kinase FA /glycogen synthase kinase-3α (kinase FA /GSK-3α) by reversible tyrosine phosphorylation/dephosphorylation was investigated. In addition to genistein, other protein tyrosine kinase (PTK) inhibitors, such as tyrphostin A47 and B42, also could induce tyrosine dephosphorylation and inactivation of kinase FA /GSK-3α in A431 cells, and this process was found to be reversible. Pretreatment of the cells with 100 μM orthovanadate, a protein tyrosine phosphatase (PTP) inhibitor, could diminish significantly the effects of PTK inhibitors on both enzyme activity and phosphotyrosine content of the kinase, suggesting that the PTK inhibitors induced tyrosine dephosphorylation/inactivation of this kinase is mediated by orthovanadate-sensitive PTP(s) in A431 cells. Moreover, the phosphotyrosine moiety of kinase FA /GSK-3α was found to be highly turned over in resting cells. Interestingly, we found that the less active, tyrosine-dephosphorylated form of kinase FA /GSK-3α immunoprecipitated from genistein-treated cells was able to reactivate partially with concomitant rephosphorylation of tyrosine residue in vitro. Taken together, these findings demonstrate that tyrosine phosphorylation and concomitant activation of kinase FA /GSK-3α can be carried out both in vitro and in vivo and an in vivo phosphatase activity may function in antagonism to PTK activation of kinase FA /GSK-3α. J. Cell. Physiol. 171:95–103, 1997. © 1997 Wiley-Liss, Inc.     

Constitutively activated neu oncoprotein tyrosine kinase interferes with growth factor-induced signals for gene activation.

The neu receptor oncoprotein tyrosine kinase, capable of transforming cultured fibroblasts and causing mammary carcinomas in transgenic mice, carries a point mutation in its transmembrane domain and shows a constitutive tyrosine kinase activity. We analyzed the neu tyrosine kinase and its substrates in transfected NIH 3T3 fibroblasts by phosphotyrosine immunoblotting. Tyrosine phosphorylated proteins were similar but not identical in epidermal growth factor (EGF)-stimulated cells expressing the human EGF receptor (EGFR) or a chimeric EGFR/neu receptor but differed from phosphotyrosyl proteins constitutively expressed in neu oncogene-transformed cells. The neu oncoprotein in the latter cells was phosphorylated in tyrosine in a ligand-independent manner and had a shortened half-life in comparison with the normal neu protein. Tumor promoter pretreatment inhibited ligand-induced receptor tyrosine phosphorylation and decreased tyrosine phosphorylated neu oncoprotein. Prolonged pretreatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also prevented the induction of immediate early growth factor-regulated genes in response to neu activation. Expression of the neu oncogene but not the protooncogene in NIH 3T3 cells was associated with enhanced levels of the jun and fos oncoproteins and loss of serum growth factor induction of immediate early mRNA responses. The constitutively activated neu oncoprotein tyrosine kinase thus deregulates cellular genomic responses to growth factors.     

Effect of vanadate on the cellular accumulation of pp15, an apparent product of insulin receptor tyrosine kinase action

The possible involvement of a 15-kDa phosphotyrosyl protein, pp15, in insulin action was investigated by using the insulin-mimetic agent, vanadate. Vanadate, a phosphotyrosine phosphatase inhibitor, was found to mimic insulin in 3T3-L1 adipocytes by three criteria. First, kinetic and concentration-dependence studies verified the insulin-like effect of vanadate in activating 2-deoxyglucose uptake. Insulin had an additive activating effect at a submaximal vanadate concentration, but showed no further activation at a saturating vanadate concentration. The trivalent arsenical, phenylarsine oxide (PAO) which forms complexes with vicinal dithiols, markedly inhibited vanadate-activated hexose transport in agreement with our previous studies in which PAO abolished the insulin-activated component of sugar uptake. Second, in situ phosphorylation experiments showed that vanadate activated tyrosine phosphorylation of the insulin receptor's beta-subunit. Exposure of vanadate-treated cells to PAO further increased the level of beta-subunit phosphorylation. The increased level of phosphorylation in the presence of PAO occurred only on tyrosyl residues. Third, vanadate caused the accumulation of a phosphorylated 15-kDa protein in the presence of PAO, but not in its absence. The characteristics of this protein were identical to those of pp15: 1) both proteins behaved identically by two-dimensional gel electrophoresis, 2) digestion of both proteins with trypsin gave rise to apparently identical phosphopeptides, and 3) both proteins contained phosphotyrosine as the only phosphoamino acid. The results indicate that both vanadate and insulin stimulate the accumulation of pp15 in the presence of PAO. The dithiol,2,3-dimercaptopropanol, but not a monothiol, reversed the effects of PAO on the inhibition of vanadate-induced hexose transport and the accumulation of pp15, thus implicating a vicinal dithiol in these actions of vanadate and insulin. Our results support the hypothesis that turnover of the phosphoryl group of pp15, a product of insulin receptor tyrosine kinase action, is coupled to signal transmission to the glucose transport system.     

Bradykinin and bombesin rapidly stimulate tyrosine phosphorylation of a 120-kDa group of proteins in Swiss 3T3 cells

We have examined the effect of bradykinin (BK) and other peptide mediators with related cellular actions on tyrosine phosphorylation in confluent Swiss 3T3 fibroblast cells using an anti-phosphotyrosine antibody. Immunoblots of extracts from cells stimulated with BK showed a major heterogeneous band centered at Mr 120,000. Three phosphorylated protein species were present within this band. The lower of these three phosphoproteins was occasionally present under basal conditions. The detection of this group of phosphoproteins by the antibody was prevented by coincubation with an excess of phosphotyrosine but not with an excess of phosphoserine or phosphothreonine. The BK-promoted increase in phosphorylation was rapid and transient with the peak response apparent following BK exposure for 1 min. The response was dose-dependent with half-maximal effect occurring at 10-30 nM BK. The antagonist Arg0, Hyp3, Thi5,8, D-Phe7-BK completely inhibited the response indicating that BK was acting via a B2 kinin receptor. Bombesin, at 0.1 microM, stimulated an increase in phosphorylation of the 120-kDa group of proteins with the same efficacy as 0.1 microM BK. On the other hand, 1 microM vasopressin was considerably less efficaceous than either of the former agonists. Short-term preexposure to 0.1 microM 12-O-tetradecanoyl-phorbol-13-acetate (1 min), a protein kinase C stimulator, or 30 microM H7 (15 min), a protein kinase C inhibitor, had no significant effect either on the basal or BK-promoted increase in tyrosine phosphorylation of these proteins. BK also stimulated inositol phosphate formation in these cells. Genistein, a tyrosine kinase inhibitor, inhibited BK stimulation of tyrosine phosphorylation. In addition, genistein partially inhibited BK stimulation of inositol phosphate formation. These results show that an increase in tyrosine phosphorylation of a 120-kDa group of proteins is an early protein kinase C-independent cellular signal elicited by both bradykinin and bombesin.     

Regulation of protein phosphotyrosine content by changes in tyrosine kinase and protein phosphotyrosine phosphatase activities during induced granulocytic and monocytic differentiation of HL-60 leukemia cells

About 1.5% of phosphorylated amino acid residues of HL-60 promyelocytic leukemia cells are phosphotyrosine. Induction of granulocytic differentiation by exposure to dimethylsulfoxide decreased tyrosine phosphorylation to 0.2%. A maximum 3-fold increase in tyrosine kinase activity and a 7-fold increase in protein phosphotyrosine phosphatase activity accompanied this change. Monocytic differentiation induced by 12-O-tetradecanoylphorbol-13-acetate, caused a decrease in phosphotyrosine levels to 0.1%; tyrosine kinase activity maximally increased 2-fold, and protein phosphotyrosine phosphatase activity increased 11-fold in these differentiated cells. Thus, although total tyrosine kinase activity markedly increased during differentiation, this was counteracted by an even greater elevation in protein phosphotyrosine phosphatase activity. The findings support the concept that tyrosine phosphorylation is important in the regulation of growth and differentiation of leukemia cells.     

A protein tyrosine phosphatase inhibitor, sodium orthovanadate, causes parthenogenetic activation of pig oocytes via an increase in protein tyrosine kinase activity.

This study was conducted to determine whether a protein tyrosine kinase (PTK) activity is involved in the initiation of the events that occur at fertilization in pig oocytes. After maturation for 47 h, a 7-h treatment of oocytes with 1 mM sodium orthovanadate, which is an inhibitor of protein tyrosine phosphatase, caused more than 90% pronuclear formation, cortical granule exocytosis, and a decrease in mitogen-activated protein kinase activity. Immunoblotting with an antibody specific for phosphotyrosine showed at least three proteins whose phosphotyrosine contents were significantly increased upon treatment of oocytes with 1 mM sodium orthovanadate. Preincubation of pig oocytes with 50 microM tyrphostin 47, a specific PTK inhibitor, completely blocked the ability of sodium orthovanadate to trigger activation events. In addition, when oocytes were pretreated with the calcium-chelating agent BAPTA-AM, sodium orthovanadate-stimulated pronuclear formation was significantly (P < 0.01) reduced (94.0% vs. 43.1%). These results suggest that PTK may be involved in pig oocyte activation in a calcium-dependent manner and that the stimulation of tyrosine kinase is able to signal a series of intracellular changes that lead to the activation events associated with fertilization.     

Heparin-binding growth factor 1 stimulates tyrosine phosphorylation in NIH 3T3 cells.

Tyrosine phosphorylation of cellular proteins induced by heparin-binding growth factor 1 (HBGF-1) was studied by using the murine fibroblast cell line NIH 3T3 (clone 2.2). HBGF-1 specifically induced the rapid tyrosine phosphorylation of polypeptides of Mr 150,000, 130,000, and 90,000 that were detected with polyclonal and monoclonal antiphosphotyrosine (anti-P-Tyr) antibodies. The concentration of HBGF-1 required for half-maximal induction of tyrosine phosphorylation of the Mr-150,000 Mr-130,000, and Mr-90,000 proteins was approximately 0.2 to 0.5 ng/ml, which was consistent with the half-maximal concentration required for stimulation of DNA synthesis in NIH 3T3 cells. HBGF-1-induced tyrosine phosphorylation of the Mr-150,000 and Mr-130,000 proteins was detected within 30 s, whereas phosphorylation of the Mr-90,000 protein was not detected until 3 min after HBGF-1 stimulation. All three proteins were phosphorylated maximally after 15 to 30 min. Phosphoamino acid analysis of the Mr-150,000 and Mr-90,000 proteins confirmed the phosphorylation of these proteins on tyrosine residues. Phosphorylation of the Mr-150,000 and Mr-90,000 proteins occurred when cells were exposed to HBGF-1 at 37 degrees C but not at 4 degrees C. Exposure of cells to sodium orthovanadate, a potent P-Tyr phosphatase inhibitor, before stimulation with HBGF-1 resulted in enhanced detection of the Mr-150,000, Mr-130,000, and Mr-90,000 proteins by anti-P-Tyr antibodies. Anti-P-Tyr affinity-based chromatography was used to adsorb the HBGF-1 receptor affinity labeled with 125I-HBGF-1. The cross-linked HBGF-1 receptor-ligand complex was eluded with phenyl phosphate as two components: Mr 170,000 and 150,000. P-Tyr, but not phosphoserine or phosphothreonine, inhibited adsorption of the (125)I-HBGF-1-receptor complex to the anti-P-Tyr antibody matrix. Treatment of cells with sodium orthovanadate also enhanced recognition of the cross-linked (125)I-HBGF-1-receptor complex by the anti-P-Tyr matrix. These data suggest that (i) the (125)I-HBGF-1-receptor complex is phosphorylated on tyrosine residues and (ii) HBGF-1-induced signal transduction involves, in part, the tyrosine phosphorylation of at least three polypeptides.     

Protein tyrosine phosphorylation during meiotic divisions of starfish oocytes.

We have used an antibody specific for phosphotyrosine to investigate protein phosphorylation on tyrosine during hormone-induced maturation of starfish oocytes. Analysis of immunoprecipitates from cortices of in vivo labeled Marthasterias glacialis oocytes revealed the presence of labeled phosphotyrosine-containing proteins only after hormone addition. Six major phosphoproteins of 195, 155, 100, 85, 45, and 35 kDa were detected. Total activity in immunoprecipitates increased until first polar body emission and was greatly reduced upon completion of meiosis but some proteins exhibited different kinetics. The labeling of the 155-kDa protein reached a maximum at germinal vesicle breakdown, while the 35-kDa appeared later and disappeared after polar body emission. Similar results were obtained with Asterias rubens oocytes. In vitro phosphorylation of cortices showed that tyrosine kinase activity is a major protein kinase activity in this fraction, the main endogenous substrate being a 68-kDa protein. The proteins phosphorylated on tyrosine in vitro were almost similar in extracts from oocytes treated or not with the hormone.