Effect of EGF on the intracellular distribution of heat-shock proteins in A431 cells

The intracellular distribution of hsp70 and hdj1 was studied using immunofluorescent method. In nonstimulated cells hsp70 and hdj1 were observed in the cytoplasm of A431 cells. When 100 ng/ml EGF was added for 15 min, both hsp70 and hdj1 were accumulated in the nuclei. Later on (up to 1 h) hsp70 was exported from the nuclei to be observed mainly in the cytoplasm, whereas hdj1 remained in the nuclei. In cells exposed to tyrphostin AG1478, this inhibitor of tyrosine kinase activity of EGF receptor prevented EGF-dependent accumulation of hsp70 and hdj1 in the nuclei. U73122, an inhibitor of phospholipase C activity, induced tyrosine phosphorylation of EGF receptor without EGF stimulation. In cells treated with U73122, both hsp70 and hdj1 were detected in the nuclei of non-stimulated cells. It is concluded that the intracellular distribution of heat shock proteins in A431 cells depends on tyrosine kinase activity of EGF receptor. Here we report for the first time the influence of EGF on the intracellular redistribution of heat shock proteins.     

Prolonged induction of p21Cip1/WAF1/CDK2/PCNA complex by epidermal growth factor receptor activation mediates ligand-induced A431 cell growth inhibition

Proliferation of some cultured human tumor cell lines bearing high numbers of epidermal growth factor (EGF) receptors is paradoxically inhibited by EGF in nanomolar concentrations. In the present study, we have investigated the biochemical mechanism of growth inhibition in A431 human squamous carcinoma cells exposed to exogenous EGF. In parallel, we studied a selected subpopulation, A431-F, which is resistant to EGF-mediated growth inhibition. We observed a marked reduction in cyclin-dependent kinase-2 (CDK2) activity when A431 and A431-F cells were cultured with 20 nM EGF for 4 h. After further continuous exposure of A431 cells to EGF, the CDK2 activity remained at a low level and was accompanied by persistent G1 arrest. In contrast, the early reduced CDK2 activity and G1 accumulation in A431-F cells was only transient. We found that, at early time points (4-8 h), EGF induces p21Cip1/WAF1 mRNA and protein expression in both EGF-sensitive A431 cells and EGF-resistant A431-F cells. But only in A431 cells, was p21Cip1/WAF1 expression sustained at a significantly increased level for up to 5 d after addition of EGF. Induction of p21Cip1/WAF1 by EGF could be inhibited by a specific EGF receptor tyrosine kinase inhibitor, tyrphostin AG1478, suggesting that p21Cip1/WAF1 induction was a consequence of receptor tyrosine kinase activation by EGF. We also demonstrated that the increased p21Cip1/WAF1 was associated with both CDK2 and proliferating cell nuclear antigen (PCNA). Taken together, our results demonstrate that p21Cip1/WAF1 is an important mediator of EGF-induced G1 arrest and growth inhibition in A431 cells.     

Effect of EGF on nuclear-cytoplasmic distribution of proteasomes in A-431 cells

The intracellular distribution of proteasomes was studied using immunofluorescent method. In nonstimulated cells proteasomes were observed both in the cytoplasm and nuclei of A-431 cells. When 100 ng/ml EGF was added for 15 min, proteasomes were located mainly in the nuclei. Later (up to 1 h) proteasomes released from the nuclei and were observed mainly in the cytoplasm. Tyrphostin AG1478, an inhibitor of tyrosine kinase, and U73122, an inhibitor of phospholipase C, prevent, proteasome export from the nuclei after EGF treatment. In contrast, a proteasome inhibitor--lactacystin has no effect on this process. The EGF-dependent tyrosine phosphorylation of EGF receptor is blocked by tyrhostin AG1478 and U733122. Lactacystin did not alter the induction of EGF receptor tyrosine phosphorylation, triggered by EGF. It is concluded that intracellular distribution of proteasomes depends on tyrosine activity of EGF receptor.     

Regulation of vascular angiotensin II receptors by EGF

After vascular endothelial injury, angiotensin II (ANG II) playsa role in the resulting hypertrophic response, and expression ofepidermal growth factor (EGF) is enhanced. Therefore, we tested thepossibility that EGF regulates vascular ANG II action and receptorexpression. Incubation of cultured aortic vascular smooth muscle cells(VSMC) with EGF (or basic fibroblast growth factor but notplatelet-derived growth factor isoforms) resulted inconcentration-dependent (1-50 ng/ml EGF), time-dependent (>8 h),and reversible decreases in ANG II surface receptor density. Forexample, a 50% reduction was observed after exposure to 50 ng/ml EGFfor 24 h. Incubation of cultured VSMC with 50 ng/ml EGF for 24 hresulted in a 77% reduction in ANG II-stimulated inositol phosphateformation. EGF not only prevented but also reversed ANG II receptorupregulation by 100 nM corticosterone. The specifictyrosine kinase inhibitor tyrphostin A48 (50 µM) reducedEGF-stimulated thymidine incorporation and EGF-stimulatedphosphorylation of mitogen-activated protein kinase but did not preventEGF from reducing ANG II receptor density. Neither pertussis toxin (100 ng/ml) nor downregulation of protein kinase C by phorbol myristateacetate (100 nM for 24 h) prevented EGF from reducing ANG II receptordensity. In summary, EGF is a potent negative regulator of vascular ANGII surface receptor density and ANG II action by mechanisms that do notappear to include tyrosine phorphorylation, pertussis toxin-sensitive G proteins, or phorbol ester-sensitive protein kinase C. The possibility that EGF shifts the cell culture phenotype to one that exhibits reducedsurface ANG II density cannot be eliminated by the present studies.

     

Epidermal growth factor (EGF) promotes phosphorylation at threonine-654 of the EGF receptor: possible role of protein kinase C in homologous regulation of the EGF receptor

Treatment of cells with tumor-promoting phorbol diesters, which causes activation of protein kinase C, leads to phosphorylation of the epidermal growth factor (EGF) receptor at threonine-654. Addition of phorbol diesters to intact cells causes inhibition of the EGF-induced tyrosine-protein kinase activity of the EGF receptor and it has been suggested that this effect of phorbol diesters is mediated by the phosphorylation of the receptor by protein kinase C. We measured the activity of protein kinase C in A431 cells by determining the incorporation of [32P]phosphate into peptides containing threonine-654 obtained by trypsin digestion of EGF receptors. After 3 h of exposure to serum-free medium, A431 cells had no detectable protein kinase C activity. Addition of EGF to these cells resulted in [32P] incorporation into threonine-654 as well as into tyrosine residues. This indicates that EGF promotes the activation of protein kinase C in A431 cells. The phosphorylation of threonine-654 induced by EGF was maximal after only 5 min of EGF addition and the [32P] incorporation into threonine-654 reached 50% of the [32P] in a tyrosine-containing peptide. This indicates that a significant percentage of the total EGF receptors are phosphorylated by protein kinase C. A variety of external stimuli activate Na+/H+ exchange, including EGF, phorbol diesters, and hypertonicity. To ascertain whether activation of protein kinase C is an intracellular common effector of all of these systems, we measured the activity of protein kinase C after exposure of A431 cells to hyperosmotic conditions and observed no effect on phosphorylation of threonine-654, therefore, activation of Na+/H+ exchange by hypertonic medium is independent of protein kinase C activity. Since stimulation of protein kinase C by phorbol diesters results in a decrease in EGF receptor activity, the stimulation of protein kinase C activity by addition of EGF to A431 cells contributes to a feedback mechanism which results in the attenuation of EGF receptor function.     

Cytoskeletal and Morphological Changes Associated with the Specific Suppression of the Epidermal Growth Factor Receptor Tyrosine Kinase Activity in A431 Human Epidermoid Carcinoma

The epidermal growth factor (EGF) receptor is well known as a mediator of mitogenic signaling and its tyrosine kinase activity has been suggested as a viable target in cancer chemotherapy. To explore the consequences of abolishing the kinase activity of this receptor, we have utilized a potent and specific inhibitor of the enzyme, PD 153035, to sustain a long-term suppression of its activity. This compound inhibits EGF receptor autophosphorylation in cells with an IC₅₀in the low nanomolar range and does not block PDGF or FGF receptor kinase until concentrations are greater than 10 μM.[1] Human epidermoid carcinoma A431 cells were grown in the presence of PD 153035 and were passed weekly until cells grew in the presence of 1 μMinhibitor. These cells, referred to as A431R, showed a remarkable change in morphology, becoming flattened and spread out. A comparison of the sensitivity of EGF receptor autophosphorylation to PD 153035 between A431 and A431R showed a similar dose response, indicating that the cells had not developed any defect in the kinase which might make it resistant to the inhibitor. Likewise, EGF receptor autophosphorylation in response to exogenously added EGF, as well as receptor internalization, was similar between the two cell lines. Furthermore, analysis of A431R cells by flow cytometry showed no significant change in DNA content or percentage of cells in any one phase of the cell cycle compared to the parent line.¹²⁵I-labeled EGF/receptor binding studies showed that receptor number in the A431R cells was equivalent to that of the parent line; however, the Scatchard plot was linear, in contrast to the typical biphasic plot obtained with the parent cells, implying a loss of high-affinity receptors. Cytoskeletal preparations from both cell lines indicated that the A431R had fourfold less EGF receptor associated with the cytoskeleton than A431. This was accompanied by a remarkable increase in polymerized actin stress fibers throughout the A431R cells, which most likely accounts for their flattened morphology. The A431R cells also exhibited a twofold increase in the expression of focal adhesion kinase, which is consistent with a greater contact area for their cell surface and increase in focal adhesions. Finally, although the A431R cells have a doubling time of 24 h, similar to that of the parent line, these cells stop growing as the monolayer approaches confluence, reminiscent of the contact inhibition seen in nontransformed cells. These data indicate that long-term suppression of the EGF receptor tyrosine kinase activity in A431 human epidermoid carcinoma results in certain cellular properties which are more consistent with a differentiated and nontransformed phenotype.     

Effect of EGF on the activity of nuclear and cytoplasmic 26S proteasomes in A431 cells

It has been first shown that EGF regulates a proteolytic activity of proteasomes. Following a 15 min action with 100 ng/ml EGF, three types of peptidase activity of both cytoplasmic and nuclear proteasomes were induced in A431 cells, although, this effect on different populations of proteasomes was selective. EGF preferentially stimulates chymotrypsin-like activity of cytoplasmic proteasomes, and induces a similar increase of chymotrypsin-like, trypsin-like and peptydylglutamyl peptide hydrolase activities of nuclear particles. Tyrphostin, an inhibitor of tyrosine kinase activity of EGF receptor, prevents the EGF effect on both proteolytic and RNase activity of nuclear and cytoplasmic proteasomes. It is concluded that EGF may rapidly and selectively stimulate enzymatic activity of EGF receptor.     

EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signaling

Binding of EGF to cells expressing human EGF receptor stimulated rapid tyrosine phosphorylation of phospholipase C-II (PLC-II), as revealed by immunoblotting analysis with phosphotyrosine-specific antibodies. Tyrosine phosphorylation of PLC-II was stimulated by low physiological concentrations of EGF (1 nM), was quantitative, and was already maximal after a 30 sec incubation with 50 nM EGF at 37 degrees C. Interestingly, antibodies specific for PLC-II were able to coimmunoprecipitate the EGF receptor and antibodies against EGF receptor also coimmunoprecipitated PLC-II. According to this analysis, approximately 1% of EGF receptor molecules were associated with PLC-II molecules. The protein tyrosine kinase inhibitor tyrphostin RG50864, which blocks EGF-dependent cell proliferation, blocked EGF-induced tyrosine phosphorylation of PLC-II, its association with EGF receptor, and EGF-induced Ca2+ release. Hence, EGF-induced tyrosine phosphorylation of PLC-II may be a regulatory event linking the tyrosine kinase activity of EGF receptor to the PIP2 hydrolysis signaling pathway.     

Enhancement of epidermal growth factor (EGF) and insulin-stimulated tyrosine phosphorylation of endogenous substrates by sodium selenate.

Sodium selenate stimulated tyrosine phosphorylation of the epidermal growth factor (EGF) receptor in A431 cells and enhanced the tyrosine phosphorylation of endogenous proteins in response to EGF in A431 cells and insulin in NIH 3T3 HIR3.5 cells. These effects occurred without changes in ligand binding, were not abolished by mercaptoethanol in the case of the EGF receptor, and appeared distinct from the effects of vanadate. These results support a role for selenium or selenoproteins in regulating EGF and insulin receptor tyrosine kinase activity and suggest a mechanism whereby selenium-containing compounds contribute to cell growth.     

Integrins induce activation of EGF receptor: role in MAP kinase induction and adhesion-dependent cell survival.

Adhesion of human primary skin fibroblasts and ECV304 endothelial cells to immobilized matrix proteins, beta1 or alphav integrin antibodies stimulates tyrosine phosphorylation of the epidermal growth factor (EGF) receptor. This tyrosine phosphorylation is transiently induced, reaching maximal levels 30 min after adhesion, and it occurs in the absence of receptor ligands. Similar results were observed with EGF receptor-transfected NIH-3T3 cells. Use of a kinase-negative EGF receptor mutant demonstrates that the integrin-stimulated tyrosine phosphorylation is due to activation of the receptor's intrinsic kinase activity. Integrin-mediated EGF receptor activation leads to Erk-1/MAP kinase induction, as shown by treatment with the specific inhibitor tyrphostin AG1478 and by expression of a dominant-negative EGF receptor mutant. EGF receptor and Erk-1/MAP kinase activation by integrins does not lead per se to cell proliferation, but is important for entry into S phase in response to EGF or serum. EGF receptor activation is also required for extracellular matrix-mediated cell survival. Adhesion-dependent MAP kinase activation and survival are regulated through EGF receptor activation in cells expressing this molecule above a threshold level (5x10(3) receptors per cell). These results demonstrate that integrin-dependent EGF receptor activation is a novel signaling mechanism involved in cell survival and proliferation in response to extracellular matrix.     

EGF receptor phosphorylation is affected by ionizing radiation

Eukaryotic cells respond to ionizing radiation with cell cycle arrest, activation of DNA repair mechanisms, and lethality. However, little is known about the molecular mechanisms that constitute these responses. Here we report that ionizing radiation enhances epidermal growth factor (EGF) receptor tyrosine phosphorylation in intact cells as well as in isolated membranes of A431 cells. Phosphoamino acid analysis revealed that ionizing radiation preferentially enhances tyrosine phosphorylation, while EGF enhances the phosphorylation of all three phosphoamino acids (serine, threonine and tyrosine) of the EGF receptor. In addition, radiation reduces the turnover rate of the EGF receptor, while EGF increases the rate of the receptor turnover and down-regulation. Moreover, the confined radiation-induced phosphorylation of tyrosine residues is inhibited by genistein, indicating that this phosphorylation of EGF receptor is due to protein tyrosine kinase activation. These studies provide novel insights into the capacity of radiation to modulate EGF receptor phosphorylation and function. The radiation-induced elevation in the EGF receptor tyrosine phosphorylation and the receptor's slower rate of turnover are discussed in terms of their possible role in cell growth and apoptosis modulation.     

Autophosphorylation of EGF receptors in the A431 cell line with an increased intracellular concentration of Ca(2+) ions

The increase in the intracellular concentration of Ca2+ in A431 cells induced by the calcium ionophore A23187 leads to phosphorylation of epidermal growth factor (EGF) receptors at serine and/or threonine residues. This process is accompanied by the decrease in the level of EGF receptor autophosphorylation at tyrosine residues. Preincubation of cells in a A23187-containing medium in the presence of phorbol-12-myristoyl-13-acetate leads to a further decrease of the phosphotyrosine content in EGF receptors. At increased intracellular concentrations of Ca2+ preincubation of A431 cells with the protein kinase C inhibitor H-7 has no effect on the degree of EGF receptor autophosphorylation. Down-regulation of cellular protein kinase C does not change the A23187-induced effect either. The data obtained suggest that the decreased autophosphorylation of EGF receptors induced by Ca2+ is not due to the activation of cellular protein kinase C.     

STAT1 and STAT3 activation by oxidative stress in A431 cells involves Src-dependent EGF receptor transactivation

Different cellular signal transduction cascades are affected by environmental stressors (UV-radiation, gamma-irradiation, hyperosmotic conditions, oxidants). In this study, we examined oxidative stress-evoked signal transduction pathways leading to activation of STATs in A431 carcinoma cells. Oxidative stress, initiated by addition of H2O2 (1-2 mM) to A431 cells, activates STAT3 and, to a lesser extent, STAT1 in dose- and time-dependent manner. Maximum phosphorylation levels were observed after a 2 minutes stimulation at 1-2 mM H2O2. Phosphorylation was blocked by AG1478, a pharmacological inhibitor of the epidermal growth factor receptor tyrosine kinase, implicating intrinsic EGF receptor tyrosine kinase in this process. Consistent with this observation, H2O2-stimulated EGFR tyrosine phosphorylation was abolished by specific Src kinase family inhibitor CGP77675, implicating Src in H2O2-induced EGFR activation. An essential role for Src and JAK2 in STATs activation was suggested by three findings. 1. Src kinase family inhibitor CGP77675 blocked STAT3 and STAT1 activation by H2O2 in a concentration-dependent manner. 2. In Src-/-fibroblasts, activation of both STAT3 and STAT1 by H2O2 was significantly attenuated. 3. Inhibiting JAK2 activity with the specific inhibitor AG490 reduced the level of H2O2-induced STAT3 phosphorylation, but not STAT1 in A431 cells. These data show essential roles for Src and JAK2 inactivation of STAT3. In contrast, H2O2-mediated activation of STAT1 requires only Src kinase activity. Herein, we postulate also that H2O2-induced STAT activation in carcinoma cells involves Src-dependent EGFR transactivation.     

Loss of three major auto phosphorylation sites in the EGF receptor does not block the mitogenic action of EGF

The EGF receptor cDNA has been transfected into receptor-negative Chinese hamster ovary (CHO) cells. A mutant cell line (CHO 11) was isolated that expresses a receptor of lower molecular weight than the EGF receptor from A431 cells (150,000 daltons compared to 170,000 daltons) and which appeared as a doublet on SDS-PAGE. By digestion of the receptor with endoglycosidase F it was shown that an altered pattern of glycosylation could not account for the smaller size of the protein, although it could explain the appearance of the CHO 11 receptor as a doublet protein. A deletion was located to the transfected cDNA and shown to involve the removal of coding sequences for the most C-terminal 20,000 daltons of the EGF receptor, which contains the three major autophosphorylation sites. Despite the loss of these sites the EGF receptor from CHO 11 cells binds EGF, demonstrates protein tyrosine kinase activity in response to EGF, and transduces a mitogenic signal. The CHO 11 receptor protein is still autophosphorylated on alternative tyrosine residues. We conclude that phosphorylation of the three tyrosines (P1, P2, and P3) in the C-terminal domain of the receptor is not required for signal transduction by the EGF receptor in these cells.     

Regulation of polyamine synthesis and transport by retinoic acid and epidermal growth factor in cultured adult rat type II pneumocytes

During injury of lung epithelial cells, the type II pneumocyte proliferates and differentiates into a type I pneumocyte to restore the epithelium. Polyamines, which constitute a family of small organic polycations, are required for this process of cell repair. Because retinoic acid (RA) and epidermal growth factor (EGF) also are involved, the purpose of this research was to determine their effect on polyamine transport and synthesis in cultured type II pneumocytes. Rat type II pneumocytes were isolated, cultured overnight, and treated with RA and/or EGF for 24 hours. Polyamine transport was determined by [(3)H]spermidine uptake, and polyamine synthesis was assessed by the activity of the initial rate-limiting enzyme ornithine decarboxylase. EGF (100 ng/mL) significantly increased spermidine transport, but RA did not. At low concentrations of spermidine (2 microM), the combined effect of RA and EGF on spermidine transport was additive. Both EGF (25 ng/mL) and RA (1 microM) increased polyamine synthesis, and cotreatment resulted in an additive effect (a fourfold increase over the control). We also found that ornithine decarboxylase activity is greatly diminished in the presence of tyrphostin B56, which is a specific inhibitor for the tyrosine kinase of the EGF receptor, suggesting that polyamine synthesis within the type II pneumocyte may depend on activation of tyrosine kinase of the EGF receptor. These results indicate that RA and EGF increase the availability of polyamines, which may be important in the lung cell repair process.     

Interferon-gamma activates EGF receptor and increases TGF-alpha in T84 cells: implications for chloride secretion

IFN-gamma inhibits intestinal Cl(-) secretion, in part via downregulation of CFTR and Na(+)-K(+)-ATPase activity and expression, but the proximal signaling events were unknown. We have shown that transforming growth factor-alpha (TGF-alpha) inhibits calcium-activated Cl(-) secretion, and effects of IFN-gamma in other systems are mediated via EGF family members. We tested whether IFN-gamma inhibits Cl(-) secretion via EGF receptor (EGFr) activation. IFN-gamma increased tyrosine phosphorylation in T84 cells at 24 h, including the EGFr. IFN-gamma also increased cell-associated pro-TGF-alpha, as well as free TGF-alpha in the bathing media. However, whereas IFN-gamma significantly inhibited carbachol-induced Cl(-) secretion, neither neutralizing antibodies to TGF-alpha nor an EGFr inhibitor (1 microM tyrphostin AG 1478) were able to reverse this inhibitory effect. AG 1478 also failed to reverse IFN-gamma-induced tyrosine phosphorylation of the EGFr, but receptor phosphorylation was attenuated by both the neutralizing antibody to TGF-alpha and PP2, a Src kinase inhibitor. Moreover, PP2 reversed the inhibitory effect of IFN-gamma on Cl(-) secretion. In total, our findings suggest an increase in functional TGF-alpha and activation of the EGFr in response to IFN-gamma. The release of TGF-alpha and intracellular Src activation likely combine to mediate EGFr phosphorylation, but only Src appears to contribute to the inhibition of transport. Nevertheless, because TGF-alpha plays a role in restitution and repair of the intestinal epithelium after injury, we speculate that these findings reflect a feedback loop whereby IFN-gamma modulates the extent of cytokine-induced intestinal damage.     

pp60cSrc Is a Caspase-3 Substrate and Is Essential for the Transformed Phenotype of A431 Cells

The protein tyrosine kinase c-Src is a major signal transduction element in many growth factor receptor signals for proliferation and transformation. We showed recently that c-Src is a mediator of antiapoptotic signals through regulation of the antiapoptotic gene Bcl-X_L. A431 cells overexpress the EGF receptor (EGFR) and possess high Src activity. In A431 cells, Src is activated by the EGFR, and inhibition of the EGF receptor results in c-Src inhibition. In this study we show that (i) inhibition of the EGFR kinase or Src kinase by specific inhibitors results in growth inhibition and inhibition of colony formation in soft agar. The relative efficacies of the EGFR kinase inhibitor and of the Src kinase inhibitor are similar suggesting the major role src plays in the oncogenic signaling of EGFR in A431 cells. (ii) The Src kinase inhibitor PP1 sensitizes A431 cells to CDDP-induced apoptosis. (iii) CDDP induces caspase-3-dependent cleavage of the c-Src C-terminal portion and a concomitant reduction in Bcl-X_L levels. We conclude that c-Src is an important antiapoptotic signaling molecule downstream of the EGF receptor that contributes to the transformed phenotype of A431 cells.     

Phospholipase C negatively regulates tyrosine phosphorylation of EGF receptor in A-431 cells

It is known that EGF induces tyrosine phosphorylation and internalization of the EGF receptor in A-431 cells. U73122, an inhibitor of phospholipase C, induces tyrosine phosphorylation of the EGF receptor and its association with phospholipase C still in nonstimulated cells. In U73122 treated cells EGF exerted no effect on these processes. Receptor-mediated endocytosis was not observed in A-431 cells treated with U73122. The reorganization of actin cytoskeleton was detected in U73122 cells.     

Effects of protein kinase C activation after epidermal growth factor binding on epidermal growth factor receptor phosphorylation

The possible role of epidermal growth factor (EGF) receptor phosphorylation at threonine 654 in modulating the protein-tyrosine kinase activity of EGF-treated A431 cells has been studied. It has been suggested that EGF could indirectly activate a protein-serine/threonine kinase, protein kinase C, that can phosphorylate the EGF receptor at threonine 654. Protein kinase C is known to be activated, and threonine 654 is phosphorylated, when A431 cells are exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). The protein-tyrosine kinase activity of EGF receptors is normally evidenced in EGF-treated cells by phosphorylation of the receptor at tyrosine. This is inhibited when TPA-treated cells are exposed to EGF. We now show that receptor phosphorylation at threonine 654 can also be detected in EGF-treated A431 cells, presumably due to indirect stimulation of protein kinase C or a similar kinase. Some receptor molecules are phosphorylated both at threonine 654 and at tyrosine. Since prior phosphorylation at threonine 654 inhibits autophosphorylation, we propose that protein kinase C can phosphorylate the threonine 654 of autophosphorylated receptors. This provides evidence for models in which protein kinase C activation, consequent upon EGF binding, could reduce the protein-tyrosine kinase activity of the EGF receptor. Indeed, we find that 12-O-tetradecanoylphorbol-13-acetate, added 10 min after EGF, further increases threonine 654 phosphorylation and induces the loss of tyrosine phosphate from A431 cell EGF receptors.