Stimulation of a MR 80,000 protein phosphorylation by EGF in EGF receptor-hyperproducing human tumor cells

NA and Ca9-22 cells derived from squamous cell carcinomas of the tongue possess a large number of epidermal growth factor (EGF) receptors (2.0 X 10(6) and 1.3 X 10(6) receptors/cell, respectively). In these cell lines, EGF stimulated receptor autophosphorylation and phosphatidylinositol (PI) turnover. Furthermore, EGF enhanced the phosphorylation of an acidic protein of Mr 80,000. Phosphorylation of this protein was also stimulated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a phorbol ester tumor promoter, and was mainly at serine residues. Phosphopeptide mapping using protease V8 or trypsin indicated that Mr 80,000 proteins isolated from the EGF- and TPA-treated cells were identical. The Mr 80,000 protein was present mainly in the cytosol, but it became closely associated with the membrane as a phosphorylated form upon EGF or TPA stimulation. These results suggest that the EGF-stimulated phosphorylation of the Mr 80,000 acidic phosphoprotein in EGF receptor-hyperproducing tumor cells is mediated through the activation of PI turnover and protein kinase C.     

Epidermal growth factor (EGF) and hormones stimulate phosphoinositide hydrolysis and increase EGF receptor protein synthesis and mRNA levels in rat liver epithelial cells. Evidence for protein kinase C-dependent and -independent pathways

Epidermal growth factor (EGF) stimulated the rapid accumulation of inositol trisphosphate in WB cells, a continuous line of rat hepatic epithelial cells. Since we previously had shown that EGF stimulates EGF receptor synthesis in these cells, we tested whether hormones that stimulate PtdIns(4,5)P2 hydrolysis would increase EGF receptor protein synthesis and mRNA levels. Epinephrine, angiotensin II, and [Arg8]vasopressin activate phospholipase C in WB cells as evidenced by the accumulation of the inositol phosphates, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. A 3-4-h treatment with each hormone also increased the rate of EGF receptor protein synthesis by 3-6-fold as assessed by immunoprecipitation of EGF receptor from [35S]methionine-labeled cells. Northern blot analyses of WB cell EGF receptor mRNA levels revealed that agents linked to the phosphoinositide signaling system increased receptor mRNA content within 1-2 h. A maximal increase of 3-7-fold was observed after a 3-h exposure to EGF and hormones. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), which activates protein kinase C also stimulated EGF receptor synthesis. Pretreatment of WB cells for 18 h with high concentrations of TPA "down-regulated" protein kinase C and blocked TPA-directed EGF receptor mRNA synthesis. In contrast, the effect of EGF on EGF receptor mRNA levels was not significantly decreased by TPA pretreatment. Epinephrine-induced increases in EGF receptor mRNA were reduced from 4- to 2-fold. Similarly, 18 h TPA pretreatment abolished the effect of TPA on EGF receptor protein synthesis but did not affect EGF-dependent EGF receptor protein synthesis. The 18-h TPA pretreatment diminished by 30-50% the induction of receptor protein synthesis by epinephrine or angiotensin II. We conclude that in WB cells EGF receptor synthesis can be regulated by EGF and other hormones that stimulate PtdIns(4,5)P2 hydrolysis. In these cells, EGF receptor synthesis appears to be regulated by several mechanism: one pathway is dependent upon EGF receptor activation and can operate independently of protein kinase C activation; another pathway is correlated with PtdIns(4,5)P2 hydrolysis and is dependent, at least in part, upon protein kinase C activation.     

Regulation of VL30 gene expression by activators of protein kinase C

The mouse genome contains a retrovirus-like sequence, designated VL30, which is expressed at high levels in transformed cells and which can be induced by exogenously supplied epidermal growth factor (EGF). Binding of EGF to the EGF receptor produces changes in intracellular calcium levels and phospholipase activity which indirectly lead to activation of protein kinase C. We treated AKR-2B cells, Swiss 3T3 cells, and the 3T3 variants NR6 (EGF receptorless) and TNR9 (phorbol ester nonresponsive) with various phorbol ester tumor promoters and with the synthetic diacylglycerol sn-1,2-dioctanoylglycerol. Tumor-promoting phorbol esters (e.g. 12-O-tetradecanoyl phorbol acetate (TPA] increased the level of VL30 expression. Stimulation with either TPA or EGF produced a similar time course of VL30 expression. TPA induced VL30 expression in the EGF-receptorless NR6 cell line, indicating that neither EGF ligand-receptor binding nor phosphorylation of the EGF receptor was required for induction of VL30 expression. Protein synthesis was not required for the TPA-mediated increase in VL30 expression, as pretreatment with cycloheximide did not block or reduce the TPA effect. VL30 expression was also stimulated by treatment with sn-1,2-dioctanoylglycerol, an analog of a probable endogenous activator of protein kinase C. These results suggest that activation of protein kinase C plays a direct role in regulating VL30 expression.     

Epidermal growth factor and phorbol ester actions on the TSH induced down regulation of the isoenzyme I (PKA I) of cyclic AMP-dependent protein kinases in dog thyroid cell primary cultures

In dog thyroid cell primary cultures the prolonged presence (up to 4-6 days) of TSH induced down regulation of the isoenzyme I (PKA I) of cAMP-dependent protein kinases. In the simultaneous presence of TSH and EGF this down regulation of PKA I was maintained, although it was slightly smaller than in assays without EGF. In contrast, the simultaneous presence of TPA, totally inhibited the TSH induced down regulation of PKA I. These results partly explain the previously observed additivity of TSH and EGF, and the non-additivity of TSH and TPA actions on cell proliferation in these cells.     

Inhibition of stimulus-dependent epidermal growth factor receptor and transforming growth factor-alpha mRNA accumulation by the protein kinase C inhibitor staurosporine

The ability of staurosporine, a potent inhibitor of protein kinase C, to block certain cellular events initiated by 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) was examined. Treatment of MDA468 breast cancer cells with TPA decreases EGF binding to the cell surface and this effect is blocked by pretreatment with staurosporine with an IC50 of 30 nM. Either 10(-9) M EGF or 100 ng/ml TPA stimulated the accumulation of both EGF receptor and TGF-alpha mRNA and staurosporine (50 nM) completely abolished these mRNA accumulations. Staurosporine did not block EGF-stimulated tyrosine phosphorylation of its receptor as measured by immunoblotting with anti-phosphotyrosine antibodies. The ability of staurosporine to block the mRNA responses of either EGF or TPA suggests that these two agents have common signaling pathways and it implies a role for protein kinase C in the control of EGF receptor and TGF-alpha expression.     

Phorbol ester or epidermal growth factor (EGF) stimulates the concurrent accumulation of mRNA for the EGF receptor and its ligand transforming growth factor-alpha in a breast cancer cell line

We have previously reported that both 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) can stimulate the synthesis rate of EGF receptors. We now show that the MDA468 breast cancer cells express the mRNA for the EGF-like molecule, transforming growth factor-alpha (TGF-alpha), and demonstrate that TPA or EGF cause an accumulation of both EGF receptor and TGF-alpha mRNA. The levels of EGF receptor mRNA paralleled our earlier protein data, with peak accumulations of 2-3-fold with 10(-9) M EGF and 3-5-fold with 100 ng/ml TPA seen between 6 and 8 h. A 7-fold accumulation of TGF-alpha mRNA was seen following 4 h of treatment with TPA, and a 2-fold accumulation was seen after 8 h with EGF. These changes in EGF receptor and TGF-alpha mRNAs were observed in the absence of any change in the mRNA level of the alpha-subunit of hexosaminidase A (a lysosomal enzyme), demonstrating some degree of specificity. Detectable quantities of immunoreactive TGF-alpha accumulated in the cell culture medium of MDA468 cell treated with the blocking anti-EGF receptor monoclonal antibody B1D8 while no immunoreactive TGF-alpha was detected in the medium of cells with unblocked receptors. The concentration of B1D8 used was sufficient to block the binding of exogenously added 125I-EGF to undetectable levels but had only minor effects on cell growth and no effect on the expression of the TGF-alpha and EGF receptor mRNA.     

Regulation of plasminogen activators in human thyroid follicular cells and their relationship to differentiated function

Human thyroid cells in culture take up and organify (125)I when cultured in TSH (acting through cAMP) and insulin. They also secrete urokinase (uPA) and tissue-type (tPA) plasminogen activators (5-100 IU/10(6)cells/day). TSH and insulin both decreased secreted PA activity (PAA), uPA and tPA protein and their mRNAs. Autocrine fibroblast growth factor increased secreted PAA and inhibited thyroid cell (125)I uptake. Epidermal growth factor (EGF) and the protein kinase C (PKC) activator, TPA significantly increased PAA and inhibited thyroid differentiated function, (TPA > EGF). For TPA, effects were rapid, increased PAA secretion and decreased (125)I uptake being seen at 4 h whereas for EGF, a 24 h incubation was required. qRT-PCR showed significantly increased mRNA expression of uPA with lesser effects on tPA. Aprotinin, which inhibits PAA, increased (125)I uptake but did not abrogate the effects of TPA and EGF. The MEKK inhibitor, PD98059 partially reversed the effects of EGF and TPA on PAA, and largely reversed the effects of EGF but not TPA on differentiated function. PKC inhibitors bisindoylmaleimide 1, and the specific PKCbeta inhibitor, LY379196 completely reversed the effects of TPA on (125)I uptake and PAA whereas EGF effects were unaffected. TPA inhibited follicle formation and this effect was blocked by LY379196 but not PD98059. We conclude that in thyroid cells, MAPK activation inversely correlates with (125)I uptake and directly correlates with PA expression, in contrast to the effects of cAMP. TPA effects on iodide metabolism, dissolution of follicles and uPA synthesis are mediated predominantly through PKCbeta whereas EGF exerts its effects through MAPK but not PKCbeta.     

Okadaic acid-induced hyperphosphorylation of the epidermal growth factor receptor. Comparison with receptor phosphorylation and functions affected by another tumor promoter, 12-O-tetradecanoylphorbol-13-acetate.

Okadaic acid, a potent tumor promoter and inhibitor of phosphoserine/threonine protein phosphatases 1 and 2A, produces a large increase in epidermal growth factor (EGF) receptor phosphorylation in several cell types. The increases are limited to phosphoserine and phosphothreonine residues. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a distinct tumor promoter and protein kinase C activator, also induces serine/threonine phosphorylation of the EGF receptor and is known to modulate receptor functions. Comparison of okadaic acid and TPA influences on the EGF receptor show significant differences. Okadaic acid did not promote phosphorylation of Thr-654, a major site of TPA-induced phosphorylation. However, other sites of phosphorylation were similar for the two tumor promoters. In vitro experiments with purified protein phosphatase 2A demonstrate the insensitivity of Thr-654 phosphorylation, which regulates EGF receptor function, to dephosphorylation by this okadaic acid-sensitive protein phosphatase. In contrast to TPA, okadaic acid did not attenuate the tyrosine kinase activity or ligand binding capacity of the EGF receptor. However, okadaic acid did produce a decrease in EGF-stimulated inositol phosphate formation in a manner distinct from that of TPA.     

Epidermal growth factor (EGF) and tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulate PG synthesis and thymidine incorporation in cultured porcine thyroid cells

This is the first report to show that epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulate the production of PGE2 and 6-keto PGF1 alpha, an end metabolite of PGI2, in the thyroid gland. In cultured porcine thyroid cells, EGF and TPA stimulate PGE2 and 6-keto PGF1 alpha production; the maximum PG levels were obtained after 3-4 h incubation with EGF or TPA; the addition of as little as 10(-11) M EGF or 5 X 10(-11) M TPA resulted in increases in PGE2 and 6-keto PGF1 alpha, and the maximum levels were obtained with 10(-8)-10(-7) M EGF or TPA. This report also shows that EGF and TPA stimulate [3H] thymidine incorporation.     

Protein kinase C and limited substrates for tyrosine kinase are involved in epidermal growth factor (EGF)-dependent growth of a human squamous cell carcinoma cell variant.

Human squamous cell carcinoma cells (NA cells) possess a large number of epidermal growth factor (EGF) receptors and their growth is inhibited by EGF. Recently, we isolated a series of variants which escaped EGF-mediated growth inhibition. The variant ER11 cells expressed a decreased level of EGF receptors and grew in an EGF-dependent fashion. Treatment of ER11 cells with EGF resulted in the activation of protein kinase C, which was followed by the enhancement of 80-kDa protein phosphorylation as observed in NA cells. Thus, EGF can activate not only tyrosine kinase but also protein kinase C in both NA and ER11 cells. The EGF-dependent growth stimulation in ER11 cells was inhibited by 12-O-tetradecanoylphorbol 13-acetate (TPA). Exposure of NA and ER11 cells to TPA for 30 h resulted in the down-regulation of protein kinase C. In these protein kinase C-deficient cells, EGF was able to activate autophosphorylation of the EGF receptor. The EGF-activated EGF receptor kinase phosphorylated numerous cellular proteins even in the protein kinase C-deficient cells. However, there were less tyrosine-phosphorylated proteins in ER11 cells than in NA cells. These results suggested that protein kinase C is necessary for the EGF-dependent growth stimulation of ER11 cells and that several tyrosine-phosphorylated proteins commonly observed in both NA and ER11 cells seem essential for cell proliferation.     

Possible contribution of protein kinase C activation to priming for DNA synthesis induced by epidermal growth factor with insulin and its inhibition by plasma membrane in primary cultured rat hepatocytes

In primary cultured rat hepatocytes, DNA synthesis was markedly induced 48 h after plating by epidermal growth factor (EGF) and insulin added at 24 h, but not by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). When EGF and insulin were added at 6 h, DNA synthesis at 30 h was 7% of DNA synthesis seen at 48 h, but became 27% by pretreatment with TPA. The similar pretreatment effect was also seen with vasopressin. Such induction at 30 h was inhibited by rat liver plasma membrane added at 2 h even in the presence of TPA or vasopressin, and also by 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine more extensively than N-(2-guanidinoethyl)-5-isoquinolinesulfonamide. These results suggest that DNA synthesis induction by EGF and insulin may require a priming period related to protein kinase C activation in primary cultured rat hepatocytes, which is inhibited by plasma membrane.     

Regulation of the phosphorylation state and function of the epidermal growth factor receptor by vitamin K-3

Vitamin K-3 or 12-O-tetradecanoylphorbol 13-acetate (TPA) reduced the binding of epidermal growth factor (EGF) to its receptor by more than 90% in human foreskin fibroblasts. After the equilibration of fibroblasts with [32P]orthophosphate, vitamin K-3 or TPA markedly increased the amount of 32P found in the receptor; the increase was principally due to serine and threonine phosphorylation. By the use of two-dimensional tryptic phosphopeptide mapping, using a synthetic phosphopeptide as a standard, threonine-654 was identified as one of the residues whose phosphorylation state was elevated by vitamin K-3 or TPA. Because of the large amounts of EGF receptor present on A431 human carcinoma cells, these cells were used to study further the relationship between the phosphorylation state of threonine-654, the tyrosine phosphorylation state of the receptor, and the receptor's protein tyrosine kinase activity toward exogenous substrates. Vitamin K-3 and TPA both increased the amount of phosphate on threonine-654 in A431 cells. However, whereas receptor from TPA-treated cells lacked phosphotyrosine, vitamin K-3-treated cells contained receptor with markedly elevated levels of phosphotyrosine. The addition of vitamin K-3, TPA or EGF to intact A431 cells followed by homogenization of the cells and the assay of EGF receptor protein tyrosine kinase activity by the use of a synthetic peptide substrate resulted in marked decreases in apparent receptor kinase activity. Therefore, assuming that the activity measured in the peptide assay reflects the protein tyrosine kinase activity of the receptor in the intact cell, the activity of the EGF receptor kinase cannot be deduced from the amount of phosphotyrosine associated with the receptor.     

A negative feedback loop attenuates EGF-induced morphological changes

Activation of the EGF receptor tyrosine kinase by ligand indirectly activates a series of other cellular enzymes, including protein kinase C. To test the hypothesis that phosphorylation of the EGF receptor by protein kinase C provides an intracellular negative feedback loop to attenuate EGF receptor signaling, we used scanning EM to follow the characteristic EGF-induced retraction of lamellipodia and concomitant cell shape changes. Wild type and mutant EGF receptors were expressed in receptor-deficient NR6 cells. The mutant receptors were prepared by truncation at C' terminal residue 973 (c'973) to provide resistance to ligand-induced down regulation that strongly attenuates receptor signaling and by replacement of threonine 654 (T654) with alanine (A654) to remove the site of phosphorylation by protein kinase C. Cells expressing WT and c'973 EGF receptors demonstrated characteristic lamellipodial retraction after exposure to EGF, with the non-down regulating c'973 EGF receptors responding more rapidly. Exposure of cells to TPA blocked this response. Replacement of T654 by alanine resulted in EGF receptors that were resistant to TPA. Cells expressing the A654 mutation underwent more rapid and more extensive morphologic changes than cells with the corresponding T654 EGF receptor. In cells expressing T654 EGF receptors, down regulation of protein kinase C resulted in more rapid and extensive EGF-induced changes similar to those seen in cells expressing A654 EGF receptors. These data indicate that activation of protein kinase C and subsequent phosphorylation of the EGF receptor at T654 lead to rapid physiological attenuation of EGF receptor signaling.     

Epidermal growth factor receptor synthesis is stimulated by phorbol ester and epidermal growth factor. Evidence for a common mechanism

Previously, we and others have shown that epidermal growth factor (EGF) stimulates the synthesis of its own receptor and the accumulation of EGF receptor mRNA. Here, we demonstrate that the tumor promotor, 12-O-tetradecanoylphorbol-13-acetate (TPA), like EGF, also stimulates receptor synthesis in the human breast carcinoma cell line, MDA468 cells. The receptor synthesis rate increased 5-fold with a peak at 8 h after exposure to TPA with half-maximal stimulation at a dose of 5 ng/ml TPA. This stimulation of receptor synthesis occurred despite a 30% decrease in general cellular protein synthesis. The increased receptor synthesis rate resulted in the accumulation of 60% more receptor protein as determined by quantitative immunoblotting using a newly developed monoclonal antibody, H9B4. Although TPA treatment resulted in an immediate loss of high affinity EGF-binding sites, the long-term effect was an increase in both the low and high affinity binding sites. The effects of EGF and TPA on receptor synthesis were not additive. Furthermore, down-regulation of protein kinase C (the Ca2+/phospholipid-dependent enzyme) by long-term TPA treatment resulted in cells unable to respond to the stimulatory effects of both TPA and EGF on receptor synthesis. Nevertheless, the TPA-pretreated cells were still growth-inhibited by EGF. These results suggest that the stimulatory effect of EGF on receptor synthesis requires protein kinase C, whereas the inhibitory effect of EGF on the proliferation of these cells does not. Although we confirmed that EGF stimulated the incorporation of phosphate into phosphatidylinositol in A431 cells, it failed to do so in the MDA468 cells. Thus, in MDA468 cells, EGF may require protein kinase C for part of its action, but we could not demonstrate an associated activation of phosphatidylinositol turnover by EGF. The exact mechanism of involvement of protein kinase C in EGF action is still not clear.     

Tumor promoter and epidermal growth factor stimulate phosphorylation of the c-erbB-2 gene product in MKN-7 human adenocarcinoma cells.

Treatment of human adenocarcinoma MKN-7 cells with epidermal growth factor (EGF) or phorbol tetradecanoate acetate (TPA) stimulated phosphorylation of the c-erbB-2 gene product. EGF induced a rapid increase in phosphotyrosine followed by relatively gradual increases in phosphoserine and phosphothreonine. On the other hand, the TPA-induced increase in phosphorylations occurred exclusively on serine and threonine residues. Tryptic phosphopeptide mapping analysis suggested that treatments with EGF and TPA induced phosphorylation of many common sites in the c-erbB-2 gene product. However, in contrast to TPA, EGF increased the phosphorylation of the c-erbB-2 protein in cells whose protein kinase C had been down-regulated by long-term pretreatment with TPA, suggesting that EGF and TPA induce phosphorylation by different mechanisms. Since the c-erbB-2 gene product did not show detectable EGF-binding activity, phosphorylation of tyrosine of the c-erbB-2 gene product might be catalyzed directly by the EGF receptor kinase that was activated by EGF.     

Reduced tyrosine phosphorylation and nonresponsiveness to EGF-mediated cytotoxicity in EGF receptor-hyperproducing UCVA-1 cells

UCVA-1 cells, derived from human pancreas adenocarcinoma, have a high number of epidermal growth factor (EGF) receptors (1.0 x 10(6) per cell) but their growth is not inhibited by EGF, unlike other EGF receptor-hyperproducing tumour cells. In UCVA-1 cells EGF activates neither the phosphatidylinositol turnover nor protein kinase C. EGF, however, enhances the phosphorylation of EGF receptors at specific tyrosine residues, indicating that the EGF receptor kinase is active and subject to autophosphorylation. Downmodulation of EGF receptors by 12-O-tetradecanoylphorbol 13-acetate (TPA) is also observed. Using an anti-phosphotyrosine antibody several phosphoproteins, including EGF receptors, were immunoprecipitated from UCVA-1 cell lysates, whereas more than 20 phosphoproteins were detected in other EGF receptor-hyperproducing tumour cells (NA), indicating that tyrosine-phosphorylation of endogenous substrates by EGF receptor kinase is significantly reduced in UCVA-1 cells. Thus, non-responsiveness of UCVA-1 cells to EGF is correlated with the reduced tyrosine phosphorylation.     

Specific association of annexin 1 with plasma membrane-resident and internalized EGF receptors mediated through the protein core domain

Phosphorylation of the Ca2+ and membrane-binding protein annexin 1 by epidermal growth factor (EGF) receptor tyrosine kinase has been thought to be involved in regulation of the EGF receptor trafficking. To elucidate the interaction of annexin 1 during EGF receptor internalization, we followed the distribution of annexin 1-GFP fusion proteins at sites of internalizing EGF receptors. The observed association of annexin 1 with EGF receptors was confirmed by immunoprecipitation. We found that this interaction was independent of a functional phosphorylation site in the annexin 1 N-terminal domain but mediated through the Ca2+ binding core domain.     

Effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the growth inhibitory and increased phosphatidylinositol (PI) responses induced by epidermal growth factor (EGF) in A431 cells

Epidermal growth factor (EGF) inhibited the growth of A431 human epidermoid carcinoma cells. The tumor promoting, phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) also retarded A431 cell growth. Addition of both TPA and EGF inhibited cell growth in an additive or synergistic manner depending upon the initial plating density of the cultures. EGF increased the production of diacylglycerol (60-70%) and stimulated the synthesis of phosphatidylinositol (PI) from 3H-inositol (three- to fourfold increase). Both of these responses were attenuated in the presence of TPA. TPA alone stimulated the production of diacylglycerol (DG) but had little effect on PI synthesis. The biological effect of TPA appeared to be mediated by the presence of a high-affinity receptor for phorbol esters on A431 cells. Moreover, the binding of 125I-EGF to A431 cells was unaffected by TPA, suggesting that the antagonistic effects of TPA were occurring distal to the EGF receptor. These findings also indicated that although TPA and EGF both inhibited A431 cell growth, this effect could be dissociated from changes in PI synthesis but may be dependent upon transient changes in DG production.