Differential influence of cAMP on the expression of the three subtypes (ATA1, ATA2, and ATA3) of the amino acid transport system A

Treatment of HepG2 cells with forskolin led to 60-100% stimulation of system A activity, measured as the Na+-dependent uptake of alpha-(methylamino)isobutyric acid. The stimulation was reproducible with cholera toxin and dibutyryl cAMP, and inhibitable by H7, a non-specific protein kinase inhibitor. The stimulatory effect was eliminated by cycloheximide and actinomycin D. The forskolin effect was associated with an increase in the maximal velocity of the transport system, with no change in substrate affinity. These cells express three different subtypes of system A (ATA1, ATA2, and ATA3). Treatment with forskolin increased the steady-state levels of ATA1 and ATA2 mRNAs, but decreased that of ATA3 mRNA.     

Expression and cyclic AMP-dependent regulation of a high affinity serotonin transporter in the human placental choriocarcinoma cell line (JAR).

The JAR human placental choriocarcinoma cell line transports serotonin, accumulating the monoamine inside the cell against a concentration gradient. The transport is energized by an NaCl gradient. Tricyclic (imipramine and desipramine) and non-tricyclic (paroxetine and fluoxetine) antidepressants inhibit the transporter markedly, but reserpine and 5-hydroxytryptophan do not. Ouabain, gramicidin, and nigericin, which reduce or abolish the transmembrane Na+ gradient, and phloridzin, which interferes with glucose transport into the cells, inhibit the transport. Preincubation of the cells with glucose-free medium also causes similar inhibition. The activity of the serotonin transporter in this cell line is stimulated in response to overnight (16-h) incubation with increasing concentrations of cholera toxin (0.1-1,000 ng/ml). Under these conditions the stimulation is maximal at 10 ng/ml cholera toxin (3.1 +/- 0.2-fold). Cholera toxin increases the cAMP content of these cells by several hundredfold within 2 h. Isobutylmethylxanthine (100 microM), dibutyryl cAMP (100 microM), and forskolin (100 microM) mimic the action of cholera toxin, eliciting a 1.6-2.5-fold stimulation of the serotonin transporter activity. The stimulatory effect of cholera toxin is antagonized significantly by simultaneous incubation of the cells with 50 microM N-(2-aminoethyl)-5-isoquinolinesulfonamide, a protein kinase inhibitor. The effect of cholera toxin on serotonin transport is specific because, under similar conditions, cholera toxin inhibits 3-O-methyl-D-glucose transport and does not influence taurine transport in this cell line. There is also no significant change in the protein content of the cells after cholera toxin treatment. Kinetic analysis reveals that cholera toxin causes an increase in the maximal velocity (7.89 +/- 0.67 to 17.55 +/- 1.06 pmol/mg of protein/5 min) and a decrease in the Michaelis-Menten constant (0.52 +/- 0.09 to 0.29 +/- 0.04 microM). These data show that the JAR human placental choriocarcinoma cell line expresses a high affinity serotonin transporter that is sensitive to inhibition by antidepressants and that the activity of the transporter is under cAMP-dependent regulation.     

Antigen- and ionophore-induced signal transduction in rat basophilic leukemia cells involves protein tyrosine phosphorylation

Treatment of rat basophilic leukemia cells (RBL-2H3) with antigen or ionophore leads to an increase in cellular protein tyrosine phosphorylation. Three major proteins of molecular mass of 72, 92, and 110 kDa are targeted by antigen and a 110-kDa species by ionophore, A23187. The antigen- and ionophore-induced tyrosine phosphorylation responses are dose-dependent and correlate with increases in serotonin release from activated cells. The presence of extracellular Ca2+ is required to sustain the antigen- and ionophore-stimulated tyrosine phosphorylation as well as mediator release. A protein tyrosine kinase inhibitor, RG 50864, differentially inhibits the antigen-stimulated tyrosine phosphorylation in the decreasing order of 72, 91, and 110-kDa proteins. The compound inhibition of the 72-kDa protein tyrosine phosphorylation correlates with that of serotonin release. In ionophore-stimulated cells, the inhibition of the 110-kDa protein tyrosine phosphorylation and serotonin release by RG 50864 occurs in parallel. These results suggest that the 72- and 110-kDa phosphoproteins may represent the respective regulators of serotonin release in antigen- and ionophore-activated cells. The 110-kDa tyrosine phosphorylated proteins from antigen- and ionophore-stimulated cells exhibit identical electrophoretic mobility and V8 protease-generated phosphopeptide maps, suggesting that these two proteins may be the same. These results provide new evidence that both the stimulatory actions of antigen and ionophore on mediator release are mediated through enhanced protein tyrosine phosphorylation in RBL-2H3 cells. Significantly, the present study suggests the presence of multiple tyrosine phosphorylation signaling pathways in RBL cells and that their selective utility may be determined by the nature of the stimulus.     

肝刺激因子对肝癌细胞增殖的调节作用

初断乳雄性ＳＤ大鼠的肝匀浆以超速离心和柱层析法分离纯化肝刺激因子（ＨＳＳ）,观察其对肝癌细胞增殖、细胞表皮生长因子（ＥＧＦ）受体表达及受体磷酸化的影响。结果表明,ＨＳＳ具有明显的促肝癌细胞分裂增殖能力,提高细胞周期中Ｓ期细胞所占比例。ＨＳＳ促肝癌细胞增殖作用与其促ＥＧＦ受体表达有关,表现为：（１）ＨＳＳ上调７０ｋＤ ＥＧＦ受体蛋白表达,此作用与ＥＧＦ合用后明显加强,即呈协同效应;（２）ＨＳＳ上调Ｅ     

A combination of H2O2 and vanadate concomitantly stimulates protein tyrosine phosphorylation and polyphosphoinositide breakdown in different cell lines

Treatment of four cell lines [rat hepatoma (Fao), murine muscle (BC3H-1), Chinese hamster ovary (CHO), and rat basophilic leukemia (RBL)] with a combination of 3 mM H2O2 and 1 mM sodium orthovanadate markedly stimulates protein tyrosine phosphorylation, which is accompanied by a dramatic increase (5-15-fold) in inositol phosphate (InsP) formation. H2O2/vanadate stimulate best formation of inositol triphosphate while their effects on the mono and di derivatives are more moderate. In the presence of 3 mM H2O2, both protein tyrosine phosphorylation and InsP formation are highly correlated and manifest an identical dose-response relationship for vanadate. Half-maximal and maximal effects are obtained at 30 and 100 microM, respectively. This stimulatory effect of H2O2/vanadate is not mimicked by other oxidants such as spermine, spermidine, KMnO4, and vitamin K3. In RBL cells, the kinetics of inositol triphosphate formation correlate with tyrosine phosphorylation of a 67-kDa protein, while tyrosine phosphorylation of a 55-kDa protein is closely correlated with both inositol monophosphate formation and serotonin secretion from these cells. Taken together, these results suggest a causal relationship between tyrosine phosphorylation triggered in a nonhormonal manner and polyphosphoinositide breakdown. Furthermore, these results implicate protein tyrosine phosphorylation in playing a role in the stimulus-secretion coupling in RBL cells.     

Phorbol ester reduces phosphorylation of epidermal growth factor receptor in pancreatic cancer cells by activation of a tyrosine phosphatase

In this study we report that phorbol 12-myristate 13-acetate (PMA) transiently reduced the level of EGF receptor tyrosine phosphorylation in three pancreatic cancer cell lines (HPAC, SW1990, and UCVA-1) in response to EGF. The effect was maximal at 40-90 min. Pretreatment with the protein kinase C inhibitor GF 109203X reduced the PMA effect. Flow cytometry experiments showed that PMA produced only a slight reduction in the surface expression of EGF-R. The phosphotyrosine phosphatase inhibitor bpV(phen) returned phosphorylation to almost control levels. Moreover, homogenates of PMA treated pancreatic cells reduced the phosphorylation of activated receptor that was immunoprecipitated from A431 epidermoid cells. A combination of orthovanadate and NaF or bpV(phen) inhibited the effect of the homogenates. These results suggest that PMA activates a phosphotyrosine phosphatase activity that reduces the steady-state level of tyrosine phosphorylation of the receptor that is induced by EGF.     

Stimulation of ultraviolet-induced apoptosis of human fibroblast UVr-1 cells by tyrosine kinase inhibitors

Damnacanthal is an anthraquinone compound isolated from the root of Morinda citrifolia and was reported to have a potent inhibitory activity towards tyrosine kinases such as Lck, Src, Lyn and EGF receptor. In the present study, we have examined the effects of damnacanthal on ultraviolet ray-induced apoptosis in ultraviolet-resistant human UVr-1 cells. When the cells were treated with damnacanthal prior to ultraviolet irradiation, DNA fragmentation was more pronounced as compared to the case of ultraviolet irradiation alone. The other tyrosine kinase inhibitors, herbimycin A and genistein, also caused similar effects on ultraviolet-induced apoptosis but to a lesser extent. Serine/threonine kinase inhibitors, K252a, staurosporine and GF109203X, rather suppressed the ultraviolet-induced DNA cleavage. Immunoblot analysis showed that pretreatment with damnacanthal followed by ultraviolet irradiation increased the levels of phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases. However, the other tyrosine kinase inhibitors did not increase the phosphorylation of extracellular signal-regulated kinases but stimulated phosphorylation of stress-activated protein kinases. Consequently, the ultraviolet-induced concurrent increase in both phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases after pretreatment with damnacanthal might be characteristically related to the stimulatory effect of damnacanthal on ultraviolet-induced apoptosis.     

Rapid uptake of tyrphostin into A431 human epidermoid cells is followed by delayed inhibition of epidermal growth factor (EGF)-stimulated EGF receptor tyrosine kinase activity.

Treatment of A431 human epidermoid cells with epidermal growth factor (EGF; 20 nM) results in decreased proliferation. This is associated with blockage of the cells in the S and/or G2 phases of the cell cycle. We found that tyrphostin, a putative tyrosine kinase inhibitor, in the range of 50 to 100 microM, partially reversed the growth-inhibitory and cell cycle changes induced by EGF. By using high-pressure liquid chromatography with electrochemical detection, we found that tyrphostin was readily incorporated into A431 cells, reaching maximal levels within 1 h. Although tyrphostin (50 to 100 microM) had no effect on high-affinity binding of EGF to its receptor in A431 cells for up to 24 h, the compound partially inhibited EGF-stimulated EGF receptor tyrosine kinase activity. However, this effect was evident only after prolonged treatment of the cells (4 to 24 h) with the drug. When the peak intracellular concentration of tyrphostin occurred (1 h), no inhibition of tyrosine kinase activity was observed. After both 1 and 24 h, tyrphostin was a less effective inhibitor of tyrosine kinase activity than the potent tumor promoter 12-O-tetradecanoyl phorbol-13-acetate, which almost completely blocked EGF receptor autophosphorylation. On the basis of our data, we hypothesize that tyrphostin is not a competitive inhibitor of the EGF receptor tyrosine kinase in intact cells and that it functions by an indirect mechanism.     

Prostaglandin E2 upregulates EGF-stimulated signaling in mitogenic pathways involving Akt and ERK in hepatocytes

Prostaglandins (PGs) such as PGE2 enhance proliferation in many cells, apparently through several distinct mechanisms, including transactivation of the epidermal growth factor (EGF) receptor (EGFR) as well as EGFR-independent pathways. In this study we found that in primary cultures of rat hepatocytes PGE2 did not induce phosphorylation of the EGFR, and the EGFR tyrosine kinase blockers gefitinib and AG1478 did not affect PGE2-stimulated phosphorylation of ERK1/2. In contrast, PGE2 elicited EGFR phosphorylation and EGFR tyrosine kinase inhibitor-sensitive ERK phosphorylation in MH1C1 hepatoma cells. These findings suggest that PGE2 elicits EGFR transactivation in MH1C1 cells but not in hepatocytes. Treatment of the hepatocytes with PGE2 at 3 h after plating amplified the stimulatory effect on DNA synthesis of EGF administered at 24 h and advanced and augmented the cyclin D1 expression in response to EGF in hepatocytes. The pretreatment of the hepatocytes with PGE2 resulted in an increase in the magnitude of EGF-stimulated Akt phosphorylation and ERK1/2 phosphorylation and kinase activity, including an extended duration of the responses, particularly of ERK, to EGF in PGE2-treated cells. Pertussis toxin abolished the ability of PGE2 to enhance the Akt and ERK responses to EGF. The results suggest that in hepatocytes, unlike MH1C1 hepatoma cells, PGE2 does not transactivate the EGFR, but instead acts in synergism with EGF by modulating mitogenic mechanisms downstream of the EGFR. These effects seem to be at least in part G(i) protein-mediated and include upregulation of signaling in the PI3K/Akt and the Ras/ERK pathways.     

Temperature-dependent tyrosine phosphorylation of microtubule-associated protein kinase in epidermal growth factor-stimulated human fibroblasts.

Treatment of normal human fibroblasts with epidermal growth factor (EGF) results in the rapid (0.5 min) and simultaneous tyrosine phosphorylation of the EGF receptor (EGFr) and several other proteins. An exception to this tyrosine phosphorylation wave was a protein (42 kDa) that became phosphorylated on tyrosine only after a short lag time (5 min). We identified this p42 kDa substrate as the microtubule-associated protein (MAP) kinase using a monoclonal antibody to a peptide corresponding to the C-terminus of the predicted protein (Science 249, 64-67, 1990). EGF treatment of human fibroblasts at 37 degrees C for 5 min resulted in the tyrosine phosphorylation of 60-70% of MAP kinase as determined by the percent that was immunoprecipitated with antiphosphotyrosine antibodies. Like other tyrosine kinase growth factor receptors, the EGFr is activated and phosphorylated at 4 degrees C but is not internalized. Whereas most other substrates were readily tyrosine phosphorylated at 4 degrees C, MAP kinase was not. When cells were first stimulated with EGF at 4 degrees C and then warmed to 37 degrees C without EGF, tyrosine phosphorylation of MAP kinase was again observed. Treatment of cells with the protein kinase C activator phorbol myristate acetate (PMA) also resulted in the tyrosine phosphorylation of MAP kinase, and again only at 37 degrees C. Tryptic phosphopeptide maps demonstrated that EGF and PMA both induced the phosphorylation of the same peptide on tyrosine and threonine. This temperature and PMA sensitivity distinguishes MAP kinase from most other tyrosine kinase substrates in activated human fibroblasts.     

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.     

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.     

Oncostatin M suppresses EGF-mediated protein tyrosine phosphorylation in breast cancer cells

The effect of oncostatin M (OM) on epidermal growth factor (EGF)-mediated protein tyrosine phosphorylation in an infiltrating ductal breast carcinoma cell line, H3922, was investigated by Western blot analysis. Pretreatment of H3922 cells with OM for 72 h suppressed EGF-stimulated protein tyrosine phosphorylation signals by 77%. Interestingly, pretreatment with OM for 6 or 48 h had little effect on these signals. EGF-mediated tyrosine phosphorylation of EGF receptor (EGFR) was suppressed by 55% in 72-h OM pretreated H3922 cells. No reduction in EGFR protein expression was detected in these cells. Flow cytometric analysis verified that OM does not suppress EGFR expression. The effect of OM could not be attributed to induction of protein tyrosine phosphatases. An H3922 subclone cell line, designated H3922-8, was found to exhibit no proliferative response to treatment with EGF. However, EGF-mediated protein tyrosine phosphorylation was detected in these cells. Radioligand EGF binding studies comparing H3922 to H3922-8 cells indicated that the clonal cells apparently lack high affinity EGF receptors. The mechanism by which OM suppresses EGF-mediated tyrosine phosphorylation has not been completely characterized. However, the suppressive effect occurs regardless of whether the cells are acutely responsive (H3922) or virtually unresponsive (H3922-8) to EGF stimulation of cell growth.     

Regulation of casein kinase II activity by epidermal growth factor in human A-431 carcinoma cells

In order to characterize more fully the mechanism by which casein kinase II is regulated in mammalian cells, the effect of epidermal growth factor (EGF) on the activity of the kinase in human A-431 carcinoma cells was examined. Treatment of cells with EGF prior to lysis consistently resulted in a transient 4-fold increase in the activity of cytosolic casein kinase II. Activity rose sharply between 20 and 30 min, peaked at approximately 50 min, and returned to basal levels by approximately 120 min. Similar results were obtained using the casein kinase II specific peptide substrate, Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu, or DNA topoisomerase II (which is specifically modified by the kinase in vivo and serves as a high affinity substrate in vitro) as the phosphate acceptor in assays. Identification of casein kinase II as the stimulated activity was confirmed by partial proteolytic mapping and phosphoamino acid analysis of modified topoisomerase II, by inhibition at nanomolar levels of heparin or micromolar levels of nonradioactive GTP, and by the ability to employ radioactive GTP as a direct phosphate donor. The EGF stimulation of casein kinase II was dependent on the availability of intracellular (but not extracellular) calcium. In addition, hormonal action was modulated by calcium/phospholipid-dependent protein kinase (protein kinase C). Casein kinase II stimulation did not require an increase in the concentration of the kinase, protein synthesis, the continual presence of a small effector molecule, or a direct interaction with the EGF receptor/tyrosine kinase. In contrast, hormonal activation of the kinase was dependent on the phosphorylation of casein kinase II or a terminal stimulatory factor.     

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.     

Effects of substitution of threonine 654 of the epidermal growth factor receptor on epidermal growth factor-mediated activation of phospholipase C

Addition of epidermal growth factor (EGF) to many cell types activates phospholipase C resulting in increased levels of diacylglycerol and intracellular Ca2+ which may lead to activation of protein kinase C. EGF treatment of cells can also lead to phosphorylation of the EGF receptor at threonine 654 (a protein kinase C phosphorylation site) which appears to attenuate some aspects of receptor signaling. Thus, a feedback loop involving the EGF receptor, phospholipase C, and protein kinase C may regulate EGF receptor function. In this report, the role of phosphorylation of threonine 654 of the EGF receptor in regulation of EGF-stimulated activation of phospholipase C was investigated. NIH-3T3 cells expressing the normal human EGF receptor or expressing EGF receptor in which an alanine residue had been substituted at residue 654 of the receptor were used. Addition of EGF to cells expressing wild-type receptor induced a rapid, but transient, increase in phosphorylation of threonine 654. EGF addition also caused the rapid accumulation of inositol phosphates in these cells. EGF-stimulated accumulation of inositol phosphates was significantly higher in cells expressing Ala-654 receptors compared to control cells. Treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), which stimulated phosphorylation of threonine 654 to a greater degree than EGF, completely inhibited EGF-dependent inositol phosphate accumulation in cells expressing wild-type receptor, but caused only a 20-30% inhibition in Ala-654 expressing cells. EGF stimulated phosphorylation of phospholipase C-gamma on serine and tyrosine residues in cells expressing wild-type of Ala-654 receptors. However, TPA treatment of cells inhibited EGF-induced tyrosine phosphorylation of phospholipase C-gamma only in cells expressing wild-type receptors. Similarly, TPA inhibited tyrosine-specific autophosphorylation of the EGF receptor and tyrosine phosphorylation of several other proteins in wild-type receptor cells, but not in Ala-654 cells. TPA treatment abolished high affinity binding of EGF to cells expressing wild-type receptors, while decreasing the number of high affinity binding sites 20-30% in Ala-654 cells. These data suggest that phosphorylation of threonine 654 can regulate early events in EGF receptor signal transduction such as phosphoinositide turnover, probably through a feedback mechanism involving protein kinase C. Subsequent dephosphorylation of threonine 654 could reactivate the EGF receptor for participation in later signaling events.     

Regulation of the epidermal growth factor receptor by phosphorylation

The receptor for epidermal growth factor (EGF) is a glycosylated transmembrane phosphoprotein that exhibits EGF-stimulable protein tyrosine kinase activity. On EGF stimulation, the receptor undergoes a self-phosphorylation reaction at tyrosine residues located primarily in the extreme carboxyl-terminal region of the protein. Using enzymatically active EGF receptor purified by immunoaffinity chromatography from A431 human epidermoid carcinoma cells, the self-phosphorylation reaction has been characterized as a rapid, intramolecular process which is maximal at 30-37 degrees C and exhibits a very low Km for ATP (0.2 microM). When phosphorylation of exogenous peptide substrates was measured as a function of receptor self-phosphorylation, tyrosine kinase activity was found to be enhanced two to threefold at 1-2 mol of phosphate per mol of receptor. Analysis of the dependence of the tyrosine kinase activity on ATP concentration yielded hyperbolic kinetics when plotted in double-reciprocal fashion, indicating that ATP can serve as an activator of the enzyme. Higher concentrations of peptide substrates were found to inhibit both the self- and peptide phosphorylation, but this inhibition could be overcome by first self-phosphorylating the enzyme. These results suggest that self-phosphorylation can remove a competitive/inhibitory constraint so that certain exogenous substrates can have greater access to the enzyme active site. In addition to self-phosphorylation, the EGF receptor can be phosphorylated on threonine residues by the calcium- and phospholipid-dependent protein kinase C. The sites on the EGF receptor phosphorylated in vitro by protein kinase C are identical to the sites phosphorylated on the receptor isolated from A431 cells exposed to the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. This phosphorylation of the EGF receptor results in a suppression of its tyrosine kinase and EGF binding activities both in vivo and in vitro. The EGF receptor can thus be variably regulated by phosphorylation: self-phosphorylation can enhance tyrosine kinase activity whereas protein kinase C-catalyzed phosphorylation can depress enzyme activity. Because these two phosphorylations account for only a fraction of the phosphate present in the EGF receptor in vivo, other protein kinases can apparently phosphorylate the receptor and these may exert additional controls on EGF receptor/kinase function.     

Regulation of the epidermal growth factor receptor phosphorylation state by sphingosine in A431 human epidermoid carcinoma cells

The regulation of protein phosphorylation by sphingosine in A431 human epidermoid carcinoma cells was examined. Sphingosine is a competitive inhibitor of phorbol ester binding to protein kinase C (Ca2+/phospholipid-dependent enzyme) and potently inhibits phosphotransferase activity in vitro. Addition of sphingosine to intact A431 cells caused an inhibition of the phorbol ester-stimulated phosphorylation of two protein kinase C substrates, epidermal growth factor (EGF) receptor threonine 654 and transferrin receptor serine 24. We conclude that sphingosine inhibits the activity of protein kinase C in intact A431 cells. However, further experiments demonstrated that sphingosine-treatment of A431 cells resulted in the regulation of the EGF receptor by a mechanism that was independent of protein kinase C. First, sphingosine caused an increase in the threonine phosphorylation of the EGF receptor on a unique tryptic peptide. Second, sphingosine caused an increase in the affinity of the EGF receptor in A431 and in Chinese hamster ovary cells expressing wild-type (Thr654) and mutated (Ala654) EGF receptors. Sphingosine was also observed to cause an increase in the number of EGF-binding sites expressed at the surface of A431 cells. Examination of the time course of sphingosine action demonstrated that the effects on EGF binding were rapid (maximal at 2 mins) and were observed prior to the stimulation of receptor phosphorylation (maximal at 20 mins). We conclude that sphingosine is a potently bioactive molecule that modulates cellular functions by: 1) inhibiting protein kinase C; 2) stimulating a protein kinase C-independent pathway of protein phosphorylation; and 3) increasing the affinity and number of cell surface EGF receptors.     

Inhibition of intrinsic protein tyrosine kinase activity of EGF-receptor kinase complex from human breast cancer cells by the marine sponge metabolite (+)-aeroplysinin-1

1. (+)-Aeroplysinin-1, a naturally occurring tyrosine metabolite from the marine sponge Verongia aerophoba, was found to inhibit the phosphorylation of lipocortin-like proteins by a highly purified preparation of the epidermal growth factor (EGF) receptor-tyrosine protein kinase complex from MCF-7 breast carcinoma cells. 2. (+)-Aeroplysinin-1 blocked the EGF-dependent proliferation of both MCF-7 and ZR-75-1 human breast cancer cells and inhibited the ligand-induced endocytosis of the EGF receptor in vitro. 3. Treatment with aeroplysinin-1 in the concentration range at 0.25-0.5 microM resulted in a time- and dose-dependent total tumor cell death in vitro. 4. At a 10-fold higher concentration the compound did not reveal any cytostatic activity in normal human fibroblasts. 5. From these data we conclude that (+)-aeroplysinin-1 represents a compound which displays a strong anti-tumor effect on EGF-dependent tumor cell lines.     

Quantitative dissociation between EGF effects on c-myc and c-fos gene expression, DNA synthesis, and epidermal growth factor receptor tyrosine kinase activity.

The exact relationship between EGF-stimulated tyrosine phosphorylation, induction of the cellular proto-oncogenes c-myc and c-fos, and DNA synthesis remains uncertain. Madin-Darby Canine Kidney (MDCK) cells possess EGF receptor sites with high binding capacity, and in contrast to A431 cells, respond to EGF by increasing DNA synthesis. Following EGF stimulation of intact MDCK cells, there was a rapid and marked increase in the autophosphorylation of the EGF receptor. This was associated with an increase in the tyrosine phosphorylation of a 120 kDa phosphoprotein believed to be an endogenous substrate of this receptor kinase. The ED50 for stimulation of phosphorylation of pp120 was approximately 0.05 nM versus 1.0 nM for receptor autophosphorylation, consistent with amplification of signalling at this step in EGF action. Stimulation of DNA synthesis occurred after 12 to 24 hours and revealed even further amplification with an ED50 of about 0.1 nM. Intermediate between these events was a time-dependent activation of c-fos and c-myc gene expression. However, the ED50 for these processes was approximately 10 nM, indicating a relatively lower sensitivity of EGF for stimulation of proto-oncogene expression. Tyrphostin (RG 50864), a compound reported to inhibit specifically the EGF receptor kinase, completely blocked EGF stimulation of proto-oncogene induction. Interestingly, under the same experimental conditions, EGF receptor autophosphorylation was decreased only 60%. These data, along with the dose-response studies, indicate that proto-oncogene induction requires near maximal stimulation of EGF receptor autophosphorylation. They also suggest that, in MDCK cells, the EGF dependent induction of the c-fos and c-myc genes is not strictly correlated to the extent of EGF receptor autophosphorylation or EGF-stimulated DNA synthesis, and that EGF stimulation of DNA synthesis likely involves additional rate-limiting intermediate steps.