Diacylglycerol treatment rapidly decreases the affinity of the epidermal growth factor receptors of Swiss 3T3 cells

The synthetic diacylglycerol 1-oleoyl-2-acetyl glycerol (OAG) and phorbol esters activate protein kinase C in intact cells. We report here that OAG inhibits the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The inhibition was detected as early as 1 min after treatment at 37 degrees C and persisted for at least 120 min. The effect of OAG was reversed upon removal of this diacylglycerol. Detailed Scatchard analysis of 125I-EGF binding to Swiss 3T3 cells at 4 degrees C after a 1 h incubation with a saturating dose of OAG at 37 degrees C, demonstrates that this OAG pretreatment does not change the apparent number of EGF receptors but causes a marked decrease in their apparent affinity for the ligand. Prolonged treatment (40 h) of the cells with phorbol dibutyrate (PBt2) which causes a marked decrease in the number of phorbol ester binding sites and in the activity of protein kinase C, prevented the inhibition of 125I-EGF binding by both PBt2 and OAG. The results support the possibility that protein kinase C plays a role in the transmodulation of the EGF receptor in intact cells.     

Pasteurella multocida toxin, a potent mitogen, stimulates protein kinase C-dependent and -independent protein phosphorylation in Swiss 3T3 cells

Pasteurella multocida toxin, either native or recombinant (rPMT), is an extremely effective mitogen for Swiss 3T3 cells and acts at picomolar concentrations (Rozengurt, E., Higgins, T. E., Chanter, N., Lax, A. J., and Staddon, J. M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 123-127). Here, we show that similar concentrations of rPMT markedly stimulated the phosphorylation of an acidic 80-kDa protein in [32P]Pi-labeled Swiss 3T3 cells. Co-migration on one- and two-dimensional gels and phosphopeptide analysis indicated that this phosphoprotein was indistinguishable from 80K, a known protein kinase C substrate. In parallel cultures, the stimulation of 80K phosphorylation by rPMT (5-10-fold) was comparable to that induced by bombesin or phorbol dibutyrate (PBt2). However, the increase in phosphorylation by rPMT occurred after a pronounced lag period (1-3 h, depending upon the concentration of rPMT) in contrast to the relatively immediate stimulation by PBt2 or bombesin. Early, but not late, addition of either PMT antiserum or the lysosomotrophic agent methylamine selectively inhibited 80K phosphorylation in response to rPMT. 80K phosphorylation persisted after removal of free toxin and was not inhibited by cycloheximide. It appears that rPMT enters the cells via an endocytotic pathway to initiate and perpetuate events leading to 80K phosphorylation. rPMT, like PBt2, also stimulated the phosphorylation of 87-kDa and 33-kDa proteins in Swiss 3T3 cells. Phosphorylation of the 80K and 87-kDa proteins by rPMT or PBt2 were greatly attenuated in cells depleted of protein kinase C. In contrast, phosphorylation of the 33-kDa protein by rPMT, but not by PBt2, persisted in the absence of protein kinase C. rPMT, like bombesin, caused a translocation of protein kinase C to the cellular particulate fraction. The toxin enhanced the cellular content of diacylglycerol. rPMT also caused a time- and dose-dependent decrease in the binding of 125I-epidermal growth factor to its receptor which was blocked by methylamine and dependent only in part upon the presence of protein kinase C. We conclude that rPMT stimulates protein kinase C-dependent and -independent protein phosphorylation in Swiss 3T3 cells.     

Phosphorylation of an acidic mol. wt. 80 000 cellular protein in a cell-free system and intact Swiss 3T3 cells: a specific marker of protein kinase C activity.

Activation of the endogenous Ca2+-activated phospholipid-dependent protein kinase (protein kinase C) by Ca2+, phosphatidylserine (PS) and phorbol dibutyrate (PBt2) in detergent-solubilized extracts of Swiss 3T3 cells resulted in a very rapid increase (detectable within seconds) in the phosphorylation of an 80 000 mol. wt. protein (termed 80 K). Neither cyclic AMP nor Ca2+ had any effect on 80 K phosphorylation. The 80 K phosphoproteins generated after activation of protein kinase C, both in cell-free conditions and in intact fibroblasts, are identical as judged by one and two-dimensional polyacrylamide slab gel electrophoresis and peptide mapping. Prolonged treatment of cells with phorbol esters causes a selective decrease in protein kinase C activity and prevents the stimulation of 80 K phosphorylation in intact fibroblasts. We now show that extracts from PBt2-treated cultures fail to stimulate 80 K phosphorylation after the addition of the protein kinase C activators. This effect was due to the lack of protein kinase C activity since the addition of exogenous protein kinase C from mouse brain stimulated 80 K phosphorylation in both control and PBt2-treated preparations. The 80 K phosphoprotein generated by activation of endogenous and exogenous protein kinase C yielded similar phosphopeptide fragments after peptide mapping by limited proteolysis. We conclude that the detection of changes in the phosphorylation of 80 K provides a useful approach to ascertain which extracellular ligands activate protein kinase C in intact cells.     

Vasopressin rapidly stimulates protein kinase C in digitonin-permeabilized Swiss 3T3 cells: involvement of a pertussis toxin-insensitive guanine nucleotide binding protein

Guanine nucleotides and pertussis toxin were used to test for the involvement of a guanine nucleotide binding protein in the vasopressin V1 receptor-mediated stimulation of protein kinase C activity in Swiss 3T3 cells. Addition of vasopressin in the presence of [gamma-32P]ATP and digitonin caused a marked and rapid increase (8 +/- 1-fold after 1 min) in the phosphorylation of an Mr = 80,000 cellular protein (80K), a specific marker for protein kinase C activation. This phosphorylation was selectively blocked by the V1 receptor antagonist Pmp1-0-Me-Tyr2 [Arg8] vasopressin, indicating that the effect was mediated through the vasopressin V1 receptor. Down regulation of protein kinase C by prior prolonged pretreatment of intact cells with phorbol 12,13-dibutyrate (PBt2) blocked the ability of vasopressin to stimulate the phosphorylation of 80K in digitonin-permeabilized cells. Addition of a submaximal concentration of vasopressin together with the GTP analogue GTP-gamma-S caused a synergistic stimulation of 80K phosphorylation. The GDP analogue GDP-beta-S caused a 50% inhibition of the phosphorylation of 80K induced by a saturating concentration of vasopressin and shifted the vasopressin dose-response curve to the right. GDP-beta-S had no effect on the dose-response for the stimulation of 80K phosphorylation induced by PBt2. Prior incubation of intact quiescent cultures of Swiss 3T3 cells with pertussis toxin did not impair either vasopressin-induced increase in cytosolic [Ca2+] or activation of protein kinase C. These findings provide functional evidence for the involvement of a pertussis toxin-insesitive G protein in the vasopressin V1 receptor-mediated stimulation of protein kinase C in Swiss 3T3 cells.     

Vasopressin rapidly stimulates protein kinase C in quiescent Swiss 3T3 cells

Addition of vasopressin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an acidic molecular weight 80,000 cellular protein (termed 80K). The effect was concentration- and time-dependent; enhancement in 80K phosphorylation could be detected as early as 30 sec after the addition of the hormone. Recently, a rapid increase in the phosphorylation of an 80K cellular protein following treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact Swiss 3T3 cells. Here we show that the 80K phosphoproteins generated in response to vasopressin and phorbol 12,13-dibutyrate (PBt2) were identical as judged by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) and peptide mapping following partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with PBt2 which leads to the disappearance of protein kinase C activity blocked the ability of vasopressin to stimulate the phosphorylation of 80K. The effect of vasopressin on 80K phosphorylation and mitogenesis was selectively blocked by the vasopressin antagonist (Pmp1-O-Me-Tyr2-Arg8) vasopressin suggesting that these responses are mediated by its specific receptor in these cells. The removal of vasopressin leads to dephosphorylation (within minutes) of the 80K phosphoprotein. We conclude that vasopressin rapidly stimulates protein kinase C activity in intact 3T3 cells.     

Epidermal growth factor increases c-myc mRNA without eliciting phosphoinositide turnover, protein kinase C activation, or calcium ion mobilization in Swiss 3T3 fibroblasts

Incubation of quiescent cultures of Swiss 3T3 cells with epidermal growth factor (EGF) caused an increase in c-myc mRNA. Under these conditions, EGF did not induce phosphoinositide turnover, formation of diacylglycerol, formation of inositol tris-, bis-, and monophosphates, protein kinase C activation, or Ca2+ mobilization. Although it has been reported that both protein kinase C and Ca2+ may be responsible for the platelet-derived growth factor- and fibroblast growth factor-induced increases in c-myc mRNA in Swiss 3T3 cells (Kaibuchi, K., Tsuda, T., Kikuchi, A., Tanimoto, T., Yamashita, T., & Takai, Y. (1986) J. Biol. Chem. 261, 1187-1192), these results indicate that neither protein kinase C nor Ca2+ is involved in the EGF-induced increase in c-myc mRNA, and that an unidentified system may be involved in this reaction.     

Epidermal growth factor and bombesin differ strikingly in the induction of early responses in Swiss 3T3 cells

Swiss 3T3 cells express receptors for both the polypeptide epidermal growth factor (EGF) and the tetradecapeptide bombesin and respond mitogenically to these substances. These cells thus provide a system to analyze potential signal transduction pathways involved in mitogenic stimulation. Here we have determined and compared the early ionic responses elicited by EGF and bombesin and their relation to diacylglycerol (DG) and inositolphosphate (InsPn) production. Whereas EGF fails to cause any significant change in intracellular Ca2+, bombesin effectively induces prompt and transient Ca2+ mobilization from intracellular stores. Further support of the idea that these receptors utilize distinct signalling pathways comes from the measurements of cytoplasmic pH (pHi). As in most target cells, EGF induces a delayed (1 min) but sustained intracellular alkalinization that reaches a new steady state after approximately 10 min. Bombesin, in contrast, elicits a biphasic response; within seconds, a rapid but transient rise in pHi is observed, followed by a further slower sustained alkalinization. Inhibition of the Na+/H+ exchanger prevents both EGF as well as bombesin-induced alkalinization. However, under these conditions, bombesin evokes a rapid and sustained acidification related to the Ca2+ response. Apparently, bombesin initiates a Ca2(+)-dependent acidifying process immediately after binding of the hormone to its receptor. Furthermore, we could demonstrate that the bombesin-induced alkalinization depends on protein kinase C activation whereas the EGF response does not. Determination of the total DG and InsPn accumulation revealed that EGF is ineffective in stimulating phospholipase C-mediated production of these second messengers. In contrast, bombesin causes a rapid DG and InsPn production coinciding with the Ca2+ response and the first phase of the rise in pHi followed by a slower DG accumulation coinciding with the second alkalinization phase. Our results show that in Swiss 3T3 cells the bombesin receptor activates the hydrolysis of inositol lipids as a mechanism of signal transduction, which consequently causes changes in Ca2+i and pHi. Clearly, the EGF receptor utilizes different pathways to evoke mitogenesis and stimulates Na+/H+ exchange independently of DG production and protein kinase C activation.     

Insulin-like growth factor 1 and insulin reduce epidermal growth factor binding to Swiss 3T3 cells by an indirect mechanism that is apparently independent of protein kinase C

Insulin-like growth factor 1 and insulin reduced the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells by 15-20% at 37 degrees C, but not at 4 degrees C. Scatchard analysis indicated that IGF-1 and insulin affected the higher-affinity component of EGF binding, an effect previously associated with the activation of protein kinase C. However, the inhibition of 125I-EGF binding by IGF-1 and insulin was increased, not reduced, when the cells were treated with high concentrations of phorbol esters to down-modulate protein kinase C. We suggest that IGF-1 and insulin activate a protein kinase with similar or overlapping specificity to that of protein kinase C.     

Phosphorylation of lamin B at the nuclear membrane by activated protein kinase C

Both bryostatin 1 and 4 beta-phorbol 12,13-dibutyrate (PBt2) activate Ca2+- and phospholipid-dependent protein kinase (protein kinase C) at the plasma membrane in HL-60 cells (Kraft, A. S., Baker, V. V., and May, W. S. (1987) Oncogene 1, 91-100). However, whereas PBt2 causes HL-60 cells to cease dividing and differentiate, bryostatin 1 antagonizes this effect and allows cells to continue proliferating. To test whether these divergent effects could be due to the differential activation of protein kinase C at the nuclear level, the phosphorylation of nuclear envelope polypeptides was evaluated in cells treated with either bryostatin 1 or PBt2. Bryostatin 1, either alone or in combination with PBt2, but not PBt2 alone, mediates rapid and specific phosphorylation of several nuclear envelope polypeptides. A major target for bryostatin-induced phosphorylation is the major nuclear envelope polypeptide lamin B (Mr = 67,000, pI 6.0). In vitro studies combining purified protein kinase C and HL-60 cell nuclear envelopes demonstrate that bryostatin activates protein kinase C to phosphorylate lamin B, whereas PBt2 does so only weakly, suggesting selective activation of this enzyme toward this substrate. Comparative phosphopeptide and phosphoamino acid analyses demonstrate that bryostatin induces phosphorylation of identical serine sites on lamin B both in whole cells and in vitro. Treatment of whole cells with bryostatin, but not PBt2, leads to specific translocation of activated protein kinase C to the nuclear envelope. Since phosphorylation of lamin B is known to be involved in nuclear lamina depolymerization at the time of mitosis, it is possible that bryostatin-activated protein kinase C activity is involved in this process. Finally, specific activation of protein kinase C at the nuclear membrane could explain, at least in part, the divergent effects of bryostatin 1 and PBt2 on HL-60 cell growth.     

Activation of signal transduction pathways protects quiescent Balb/c-3T3 fibroblasts against death due to serum deprivation.

Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin protect density-inhibited murine Balb/c-3T3 fibroblasts against death by distinctive mechanisms. Determination of the cell survival-enhancing activity of growth factors by cell enumeration and neutral red uptake measurement gives equivalent results. PDGF displays a steep dose-response relationship in the 1-5 ng/ml range. The other factors display shallow log-linear relationships in the following ranges: EGF: 0.2-5 ng/ml; IGF-1: 2-80 ng/ml; and insulin: 57-4,500 ng/ml. Agonists that lead to the activation of protein kinase A, including forskolin, 8-bromoadenosine 3':5'-cyclic monophosphate (Br-cAMP) and N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (db-cAMP), markedly increase both short-term (5-h) and long-term (20-h) survival of cells. 2-Isobutyl-1-methylxanthine (IBMX) markedly enhances short-term survival, but its effect decays with time. The protein kinase C agonist 12-O-tetradecanoyl phorbol-13-acetate (TPA) has a moderate protective effect at concentrations of 16-32 nM, and 64 nM TPA is highly effective. The synthetic diaclglycerols 1,2-dioctanoylglycerol (DiC8) and 1-oleoyl-2-acetylglycerol (OAG) and the calcium ionophore ionomycin show low activity. Supplementation of EGF with a protein kinase A or C agonist results in a varying additive increase in short-term (5-h) cell survival and supplementation of EGF + insulin or PDGF + EGF + insulin increases further the already high level of protection given by the growth factor combinations. Combining a protein kinase A and a protein kinase C agonist in the absence of growth factors gives an approximately additive increase in cell survival. Results obtained with kinase, RNA, and protein synthesis inhibitors suggest that: 1) activated protein kinase C catalyzes one or more phosphorylation events in quiescent Balb/c-3T3 cells that lead to gene expression with the protein product(s) mediating protection of quiescent cells against death, and 2) phosphorylation events catalyzed by protein kinase A largely serve to protect cells by a mechanism not requiring de novo RNA and protein biosynthesis.     

Serum, like phorbol esters, rapidly activates protein kinase C in intact quiescent fibroblasts.

Addition of serum to quiescent cultures of Swiss 3T3 cells and mouse embryo fibroblasts causes a rapid increase in the phosphorylation of an 80 000 mol. wt. cellular protein (termed 80 K). The effect is dose- and time-dependent; enhancement in 80 K phosphorylation can be detected as early as 10-15 s after adding serum. In contrast, platelet-derived growth factor elicits the response after a lag of 1.5 min suggesting that this growth factor does not mediate the response to serum. Recently a rapid increase in the phosphorylation of an 80 K cellular protein following treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact fibroblasts. The 80 K phosphoproteins generated in response to serum and to phorbol dibutyrate (PBt2) co-migrated in one- and two-dimensional PAGE and produced identical phosphopeptide fragments when subjected to partial digestion with Staphyloccocus aureus V8 protease. These observations suggest that the same 80 K protein is generated in response to serum and PBt2. We conclude that activation of protein kinase C in intact cells is one of the earliest effects elicited by serum in quiescent fibroblasts.     

CCK(B)/gastrin receptor mediates synergistic stimulation of DNA synthesis and cyclin D1, D3, and E expression in Swiss 3T3 cells.

In order to develop a model system for identifying signaling pathways and cell cycle events involved in gastrin-mediated mitogenesis, we have used high efficiency retroviral-mediated transfection of cholecystokinin (CCK)(B)/gastrin receptor into Swiss 3T3 cells. The retrovirally-transfected CCK(B)/gastrin receptor binds 125I-CCK-8 with high affinity (Kd = 1.1 nM) and is functionally coupled to intracellular signaling pathways including rapid and transient increase in Ca2+ fluxes, protein kinase C-dependent protein kinase D activation, and MEK-dependent ERK1/2 activation. In the presence of insulin, CCK-8 or gastrin induced a 66.5 +/- 8.8-fold (mean +/- SEM, n = 24 in eight independent experiments) increase in cellular DNA synthesis, reaching a level similar to that achieved by stimulation with a saturating concentration of fresh serum, and much greater than the response to each agonist added alone. CCK-8 also induced a striking increase in the expression of cyclins D1, D3, and E and hyperphosphorylation of Rb acting synergistically with insulin. Similar effects were observed when CCK(B)/gastrin receptor was activated in the presence of EGF or bombesin. Our results demonstrate that activation of CCK(B)/gastrin receptor retrovirally-transfected into Swiss 3T3 induces a potent synergistic effect on DNA synthesis, accumulation of cyclins D1, D3, and E and hyperphosphorylation of Rb in combination with insulin, EGF, or bombesin. Thus, the CCK(B)/gastrin receptor transfected into Swiss 3T3 cells provides a novel model system to elucidate mitogenic signal transduction pathways and cell cycle events activated via this receptor.     

Basic and acidic fibroblast growth factors modulate the epidermal growth factor receptor by a protein kinase C-independent pathway

Human acidic and basic fibroblast growth factors (aFGF and bFGF) inhibit epidermal growth factor (EGF) receptor binding in mouse Swiss 3T3 cells. Scatchard analysis indicates that aFGF and bFGF cause a decrease in the high affinity EGF receptor population, similar to that observed for activators of protein kinase C such as phorbol esters, platelet-derived growth factor (PDGF) and bombesin. However, unlike phorbol esters, aFGF and bFGF inhibit EGF binding in protein kinase C-deficient cells. The time course and dose response of inhibition of EGF binding by both aFGF and bFGF are very similar, with an ID50 of approximately 0.10 ng/ml. In contrast to bombesin but like PDGF, neither aFGF nor bFGF act on the EGF receptor through a pertussis toxin-sensitive G protein. These results indicate that both acidic and basic FGF depress high affinity EGF binding in Swiss 3T3 cells with similar potency through a protein kinase C/Gi-independent pathway.     

Identification of multiple epidermal growth factor-stimulated protein serine/threonine kinases from Swiss 3T3 cells

Growth factor activation of serine/threonine protein kinases was studied by treating quiescent Swiss 3T3 cells with epidermal growth factor (EGF) and examining cytosolic extracts for protein kinase activity under conditions inhibitory to calcium- and cyclic nucleotide-dependent kinases. Cytosolic extracts of cells stimulated for 5 min were fractionated by Mono Q fast protein liquid chromatography. Eight peaks of kinase activity were resolved, of which five were stimulated by EGF treatment of cells. These peaks were revealed using the synthetic peptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (S6 peptide), 40 S ribosomal S6 protein, glycogen synthase, microtubule-associated protein 2, and myelin basic protein as substrates. The peaks varied in the kinetics of their activation by EGF and in their response to insulin. Selected peaks were resolved further by sizing gel chromatography. The results together indicate that at least seven distinct fractions of cytosolic kinase activities are stimulated in Swiss 3T3 cells by EGF. One of these, which phosphorylates both S6 protein and S6 peptide, is similar to the S6 kinase characterized previously in this cell line by others. Four additional activities that also phosphorylate the S6 protein and S6 peptide appear unrelated to this enzyme. Finally, two kinase activities that phosphorylate both myelin basic protein and microtubule associated protein 2 are EGF stimulated. One is similar to an insulin-stimulated microtubule-associated protein 2 kinase described in other cell lines whereas the other seems to represent a novel activity. Several of these EGF-stimulated activities were inactivated by protein phosphatases, suggesting that they might be regulated by phosphorylation.     

Bombesin stimulation of fibroblast mitogenesis: specific receptors, signal transduction and early events

Quiescent cultures of Swiss 3T3 cells can be stimulated to recommence deoxyribonucleic acid (DNA) synthesis by polypeptide growth factors, neuropeptides and various pharmacological agents that act via multiple signal transduction pathways. Neuropeptides of the bombesin family provide novel and potent mitogens to elucidate these pathways. The peptides bind to specific receptors that have been characterized by radioligand binding and sensitivity to antagonists and identified as glycoproteins of relative molecular mass (Mr) 75,000-85,000 by chemical cross-linking. After binding, bombesin elicits a cascade of early molecular events, including stimulation of phosphorylation of the acidic Mr 80,000 cellular protein (80,000) that is a major substrate of protein kinase C; Ca2+ mobilization mediated by Ins(1,4,5)P3; Na+ and K+ fluxes; transmodulation of (EGF) receptor; enhancement of cyclic adenosine monophosphate (cAMP) accumulation and expression of the proto-oncogenes c-fos and c-myc. Studies using digitonin-permeabilized 3T3 cells show that a G protein plays a role in the transduction of the mitogenic signal triggered by the binding of bombesin to its receptor.     

Reduction of epidermal growth factor receptor affinity by heterologous ligands: evidence for a mechanism involving the breakdown of phosphoinositides and the activation of protein kinase C

The tetradecapeptide bombesin converts epidermal growth factor (EGF) receptors on Swiss 3T3 cells from a high affinity state (KD = 9.8 X 10(-11)M) to a lower affinity state (KD = 1.8 X 10(-9)M). This conversion occurs when the cells are incubated with bombesin at 37 degrees C but not when incubated at 4 degrees C. Previously, a number of other (chemically unrelated) cell growth-promoting peptides and polypeptides have been shown to induce a similar indirect, temperature-dependent reduction of EGF receptor affinity. We have now demonstrated that hormones and growth factors which cross-regulate EGF receptor affinity in Swiss 3T3 cells have a common ability to stimulate the breakdown of phosphoinositides in these cells. We propose that the reduction of EGF receptor affinity is a consequence of the activation of protein kinase C by the diacylglycerol generated by this breakdown. In support of this proposal we have found that exogenously added diacylglycerol reduces the affinity of the Swiss 3T3 cell EGF receptor.     

Bombesin induction of c-fos and c-myc proto-oncogenes in Swiss 3T3 cells: significance for the mitogenic response

Bombesin is a potent mitogen for Swiss 3T3 cells and acts synergistically with insulin and other growth factors. We show here that addition of bombesin to quiescent Swiss 3T3 cells causes a striking increase in the levels of c-fos and c-myc mRNAs. Enhanced expression of c-fos (122 +/- 14-fold) occurred within minutes of peptide addition followed by increased expression of c-myc (82 +/- 16-fold). The concentrations of peptide required for half-maximal increase in the levels of c-fos and c-myc mRNAs were 1.0 and 0.9 nM, respectively. The peptide [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P which inhibits the binding of bombesin to its receptor and bombesin-stimulated DNA synthesis in Swiss 3T3 cells blocked the increase in c-fos and c-myc mRNA levels promoted by bombesin. Down-regulation of protein kinase C by long-term exposure to phorbol esters prevented c-fos and c-myc induction by bombesin. This and other results indicate that the induction of these proto-oncogenes by bombesin could be mediated by the coordinated effects of protein kinase C activation and Ca2+ mobilization. The marked synergistic effect between bombesin and insulin was used to assess whether the increase in the induction of c-fos and c-myc is an obligatory event in cell activation. In the presence of insulin, bombesin stimulated DNA synthesis at subnanomolar concentrations but had only a small effect on c-fos and c-myc mRNA levels. This apparent dissociation of mitogenesis from proto-oncogene induction was even more dramatic in 3T3 cells with down-regulated protein kinase C. In these cells bombesin stimulated DNA synthesis in the presence of insulin but failed to enhance c-fos and c-myc mRNA levels at comparable concentrations. Thus, the induction of c-fos and c-myc may be a necessary step in the mitogenic response initiated by ligands that act through activation of protein kinase C but the expression of these proto-oncogenes may not be an obligatory event in the stimulation of mitogenesis in 3T3 cells by mitogens that utilise other signalling pathways.     

Growth factors modify the epidermal growth factor receptor through multiple pathways

Previous results have shown that tumor promoters modify the properties of the epidermal growth factor (EGF) receptor through the activation of protein kinase C. Diacylglycerol-generating factors such as platelet-derived growth factor (PDGF) and p28sis should activate protein kinase C and alter EGF receptor properties in a similar manner. To test directly the involvement of protein kinase C in the action of media from v-sis-transformed cells on the EGF receptor, Swiss 3T3 cells were first extensively treated with various concentrations of the tumor-promoter phorbol dibutyrate (PDBu) This treatment reduced levels of active protein kinase C in the cells, making them less responsive to subsequent rechallenge with the tumor promoter. The results demonstrate that there are at least two components to the action of media from v-sis transformed cells on EGF binding: a labile factor that confers protein kinase C independence and a stable factor that appears to be dependent on protein kinase C. The action of the first factor cannot be mimicked by transforming growth factor-beta or EGF in either the presence or absence of PDGF. The action of the second factor is similar to that of PDGF. These findings indicate that heterologous regulation of the EGF receptor can occur through both protein kinase C-dependent and -independent pathways.     

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.     

Role of extracellular electrolytes in the activation of ribosomal protein S6 kinase by epidermal growth factor, insulin-like growth factor 1, and insulin in ZR-75-1 cells

Activation of ribosomal protein S6 kinase by epidermal growth factor (EGF), insulin, and insulin-like growth factor 1 (IGF1) was studied in the human mammary tumor cell line ZR-75-1 in isotonic buffers. In contrast to growth factor-dependent S6 phosphorylation which is strongly dependent on extracellular pH (Chambard, J. C., and J. Pouyssegur. 1986. Exp. Cell Res. 164:282-294.) preincubation of cells in buffers with different pH values ranging from 7.5 to 6.5 had no effect on basal or EGF-stimulated S6 kinase activity. Replacement of extracellular Na+ with choline or replacement of extracellular Ca++ with EGTA also did not inhibit stimulation of S6 kinase by EGF. When intracellular Ca++ was buffered with the permeable Ca++ chelator quin2, EGF stimulation was reduced 50%. A similar inhibition of the EGF response was observed when cells were incubated in buffers with high K+ concentrations or in the presence of the K+ ionophore valinomycin. Insulin and IGF1 stimulation of S6 kinase were also inhibited by high K+ concentrations and by buffering intracellular Ca++. In contrast to the responses to EGF, insulin- and IGF1-activation of S6 kinase was enhanced when glucose was present and depended on the presence of bicarbonate in the medium. The results indicate that ionic signals generated by growth factors and insulin, such as increases in intracellular pH or Na+, do not seem to be involved in the activation of S6 kinase. However, effects of growth factors or insulin on membrane potential and/or K+ fluxes and redistribution of intracellular Ca++ may play a role in the activation process. Furthermore, the mechanism of insulin activation of S6 kinase is distinct from the growth factors by its dependency on extracellular bicarbonate.