Activation of the epidermal growth factor receptor by skin tumor promoters and in skin tumors from SENCAR mice

The present study was designed to further investigate the role of the epidermal growth factor receptor (EGFr) in mouse skin tumor promotion by evaluating the status of the EGFr in tumor promoter-treated mouse epidermis and in mouse skin tumors. Female SENCAR mice received three topical treatments of either the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or the nonphorbol esters okadaic acid and chrysarobin. Membrane proteins from SENCAR mouse epidermis were isolated 6 h after the last treatment, and the phosphotyrosine content of the EGFr and several potential substrates were examined by Western blot analysis. The results indicated that multiple applications of all three tumor promoters led to an increase in the phosphotyrosine content of the EGFr and also of several lower molecular weight proteins (M(r) approximately 80,000-85,000). Phosphorylation of PLC gamma 1 on tyrosine residues could not be detected in tumor promoter-treated mouse epidermis when the phosphotyrosine content of the EGFr was elevated or in cultured keratinocytes exposed to exogenous EGF. When two tyrosine kinase inhibitors (tyrphostins RG50864 and RG13022) were incorporated into the treatment regimens, the TPA-induced epidermal hyperplasia and cell proliferation were effectively blocked, and the TPA-stimulated EGFr tyrosine phosphorylation was significantly reduced. Examination of the phosphotyrosine content of epidermal membrane proteins isolated from skin papillomas revealed that the EGFr also had elevated phosphotyrosine levels. These results demonstrate that multiple topical treatments with both phorbol ester and nonphorbol ester tumor promoters lead to activation of the EGFr tyrosine kinase in mouse epidermis. In addition, these data suggest that signaling through the EGFr pathway plays an important role in the tumor promotion stage of multistage carcinogenesis in mouse skin.     

Excimer laser angioplasty

Increased expression of interleukin-1 (IL-1) in skin elicits a variety of responses, including inflammation and epidermal hyperplasia, which are also characteristic events elicited by tumor promoters. The goal of this study was to investigate whether various classes of tumor promoters increase expression of IL-1 alpha and whether phorbol ester-induced IL-1 alpha expression can be blocked by antitumor promoters. Northern analysis of mRNA isolated from the dorsal skins of SENCAR mice treated with 1 microgram of 4 beta-12-O-tetradecanoylphorbol-13-acetate (TPA) showed that a single application of TPA produced a significant increase in IL-1 alpha mRNA at 6 h that decreased by 24 h after treatment. Two treatments of TPA at 48-h intervals induced, at 6 h, twice as much IL-1 alpha mRNA as one treatment. Of the other promoters tested, anthralin (22.6 micrograms), mezerein (2 micrograms), calcium ionophore A23187 (120 micrograms), and benzoyl peroxide (20 mg) induced IL-1 alpha mRNA with different kinetics and to different extents. On the other hand, the non-tumor promoting phorbol ester analogue 4 alpha-12-O-tetradecanoylphorbol-13-acetate had little effect on the expression of IL-1 alpha mRNA. The effects of various antitumor promoters on TPA-induced IL-1 alpha mRNA expression were also assessed. Fluocinolone acetonide, mepacrine, and 5,8,11,14-eicosatetraynoic acid were the most effective inhibitors, and each produced about 80% inhibition. Other antitumor promoters such as retinoic acid, N-tosyl-L-phenylalanine chloromethyl ketone, and butylated hydroxytoluene inhibited approximately 35%, 65%, and 50% of TPA-induced IL-1 alpha mRNA expression, respectively. Therefore, this study suggests a possible role of IL-1 alpha in the promotion stage of skin carcinogenesis.     

Molecular cloning and functional expression of a phorbol ester-inducible 8S-lipoxygenase from mouse skin

One of the effects of topical application of phorbol ester to mouse skin is the induction of an 8S-lipoxygenase in association with the inflammatory response. Here we report the molecular cloning and characterization of this enzyme. The cDNA was isolated by polymerase chain reaction from mouse epidermis and subsequently from a mouse epidermal cDNA library. The cDNA encodes a protein of 677 amino acids with a calculated molecular mass of 76 kDa. The amino acid sequence has 78% identity to a 15S-lipoxygenase cloned recently from human skin and approximately 40% identity to other mammalian lipoxygenases. When expressed in vaccinia virus-infected Hela cells, the mouse enzyme converts arachidonic acid exclusively to 8S-hydroperoxyeicosatetraenoic acid while linoleic acid is converted to 9S-hydroperoxy-linoleic acid in lower efficiency. Phorbol ester treatment of mouse skin is associated with strong induction of 8S-lipoxygenase mRNA and protein. By Northern analysis, expression of 8S-lipoxygenase mRNA was also detected in brain. Immunohistochemical analysis of phorbol ester-treated mouse skin showed the strongest reaction to 8S-lipoxygenase in the differentiated epidermal layer, the stratum granulosum. The inducibility may be a characteristic feature of the mouse 8S-lipoxygenase and its human 15S-lipoxygenase homologue.     

Presynaptic modulation of cholecystokinin release by protein kinase C in the rat hippocampus

The role of protein kinase C (PKC) in modulating the release of the octapeptide cholecystokinin (CCK-8) was investigated in rat hippocampal nerve terminals (synaptosomes). The PKC-activating phorbol ester 4beta-phorbol 12,13-dibutyrate (beta-PDBu) dose dependently (5-5,000 nM) increased CCK-8 release in a strictly Ca2+dependent way. This effect was observed only when synaptosomes were stimulated with the K+(A) channel blocker 4-aminopyridine (4-AP; 1 mM) but not with KCl (10-30 mM). The PDBu-induced exocytosis of CCK-8 was completely blocked by the two selective PKC inhibitors chelerythrine and calphostin-C and was not mimicked by alpha-PDBu, an inactive phorbol ester. In addition, an analogue of the endogenous PKC activator diacylglycerol, oleoylacetylglycerol, dose dependently increased CCK-8 exocytosis. Beta-PDBu (50-100 nM) also stimulated the 4-AP-evoked Ca2+-dependent release of the classic transmitter GABA, which co-localizes with CCK-8 in hippocampal interneurons. As a possible physiological trigger for PKC activation, the role of the metabotropic glutamate receptor was investigated. However, the broad receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid did not stimulate, but instead inhibited, both the CCK-8 and the GABA exocytosis. In conclusion, presynaptic PKC may stimulate exocytosis of distinct types of co-localizing neurotransmitters via modulation of presynaptic K+ channels in rat hippocampus.     

Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells

Cysteine-rich domains (Cys-domains) are approximately 50-amino acid-long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-gamma (Cys1-GFP). Strikingly, stimulation of G-protein or tyrosine kinase-coupled receptors induced a transient translocation of cytosolic Cys1-GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1-GFP in the membrane, whereas DiC8 left Cys1-GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1-GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-gamma also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of proteins to selective lipid membranes.     

Cultured fucosidosis fibroblasts: a simple technique demonstrating storage of tritiated-fucose labeled material

Nonsteroidal anti-oestrogenic drugs, tamoxifen and clomiphene citrate, at concentrations higher than 0.001 mug/ml reduced the colony forming ability of cells derived from a rat uterine adenocarcinoma in vitro. The 50 per cent inhibitory dose of these drugs was about one-hundredth of that of sex steroids. When the cells were treated with combinations of these nonsteroidal anti-oestrogenic drugs and the 50 per cent inhibitory dose (8 mug/ml) of progesterone, a synergistic effect on the inhibition of colony formation was observed. In contrast to progesterone, oestradiol-17beta (the 50 per cent inhibitory dose of which was about 16 mug/ml) suppressed additively the colony formation only in combination with low doses of anti-oestrogenic drugs.     

Improved tumour yields by means of a TPA-DMBA-TPA variation of the Berenrlum-Mottram experiment on the back skin of NMRI mice. The effect of stationary hyperplasia without inflammation

Application of the phorbol ester TPA to the back skin of NMRI mice 14 times within a period of 7 weeks causes a stationary hyperplasia, with a corresponding increase in the labelling index of the basal cells from 1% to 14%. By initiation of skin, which has been pretreated with TPA in this way, with the carcinogen DMBA, followed by continued treatment with TPA (initiation-promotion corresponding to the classical Berenblum-Mottram experiment) the tumour yield (papillomas, carcinomas) is very much higher than that obtained using the scheme of the normal Berenblum-Mottram experiment. The preliminary induction of a stationary hyperplasia with high rates of nucleic acid synthesis must be considered an important co-factor in epidermal carcinogenesis.     

Intrauterine infection and the effects of inflammatory mediators on prostaglandin production by myometrial cells from pregnant women

OBJECTIVE: Our purpose was to evaluate the effects of known stimulants of prostaglandin production on cultured myometrial cells from women in labor with and without intrauterine infection. STUDY DESIGN: Myometrial segments were obtained from 16 patients between 33 and 40 weeks' gestation who had been in labor for > or = 8 hours at cesarean delivery; 8 patients had clinical chorioamnionitis and 8 did not. Myometrial cells were isolated and grown in culture. Incubations were conducted with interleukin-1 beta, tumor necrosis factor-alpha, or epidermal growth factor. Prostaglandin E2, prostaglandin F2 alpha, and 6-keto-prostaglandin F1 alpha (the stable metabolite of prostacyclin) were measured by radioimmunoassay, and cellular protein was determined. RESULTS: Cultured human myometrial cells from patients with and without prior intrauterine infection produced prostaglandins in response to interleukin-1 beta, tumor necrosis factor-alpha, and epidermal growth factor at a significantly increased rate (p<0.05 vs controls at and above 10 ng/ml of interleukin-1 beta, tumor necrosis factor-alpha, and epidermal growth factor). The major prostaglandin produced in response to each stimulant was 6-keto-prostaglandin F1 alpha; however, this response was attenuated in cells from patients with intrauterine infection. CONCLUSIONS: Cultured human myometrial cells from patients with and without prior intrauterine infection respond to known stimulants of prostaglandin production. Prior intrauterine infection has no effect on baseline prostaglandin production, but the amount of prostacyclin produced as a response to cellular stimulants is decreased with prior intrauterine infection. This effect may have a role in regulating myometrial function in intrauterine infection.     

Changes in the weight of the adrenal glands and in the concentration of plasma corticosterone in perinatal rats after prenatal treatment with oestadiol benzoate

A comparison was made of gonadotropin release after an iv bolus of large (150 mug) and small (10 mug) doses of LRF during various phases of the menstrual cycle and in primary hypogonadal women. Gonadotropin release was quantitatively similar to small and large doses of LRF in hypogonadal women with low estradiol levels. In contrast, normal women from the early to late follicular phase of the cycle, the gonadotropin response to a small dose of LRF (10 mug) was not as great as that observed with a large dose of LRF (150 mug) and did not elicit the augmented gonadotropin response of the late follicular phase. During the mid-luteal phase, a significant enhancement of responses to small doses of LRF was observed. The sustained gonadotropin release induced by the large dose of LRF in the late follicular phase was no longer apparent during the mid-luteal phase, although the dose-related discrimination of LH release was maintained. These dose-related disparities in the gonadotropin release to LRF might represent functional expressions of the two-pool dynamics of the gonadotrophs induced by ovarian steroids.     

Analysis of the ability of 12-O-tetradecanoylphorbol-13-acetate to induce epidermal hyperplasia, transforming growth factor-alpha, and skin tumor promotion in wa-1 mice

Wa-1 mutant mice possess a defect in the production of transforming growth factor-alpha (TGF-alpha) that leads to a phenotype characterized by wavy hair and curly whiskers. In light of recent evidence indicating the importance of TGF-alpha in epithelial tumorigenesis, this study characterizes the responsiveness of wa-1 mice to skin tumor promotion by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). The responsiveness of wa-1 mice to TPA was compared with that of SENCAR and C57BL/6 mice, representing mouse lines highly sensitive and resistant to skin tumor promotion, respectively. Wa-1 mice were found to be very resistant to skin tumor promotion by TPA after initiation with 10 nmol DMBA, similar to C57BL/6 mice. TPA failed to induce a dramatic increase in TGF-alpha mRNA and protein in the skin of wa-1 mice, whereas TGF-alpha mRNA and protein were dramatically induced in the skin (both epidermis and dermis) of SENCAR and C57BL/6 mice. TPA treatment dramatically increased mRNA levels of two other EGF receptor ligands, amphiregulin and heparin binding-EGF, however, in the skin of all three mouse lines. Comparison of histologic changes in skin revealed that wa-1 mice exhibited only modest sustained epidermal hyperplasia after multiple treatments with TPA, similar in magnitude to that of C57BL/6 mice and significantly lower than that of SENCAR mice. The current data indicate that wa-1 mice are relatively resistant to TPA promotion. Possible mechanisms for this resistance are discussed.     

Diminution of mouse epidermal superoxide dismutase and catalase activities by tumor promoters

The effects of phorbol ester tumor promoters and related compounds on superoxide dismutase (SOD) and catalase were examined. The treatment of adult mouse skin with 2 micrograms 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a sustained decrease in the basal levels of both SOD and catalase activities in the epidermis. A decline in SOD activity occurred within 3 h after application and the maximum effect was seen at 16--17 h. The decrease in SOD activity was always accompanied by a similar decline in the epidermal catalase activity. The alterations in both enzymes occurred against a high background of enhanced protein synthesis which indicates that the effect of TPA is selective for SOD and catalase. Other tumor promoters such as phorbol 12,13-dibutyrate and the non-phorbol tumor promoter anthraline also lowered the activities of both the enzymes. Mezerein, a resiniferonol derivative with weak promoting activity but a potent stage-II promoter, appeared to be more potent than TPA in lowering the basal levels. These results indicate that damage which favors neoplastic progression could occur in TPA-treated mouse skin due to the accumulation of free radicals resulting from low levels of SOD and catalase activity. In addition, the TPA-caused decrease in the levels of SOD and catalase was not prevented by either retinoic acid, fluocinolone acetonide, tosyl amino-2-phenylethyl chloromethyl ketone, or butylated hydroxytoluene, suggesting that inhibition of tumor promotion by these agents is not mediated through alterations in the levels of enzymatic activities which decrease free radical concentrations.     

PMA inhibits both spontaneous and glucocorticoid-mediated leptin secretion by human omental adipose tissue explants in vitro

The activation of PKC by the acute administration of the phorbol ester PMA (1 microM, 2h) to omental adipose tissue explants in vitro resulted in a marked (about 75%) and persistent (up to at least 96 h) inhibition of leptin secretion. This PKC-mediated inhibition was not observed after the administration of an inactive phorbol ester (phorbol 12,13-dicecanoate). The inhibition by PMA of leptin secretion was not restricted to the spontaneous secretion, but blocked also effectively the leptin response to a powerful stimulus, such as the glucocorticoid dexamethasone. As the PKC activity has been shown to be elevated during fasting, the negative relation here described between PKC activity and leptin secretion could be of physiological relevance.     

Usefulness of spoligotyping in molecular epidemiology of Mycobacterium bovis-related infections in South America

We studied the role of Ca++ and protein kinase C (PKC) in alpha-1A adrenergic receptor (AR)-mediated activation of mitogen-activated protein kinase pathways in PC12 cells. In PC12 cells stably transfected with the human alpha-1A AR, norepinephrine (NE) strongly activated both extracellular signal regulated kinases (ERKs) and c-jun-NH2-terminal kinases (JNK). Ten nanomolar thapsigargin (TG) increased cytoplasmic Ca++ at least as much as NE but did not activate ERKs or JNK. Higher concentrations of TG caused a small activation of ERKs but not JNK. Emptying [Ca++]i stores by pretreatment with TG prevented the NE-stimulated increase in [Ca++]i but not ERK or JNK activation. The Ca++ chelator bis(2-aminophenoxy)ethane-N-N-N'-N'-tetraacetate (BAPTA) dose dependently abolished NE-stimulated Ca++ responses but not ERK or JNK activation. NE increased tyrosine phosphorylation of Pyk2, and this response was neither blocked by BAPTA nor mimicked by TG. The phorbol ester tumor promoting agent (TPA) caused a dose-dependent activation of ERKs that was potentiated by 10 nM TG. TPA caused only a small activation of JNK relative to that caused by NE, which was not affected by TG. The potent PKC inhibitor bisindolylmaleimide I dose dependently inhibited ERK and JNK activation by TPA, but not NE. ATP and UTP activated similar mitogen-activated protein kinase responses through endogenous P2Y2 receptors, and these responses were not blocked by BAPTA or bisindolylmaleimide I, suggesting that these results may be generalizable to other Gq/11-coupled receptors. The results suggest that Ca++ release and PKC activation are neither necessary nor sufficient for alpha-1A AR-mediated activation of mitogenic responses in PC12 cells.     

Phorbol ester tumor promoters regulate insulin-like growth factor-binding protein-4 proteolysis

Cultured human fibroblasts secrete a specific protease that alters extracellular insulin-like growth factor-binding protein-4 (IGFBP-4) structure and function. This enzyme appears to be secreted in a latent form and requires IGFs for activation. To study regulation of the IGFBP-4 protease, we treated normal adult human fibroblasts with various hormones and growth regulatory factors, and collected the human fibroblast-conditioned medium (HFCM) for analysis of IGFBP-4 protease activity. The IGFBP-4 protease assay involved incubation of 50 microliters HFCM with or without 5 nM IGF-II for 6 h at 37 C under cell-free conditions; IGF-activated IGFBP-4 hydrolysis was assessed by Western ligand blotting. In HFCM from cells treated with vehicle, GH, insulin, epidermal growth factor, steroid hormones, or forskolin, IGF-II induced the select loss of detectable IGFBP-4 during the assay. In contrast, IGFBP-4 levels were maintained when HFCM from cells treated with phorbol ester tumor promoters was incubated with IGF-II under cell-free conditions. Hydrolysis of [125I]IGFBP-4 to 18,000 and 14,000 mol wt fragments also was prevented in HFCM from cells treated with phorbol esters. Phorbol esters had no effect on endogenous or exogenous IGFBP-4 proteolysis when added directly to HFCM during the assay, however. Treatment of cells with actinomycin-D or cycloheximide could prevent a phorbol ester-induced block of IGF-dependent IGFBP-4 proteolysis. These data suggest that phorbol ester tumor promoters stimulate human fibroblasts to produce and secrete an inhibitor of the IGFBP-4 proteolytic reaction. Alterations in IGFBP-4 protease activity could affect local IGF action through regulation of IGFBP-4 availability.     

The effects of phenobarbital and gonadal steroids on periovulatory serum levels of luteinizing hormone and follicle-stimulating hormone in the hamster

The occurrence of ovulation and serum levels of LH and FSH (measured by radioimmunoassay) were determined in periovulatory hamsters injected with an ovulation-blocking dose of phenobarbital (Phen) combined with progesterone (P), estradiol-17beta (E2), or testosterone (T). Proestrous hamsters were treated at 1300 h with Phen plus oil, P, P plus E2, E2, T, or a second injection of Phen at 2000 h. Each treatment group was divided into 3 subgroups, each of which was serially bled 4 times at 6 h intervals beginning at 1200, 1400, and 1600 h on proestrus. Phen blocked ovulation on the next morning in all animals, while treatments that included P (1 mg) restored the normal complement of ova in 65-75% of the animals. Neither E2 (1, 10 or 50 mug) nor T (0.1 or 1 mg) overcame the Phen block of ovulation. Control hamsters had peak levels of LH between 1400 and 1800 h and a biphasic release of FSH consisting of a peak at 1600 h on proestrus, a return to basal levels at 2200 h, and a second more sustained surge between 2400 and 0800 h on the morning of estrus. Phen completely depressed the proestrous surge of both gonadotropins but only partially inhibited the second FSH elevation on the morning of estrus. In ovulatory animals, P alone or combined with 1 or 10 mug E2 restored peak LH levels at 1600 h. FSH levels on proestrus in hamsters treated with Phen plus P peaked at 1800 h, while the addition of 1 mug E2 resulted in increased FSH levels at 1600 h; peak levels in both groups were about half of control values. No proestrous increase was detected in ovulatory animals treated with P and 10 mug E2. FSH levels on estrus in hamsters injected with P alone or in combination with E2 were intermediate between those of controls and animals given Phen only. Levels of LH and FSH in animals treated with a single or double dose of Phen or Phen plus E2 or T were not different during the periovulatory period.     

Dose-dependent prolactin releasing activity of arginine vasotocin in intact and pinealectomized estrogen-progesterone treated adult male rats

Estrogen-progesterone (EP) treated adult male rats were injected intravenously (iv) with 0.1, 1 or 10 mug arginine vasotocin (AVT) or Ringers lactate solution. A significant dose-related rise in plasma prolactin was evident 10 min after injection with AVT. In a second experiment, sham-operated or pinealectomized EP-treated male rats were injected iv with 0.1 or 1 mug AVT or diluent. Plasma prolactin was significantly elevated in both sham-operated and pinealectomized groups at both 10 and 20 min post-injection of 1 mug AVT. These results indicate that AVT has prolactin-releasing activity and that this activity is not dependent upon the presence of an intact pineal gland.     

Stimulation of human B lymphocytes by phorbol esters reported to be selective in the protein kinase C isoforms they activate

It is reported here that B cells can be stimulated by two phorbol esters which, in cell free substrate phosphorylation assays, are selective in the PKC isoforms they activate: thymeleatoxin (Thy) stimulates all of the classical (c) or Group A PKCs (alpha, beta 1, beta 2 and gamma) but not PKC delta and epsilon which belong to the novel (n) or Group B PKCs, while 12-deoxyphorbol-13-O-phenylacetate-20-acetate (dPPA) is a specific activator of PKC beta 1. By itself, phorbol 12-myristate, 13-acetate (PMA)--which activates all cPKC and nPKC--was, on a molar basis, some 40-times more potent than either Thy or dPPA which were themselves equipotent at promoting DNA synthesis in resting B cells: the peak response achieved with Thy and dPPA was higher (1.4 x) than that obtained with PMA. In the presence of calcium ionophore, PMA, Thy and dPPA all stimulated a higher (and equivalent) peak response which was achieved at a lower phorbol ester concentration in each case: however, whereas Thy now approached PMA in potency, dPPA remained some 40-times less potent.     

Regulation of RasGRP via a phorbol ester-responsive C1 domain

As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.