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.     

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.     

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.     

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.     

Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. I. Activation of protein kinase C and inhibition of epidermal growth factor binding

Addition of bombesin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an Mr 80,000 cellular protein (designated 80k). The effect was both concentration and time dependent; enhancement in 80k phosphorylation could be detected as early as 10 s after the addition of peptide. Recently, a rapid increase in the phosphorylation of an 80k cellular protein after treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact fibroblasts (Rozengurt, E., A. Rodriguez-Pena, and K. A. Smith, 1983, Proc. Natl. Acad. Sci. USA., 80:7244-7248; Rozengurt, E., A. Rodriguez-Pena, M. Coombs, and J. Sinnett-Smith, 1984, Proc. Natl. Acad. Sci. USA., 81:5748-5752). The 80k phosphoproteins generated in response to bombesin and to phorbol 12,13-dibutyrate were identical as judged by one- and two-dimensional PAGE and by peptide mapping after partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with phorbol 12,13-dibutyrate, which leads to the disappearance of protein kinase C activity, blocked the ability of bombesin to stimulate 80k. Bombesin also caused a rapid (1 min) inhibition of 125I-labeled epidermal growth factor (125I-EGF) binding to Swiss 3T3 cells. The inhibition was both concentration and temperature dependent and resulted from a marked decrease in the affinity of the EGF receptor for its ligand. Peptides structurally related to bombesin, including gastrin-releasing peptide, also stimulated 80k phosphorylation and inhibited 125I-EGF binding; both effects were selectively blocked by a novel bombesin antagonist. These results strongly suggest that these responses are mediated by specific high-affinity receptors that recognize the peptides of the bombesin family in Swiss 3T3 cells. While an increase in cytosolic Ca2+ concentration does not mediate the bombesin inhibition of 125I-EGF binding, the activation of protein kinase C in intact Swiss 3T3 cells by peptides of the bombesin family may lead to rapid inhibition of the binding of 125I-EGF to its cellular receptor.     

Bradykinin transiently activates protein kinase C in Swiss 3T3 cells. Distinction from activation by bombesin and vasopressin

The results presented here demonstrate that bradykinin, acting through a B2 subtype receptor, induces a unique pattern of early signals in quiescent Swiss 3T3 cells. Bradykinin caused a rapid mobilization of calcium from internal stores, as judged by measurements of intracellular Ca2+ concentration in fura-2-loaded cells and by 45Ca2+ efflux from radiolabeled cells. Analysis of phosphoproteins from 32P-labeled Swiss 3T3 cells by one- and two-dimensional gel electrophoresis revealed that bradykinin stimulated transient phosphorylation of an acidic cellular protein migrating with an apparent Mr = 80,000 (termed 80K), identified as a major and specific substrate of protein kinase C. Down-regulation of protein kinase C by pretreatment with phorbol 12,13-dibutyrate (PDBu) completely abolished the increase in 80K phosphorylation. In contrast to the sustained effect induced by bombesin, vasopressin, or PDBu, the stimulation of 80K phosphorylation by bradykinin reached a maximum after 1 min of incubation, and then it rapidly decreased to almost basal levels. Furthermore, bradykinin did not induce protein kinase C-mediated events such as inhibition of 125I-epidermal growth factor binding or enhancement of cAMP accumulation. Bombesin and vasopressin elicited both responses in parallel cultures. Bradykinin induced rapid accumulation of total inositol phosphates in cells labeled with myo-[3H]inositol. In contrast to bombesin and vasopressin which stimulated a linear increase in inositol phosphate accumulation over a 10-min period, the effect of bradykinin reached a plateau after 2.5 min of incubation with no further increase up to 10 min. The results demonstrate that the early signaling events triggered by bradykinin can be distinguished from those elicited by bombesin and vasopressin in Swiss 3T3 cells.     

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.     

Rapid dephosphorylation of a Mr 80,000 protein, a specific substrate of protein kinase C upon removal of phorbol esters, bombesin and vasopressin

The mitogens phorbol 12,13-dibutyrate, bombesin and vasopressin stimulate the phosphorylation of an acidic Mr 80,000 cellular protein, a specific substrate of protein kinase C, in intact Swiss 3T3 cells. Phosphorylation of this substrate was rapidly reversed upon the removal of each of these agents. Dephosphorylation occurred with a similar half-life in each of the cases studied (2.2, 1.5 and 2 minutes for phorbol 12,13-dibutyrate, bombesin and vasopressin respectively) and agreed closely with the dissociation of the ligands from their specific high-affinity binding sites in Swiss 3T3 cells.     

Stimulation of tyrosine kinase activity in anti-phosphotyrosine immune complexes of Swiss 3T3 cell lysates occurs rapidly after addition of bombesin, vasopressin, and endothelin to intact cells.

Treatment of quiescent Swiss 3T3 cells with the mitogenic peptides bombesin, vasopressin, endothelin/vasoactive intestinal contractor (VIC), and bradykinin strikingly increased the initial rate of tyrosine phosphorylation measured in anti-phosphotyrosine immunoprecipitates of a major band of Mr 115,000 (p115) and two minor components of Mr 90,000 and 75,000. Neuropeptides increased the labeling of p115 within seconds and with great potency; half-maximum concentrations were 0.1, 0.2 and 0.3 nM for bombesin, vasopressin, and VIC, respectively. Immunoblotting and peptide mapping showed that the p115 band phosphorylated in anti-phosphotyrosine immunoprecipitates is identical to a major Mr 115,000 substrate for neuropeptide-stimulated tyrosine phosphorylation in intact Swiss 3T3 cells. Furthermore, bombesin, vasopressin, and VIC markedly increased the rate of phosphorylation of Raytide, a broad specificity tyrosine kinase peptide substrate, by decreasing (8 +/- 1.3-fold) the apparent Km of the kinase for the substrate. Phorbol 12,13-dibutyrate and the Ca2+ ionophore A23187 had a weaker effect on tyrosine protein kinase activity in immune complexes compared with bombesin. Furthermore, down-regulation of protein kinase C blocked the small effect of phorbol esters but did not impair bombesin-stimulated tyrosine kinase activity. These results provide direct evidence for neuropeptide activation of a tyrosine kinase in cell-free preparations and identify a novel event in the action of this class of growth factors in Swiss 3T3 cells.     

Protein kinase D potentiates DNA synthesis and cell proliferation induced by bombesin, vasopressin, or phorbol esters in Swiss 3T3 cells

We examined whether protein kinase D (PKD) overexpression in Swiss 3T3 cells potentiates the proliferative response to either the G protein-coupled receptor agonists bombesin and vasopressin or the biologically active phorbol ester phorbol 12,13-dibutyrate (PDBu). In order to generate Swiss 3T3 cells stably overexpressing PKD, cultures of these cells were infected with retrovirus encoding murine PKD and green fluorescent protein (GFP) expressed as two separate proteins translated from the same mRNA. GFP was used as a marker for selection of PKD-positive cells. PKD overexpressed in Swiss 3T3 cells was dramatically activated by cell treatment with bombesin or PDBu as judged by in vitro kinase autophosphorylation assays and exogenous substrate phosphorylation. Concomitantly, these stimuli induced PKD phosphorylation at Ser(744), Ser(748), and Ser(916). PKD activation and phosphorylation were prevented by exposure of the cells to protein kinase C-specific inhibitors. Addition of bombesin, vasopressin, or PDBu to cultures of Swiss 3T3 cells overexpressing PKD induced a striking increase in DNA synthesis and cell number compared with cultures of Swiss 3T3-GFP cells. In contrast, stimulation of DNA synthesis in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not enhanced. Our results demonstrate that overexpression of PKD selectively potentiates mitogenesis induced by bombesin, vasopressin, or PDBu in Swiss 3T3 cells.     

Bombesin, diacylglycerols, and phorbol esters rapidly stimulate the phosphorylation of an Mr = 80,000 protein kinase C substrate in permeabilized 3T3 cells. Effect of guanine nucleotides

We have used digitonin permeabilization to study the mechanism of bombesin-induced activation of protein kinase C in Swiss 3T3 cells. Protein kinase C-mediated phosphorylations in permeabilized cells were identified using phorbol esters and diacylglycerols. Addition of phorbol 12,13-dibutyrate (PDBu) in the presence of [gamma-32P]ATP and digitonin caused a marked and rapid time- and dose-dependent increase in the phosphorylation of an Mr 80,000 cellular protein (maximum stimulation = 12.6 +/- 1.6-fold after 1 min, EC50 = 27 nM). 1-oleoyl-2-acetylglycerol substituted for PDBu in stimulating the phosphorylation of Mr 80,000 protein (EC50 = 13 microM). Bombesin also caused a striking increase in the phosphorylation of Mr 80,000 protein with a time course similar to that observed with PDBu. This phosphorylation was mimicked by mammalian bombesin-like peptides and blocked by the bombesin antagonists [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P and [Leu13 psi (CH2NH)Leu14]bombesin. Down-regulation of protein kinase C in intact cells by prolonged exposure to PDBu prevented Mr 80,000 protein phosphorylation upon subsequent bombesin addition in digitonin-permeabilized cells. Comigration on one- and two-dimensional gel electrophoresis and phosphopeptide mapping confirmed that the Mr 80,000 protein phosphorylated in permeabilized cells was indistinguishable from the Mr 80,000 protein which is the major protein kinase C substrate in intact cells. The GDP analogue guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) caused a 70% inhibition of the bombesin-induced phosphorylation of Mr 80,000 protein but had no effect on the phosphorylation induced by PDBu. Bombesin stimulated Mr 80,000 protein phosphorylation in permeabilized cells in a dose-dependent manner (EC50 = 4 nM), and GDP beta S shifted the bombesin dose response curve to higher bombesin concentrations (EC50 = 14 nM). These results demonstrate for the first time a growth factor receptor-mediated activation of protein kinase C in permeabilized cells and provide functional evidence for the involvement of a G protein in the transmembrane signaling pathway that mediates the stimulation of protein kinase C by bombesin in Swiss 3T3 cells.     

Bombesin enhancement of cAMP accumulation in Swiss 3T3 cells: evidence of a dual mechanism of action

Addition of bombesin in the presence of either forskolin or cholera toxin caused a marked (4-6 fold) enhancement of cAMP accumulation in Swiss 3T3 cells. This effect was time and concentration dependent, induced by various bombesin-like peptides and blocked by a bombesin antagonist. Enhancement of cAMP accumulation by bombesin was diminished by chronic pretreatment with phorbol dibutyrate implicating the involvement of protein kinase C in the activation. Pretreatment with pertussis toxin, which uncouples protein kinase C activation from cAMP accumulation (Proc. Natl. Acad. Sci. U.S.A., 84:2282, 1987) also inhibited bombesin enhancement of cAMP. Bombesin was also shown to release E type prostaglandins into the medium, an effect which was abolished by the cyclooxygenase inhibitor indomethacin. Low concentrations (100 nM) of indomethacin partially inhibited the accumulation of cAMP by bombesin in the presence of forskolin indicating that the release of E type prostaglandins into the medium is also involved in the accumulation of cAMP by bombesin. The additive nature of PBt2-mediated down-regulation and treatment with indomethacin suggests that activation of protein kinase C and the release of E type prostaglandins provide two distinct pathways involved in the enhancement of cAMP accumulation by bombesin. Finally, bombesin in the presence of forskolin stimulated the phosphorylation of the intermediate filament component vimentin, identified in the accompanying paper as a substrate for a cAMP dependent protein kinase in intact Swiss 3T3 cells.     

Fibroblast growth factor stimulates protein kinase C in quiescent 3T3 cells without Ca2+ mobilization or inositol phosphate accumulation

To elucidate the transmembrane signalling processes initiated by fibroblast growth factor (FGF), we have studied the effect of recombinant basic FGF (bFGF) on various early events associated with mitogenesis in Swiss 3T3 fibroblasts. bFGF, at mitogenic concentrations, neither induced Ca2+ mobilization from intracellular stores nor increased the accumulation of inositol phosphates. In contrast, bFGF stimulated the phosphorylation of the Mr 80,000 (80K) cellular protein which is a major substrate of protein kinase C. This effect was potentiated by the diacylglycerol kinase inhibitor R59022. Two-dimensional polyacrylamide gel electrophoresis and phosphopeptide mapping showed that the 80K phosphoproteins generated in response to bFGF, bombesin, and phorbol 12,13-dibutyrate were indistinguishable. Down-regulation of protein kinase C prevented bFGF stimulation of 80K phosphorylation. Other protein kinase C-dependent early events such as transmodulation of the epidermal growth factor receptor, cytoplasmic alkalinization, inhibition of vasopressin induced increase in cytosolic [Ca2+], and enhancement of cAMP accumulation in response to forskolin were also induced by bFGF. Similar results were obtained when bFGF was added to quiescent cultures of tertiary mouse embryo fibroblasts. We conclude that bFGF stimulates protein kinase C through a signal transduction pathway distinct from inositol phospholipid turnover and Ca2+ mobilization.     

Stimulation of Na+/H+ antiport activity by epidermal growth factor and insulin occurs without activation of protein kinase C

Addition of phorbol 12, 13 dibutyrate (PBt2) or a combination of epidermal growth factor (EGF) and insulin to quiescent cultures of Swiss 3T3 cells increased intracellular pH in a Na+-dependent fashion. In contrast to PBt2, EGF plus insulin failed to stimulate protein kinase C and elicited the ionic response in cells lacking this enzyme. We suggest that the stimulation of the Na+/H+ antiport in Swiss 3T3 cells is mediated by at least two separate pathways, only one of which is dependent upon the activation of protein kinase C.     

Phorbol ester-induced translocation of diacylglycerol kinase from the cytosol to the membrane in Swiss 3T3 fibroblasts

The tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, causes a rapid, partial redistribution of 1,2-sn-diacylglycerol kinase from the cytosol to the particulate fraction of quiescent, starved Swiss 3T3 fibroblasts. We utilized exogenous dioleoylglycerol as substrate for the kinase. The inactive alpha form of the phorbol ester does not cause any change in diacylglycerol kinase localization, and depletion of protein kinase C (Ca2+/phospholipid-dependent enzyme) by chronic administration of phorbol ester blocks the redistribution. Phorbol ester has no direct effect on Swiss 3T3 membrane-bound diacylglycerol kinase nor does it directly effect cytosolic diacylglycerol kinase. When phorbol ester is added to Swiss 3T3 membranes in the presence of ATP, magnesium, and calcium, there is no activation of membrane-bound kinase, indicating that phorbol ester does not activate membrane-bound kinase through phosphorylation by protein kinase C. Reconstitution studies show that the soluble rat brain diacylglycerol kinase binds to diacylglycerol-enriched membranes, produced by treatment of red cell ghosts with phospholipase C or calcium, suggesting that cytosolic diacylglycerol kinase may be capable of translocation to the membrane in response to elevated substrate concentration in the intact cell. Stimulation of the cells with phorbol ester increases the total mass of diacylglycerol. In protein kinase C-depleted cells, addition of a cell-permeable synthetic diacylglycerol, dioctanoylglycerol, results in a partial redistribution of cytosolic diacylglycerol kinase to the membrane, by 5 min, also suggesting that the translocation of diacylglycerol kinase activity is regulated primarily by substrate concentration.     

Protein kinase D potentiates DNA synthesis induced by Gq-coupled receptors by increasing the duration of ERK signaling in swiss 3T3 cells

Protein kinase D (PKD) potentiates cellular DNA synthesis in response to G protein-coupled receptor (GPCR) agonists but the mechanism(s) involved has not been elucidated. Here, we examined whether PKD overexpression in Swiss 3T3 cells regulates the activation/inactivation kinetics of the extracellular-regulated protein kinase (ERK) in response to the mitogenic GPCR agonists bombesin and vasopressin. Addition of bombesin or vasopressin to Swiss 3T3 cells overexpressing PKD induced a striking increase in the duration of MEK/ERK/RSK activation as compared with cultures of either control Swiss 3T3 cells or Swiss 3T3 cells expressing a kinase-inactive PKD mutant. In contrast, the duration of ERK activation in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not increased. Furthermore, bombesin or vasopressin promoted a striking increase in phosphorylation (at Ser-374) and accumulation of c-Fos (the c-fos proto-oncogene product) in Swiss 3T3 cells overexpressing wild-type (but not kinase-inactive) PKD. Inhibition of the sustained phase of ERK/RSK activation abrogated the increase in c-Fos accumulation and DNA synthesis induced by bombesin or vasopressin in PKD-overexpressing cells. Our results demonstrate that PKD selectively potentiates mitogenesis induced by bombesin or vasopressin in Swiss 3T3 cells by increasing the duration of MEK/ERK/RSK signaling.     

Stimulation of mitogenesis and glucose transport by 1-monooleoylglycerol in Swiss 3T3 fibroblasts

1-Monooleoylglycerol (MOG), a recently reported diacylglycerol kinase inhibitor (Bishop, W. R., Ganong, B. R., and Bell, R. M. (1986) J. Biol. Chem. 261, 6993-7000), exerts potent stimulatory effects on [3H]thymidine incorporation into DNA and glucose transport in Swiss 3T3 fibroblasts. MOG induces a rapid and sustained 2.5-fold increase in the cellular 1,2-diacylglycerol (1,2-DG) content, and phosphorylation of an acidic 80-kDa protein, a putative substrate for the protein kinase C (Ca2+/phospholipid-dependent protein kinase). The effect of MOG is additive to that of bombesin in terms of both an increase in tissue diacylglycerol content and phosphorylation of the 80-kDa proteins. In addition to these effects, MOG potently stimulates release of arachidonic acid from phospholipids. Inhibitors of cyclooxygenase and lipoxygenase have little effect, if any, on MOG-induced stimulation of glucose transport and DNA synthesis, while exogenously applied arachidonic readily stimulates both of these cellular responses. Furthermore, arachidonic acid, at its biologically active concentrations, is found to induce a rapid and sustained increase in cellular 1,2-DG content and stimulate the phosphorylation of the 80-kDa protein, although to a lesser extent than MOG. Prolonged pretreatment of the cells with phorbol 12,13-dibutyrate, which reduces the cellular protein kinase C content, markedly attenuates the effects of both MOG and arachidonic acid on glucose transport and DNA synthesis. These data indicate that MOG increases endogenous 1,2-DG content and thereby acts as a potent activator of protein kinase C, and that activation of protein kinase C is a crucial step in MOG-induced stimulation of mitogenesis and glucose transport.     

A novel vasoactive peptide endothelin stimulates mitogenesis through inositol lipid turnover in Swiss 3T3 fibroblasts

Endothelin, a novel vasoactive peptide derived from endothelial cells (Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. (1988) Nature 332, 411-415), acts as a potent mitogen in Swiss 3T3 fibroblasts. The effect is dose-dependent with a half-maximal effect obtained at approximately 3 x 10(-11) M and is synergistically enhanced by a low concentration of insulin-like growth factor-I. Endothelin specifically binds to a single class of high affinity receptors in intact Swiss 3T3 cells and stimulates phospholipase C with the production of second messengers inositol trisphosphate and 1,2-diacylglycerol, leading to biphasic increases in the intracellular free Ca2+ concentration, as measured with a fluorescent indicator fura-2, phosphorylation of a putative cellular substrate of 80 kDa for protein kinase C, and transient expression of cellular protoonocogenes, c-fos and c-myc. Mitogenic effect of endothelin is markedly attenuated in phorbol ester-pretreated, protein kinase C-depleted cells. Endothelin-induced inositol phosphates production is not affected by removal of extracellular Ca2+, suggesting that endothelin-induced phospholipase C activation is not the result of stimulation of Ca2+ influx across the plasma membrane. These composite results indicate that the inositol lipid signaling pathway plays an important role in endothelin-induced mitogenesis in Swiss 3T3 fibroblasts. The mitogenic effect of endothelin is considerably smaller than that of bombesin, another well characterized mitogen acting through the inositol lipid pathway, despite comparable potencies in eliciting initial second messenger signals. In endothelin-treated cells, an increase in cellular 1,2-diacylglycerol content is transient, and cellular cyclic AMP content is reduced. By contrast, bombesin induces a more prolonged increase in cellular 1,2-diacylglycerol content and a slight increase in cellular cyclic AMP content. Because both 1,2-diacylglycerol and cyclic AMP are thought to serve as signals for promoting DNA synthesis in Swiss 3T3 fibroblasts, these differences in the signal generation may contribute to the differences in potencies between the two mitogens.     

Translocation of diacylglycerol kinase from the cytosol to the membrane in phorbol ester-treated Swiss 3T3 fibroblasts

The tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, causes a rapid, partial redistribution of 1,2-diacylglycerol kinase from the cytosol to the particulate fraction of quiescent Swiss 3T3 fibroblasts. The inactive alpha form of the phorbol ester does not cause any change in diacylglycerol kinase localization, and depletion of protein kinase C by chronic administration of phorbol ester blocks the redistribution. Phorbol ester has no direct effect on membrane-bound diacylglycerol kinase in 3T3 cells. When phorbol ester is added to 3T3 membranes in the presence of ATP, Mg2+, and Ca2+, there is no activation of membrane-bound kinase, indicating that phorbol ester does not activate membrane-bound kinase through phosphorylation by protein kinase C. Stimulation of the cells with phorbol ester increases the total mass of diacylglycerol. In protein kinase C-depleted cells, addition of a cell-permeable synthetic diacylglycerol, dioctanoylglycerol, results in a partial redistribution of cytosolic diacylglycerol kinase to the membrane, also suggesting that the translocation of DAG kinase is regulated primarily by substrate concentration.     

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.