Acetylcholine receptor-associated 43K protein contains covalently bound myristate

Torpedo electroplaque and vertebrate neuromuscular junctions contain high levels of a nonactin, 43,000-Mr peripheral membrane protein referred to as the 43K protein. 43K protein is associated with the cytoplasmic face of postsynaptic membranes at areas of high acetylcholine receptor density and has been implicated in the establishment and/or maintenance of these receptor clusters. Cloning of cDNAs encoding Torpedo 43K protein revealed that its amino terminus contains a consensus sequence sufficient for the covalent attachment of the rare fatty acid myristate. To examine whether 43K protein is, in fact, myristoylated, mouse muscle BC3H1 cells were metabolically labeled with either [35S]cysteine or [3H]myristate and immunoprecipitated with a monospecific antiserum raised against isolated Torpedo 43K protein. In cells incubated with either precursor, a single labeled species was specifically recovered that comigrated on SDS-PAGE with 43K protein purified from Torpedo electric organ. Approximately 95% of the 3H labeled material released from [3H]myristate-43K protein by acid methanolysis was extractable in organic solvents and eluted from a C18 reverse-phase HPLC column exclusively at the position of the methyl myristate internal standard. Thus, 43K protein contains authentic myristic acid rather than an amino or fatty acid metabolite of [3H]myristate. Myristate appears to be added to 43K protein cotranslationally and cannot be released from it by prolonged incubation in SDS, 2-mercaptoethanol, or hydroxylamine (pH 7.0 or 10.0), characteristics consistent with amino terminal myristoylation. Covalently linked myristate may be responsible for the high affinity of purified 43K protein for lipid bilayers despite the absence of a notably hydrophobic amino acid sequence.     

N-myristoylation of the rod outer segment G protein, transducin, in cultured retinas.

Bovine retinas incubated with [3H]myristic acid incorporated detectable radiolabel into only a few proteins. The most heavily labeled was the alpha subunit of the rod outer segment G protein transducin (Gt alpha). The radiolabeled protein was specifically eluted from illuminated membranes in the presence of GTP, displaying the unique solubility properties of Gt alpha. It comigrated with Gt alpha in electrophoresis and chromatography and was immunoprecipitated by Gt alpha-specific antibodies. The radiolabel was confirmed by hydrolysis, chemical derivatization, and chromatography to be amide-linked myristic acid. The solubility of the myristoylated Gt alpha indicates that myristoylation is not sufficient to cause tight membrane association of this normally membrane-bound subunit. Incorporation of [3H]myristate was blocked by the protein synthesis inhibitor cycloheximide, suggesting that that fatty acid group is introduced during or soon after translation in the rod inner segment.     

Myristoylation of neutrophil proteins and their biological characteristics

We have studied protein acylation in neutrophils of guinea pigs using [3H]myristate. A large number of neutrophil proteins were acylated with exogenously added myristic acid. The myristoylation was detected on 110, 77, 56, 54, 52, 42, and 37 kDa proteins. These myristoylations were stronger in peripheral blood than in peritoneal cells. Myristic acid was found to be covalently linked by an amid bond to these proteins since the proteins were resistant to boiling, chloroform/methanol and hydroxylamine treatment. Most myristoylated proteins appeared to be associated with the membrane fraction, while some of the proteins such as 77 kDa one was distributed also in the cytoplasm and translocated from the cytoplasm to the plasma membrane by stimulation. Lysozyme was myristoylated in vitro by the N-hydroxysuccinimide ester of myristic acid. The myristoylated lysozyme had an ability to be associated with phospholipid liposomes, and the membrane-associated lysozyme became a substrate of the rat brain Ca2+- and phospholipid dependent protein kinase (protein kinase C). These results indicate that myristoylation in neutrophil proteins may have an important role in metabolic regulation through their membrane association.     

The majority of cellular fatty acid acylated proteins are localized to the cytoplasmic surface of the plasma membrane

The BC3Hl muscle cell line was previously reported to contain a broad array of fatty acid acylated proteins [Olson, E. N., Towler, D. A., & Glaser, L. (1985) J. Biol. Chem. 260, 3784-3790]. Palmitate was shown to be attached to membrane proteins posttranslationally through thiol ester linkages, whereas myristate was attached cotranslationally, or within seconds thereafter, to soluble and membrane-bound proteins through amide linkages [Olson, E. N., & Spizz, G. (1986) J. Biol. Chem. 261, 2458-2466]. The temporal and subcellular differences between palmitate and myristate acylation suggested that these two classes of acyl proteins might follow different intracellular pathways to distinct subcellular membrane systems or organelles. In this study, we examined the subcellular localization of the major fatty acylated proteins in BC3Hl cells. Palmitate-containing proteins were localized to the plasma membrane, but only a subset of myristate-containing proteins was localized to this membrane fraction. The majority of acyl proteins were nonglycosylated and resistant to digestion with extracellular proteases, suggesting that they were not exposed to the external surface of the plasma membrane. Many proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins face the cytoplasm. Two-dimensional gel electrophoresis of proteins labeled with [3H]palmitate and [3H]myristate revealed that individual proteins were modified by only one of the two fatty acids and did not undergo both N-linked myristylation and ester-linked palmitylation. Together, these results suggest that the majority of cellular acyl proteins are routed to the cytoplasmic surface of the plasma membrane, and they raise the possibility that fatty acid acylation may play a role in intracellular sorting of nontransmembranous, nonglycosylated membrane proteins.     

Dissociation of Phosphorylation and Translocation of a Myristoylated Protein Kinase C Substrate (MARCKS Protein) in C6 Glioma and N1E-115 Neuroblastoma Cells

Abstract: An 80-kDa protein labeled with [³H]myristic acid in C6 glioma and N1E-115 neuroblastoma cells has been identified as the myristoylated alanine-rich C kinase substrate (MARCKS protein) on the basis of its calmodulin-binding, acidic nature, heat stability, and immunochemical properties. When C6 cells preincubated with [³H]myristate were treated with 200 n M 4β-12- O -tetradecanoylphorbol 13-acetate (β-TPA), labeled MARCKS was rapidly increased in the soluble digitonin fraction (maximal, fivefold at 10 min) with a concomitant decrease in the Triton X-100–soluble membrane fraction. However, phosphorylation of this protein was increased in the presence of β-TPA to a similar extent in both fractions (maximal, fourfold at 30 min). In contrast, β-TPA–stimulated phosphorylation of MARCKS in N1E-115 cells was confined to the membrane fraction only and no change in the distribution of the myristoylated protein was noted relative to α-TPA controls. These results indicate that although phosphorylation of MARCKS by protein kinase C occurs in both cell lines, it is not directly associated with translocation from membrane to cytosol, which occurs in C6 cells only. The cell-specific translocation of MARCKS appears to correlate with previously demonstrated differential effects of phorbol esters on stimulation of phosphatidylcholine turnover in these two cell lines.     

Ubenimex (Bestatin), an aminopeptidase inhibitor, modulates protein kinase C in K562 cells.

Ubenimex (Bestatin) is a potent inhibitor of aminopeptidases (APase) including APase N (EC 3.4.11.2), a widely distributed membrane-bound metalloprotease. Binding of Ubenimex (UBX) to cells has been implicated in a variety of its biological activities, while little evidence has yet been provided as to any subsequent mechanisms of intracellular signal transduction. We now examined the possible involvement of protein kinase C (PKC), a key regulator in transmembrane signaling. Human leukemia K562 cells were cultured in the presence or absence of UBX (1 to 50 micrograms.ml-1, 1 to 72 h), and the subcellular distribution as well as phorbol-12, 13-dibutyrate (PDBu)-induced redistribution of PKC activities were assessed. The membrane-bound enzymatic activity tended to increase in the presence of UBX, while a significant loss of the activity was demonstrable upon subsequent exposure to PDBu (100 nM, 10 min) in both the cytosolic and membrane fractions. Specific binding of [3H]PDBu to intact K562 cells was also down-modulated with UBX concentration- and time-dependently, suggesting loss of PKC enzyme protein on the cell surface. Western blot analysis of the total cell extracts disclosed no appreciable alteration in the amount of PKC protein. APase inhibition with UBX was observable independently of PKC modulation. The present findings were discussed with reference to the possible differential mechanisms of PKC-mediated regulation of cellular responses depending on cell types.     

Myristoylation of flagellar creatine kinase in the sperm phosphocreatine shuttle is linked to its membrane association properties.

TCK, the flagellar creatine kinase (ATP:creatine N-phosphotransferase) of sperm from the sea urchin Strongylocentrotus purpuratus is a membrane-associated lipophilic protein involved in energy transport. The cDNA derived protein sequence contains a consensus site sufficient for the covalent attachment of myristate. To examine whether TCK was myristoylated, mouse fibroblast Swiss 3T3 and baby hamster kidney cell lines were transfected with a cDNA encoding the entire TCK protein linked to a metallothionein promotor. TCK expression was induced by zinc and paralleled by incorporation of [3H]myristic acid derived label into the protein. 3H Label incorporated into TCK was resistant to hydroxylamine treatment. The 3H-labeled material released from TCK by acid methanolysis eluted from a C18 reverse phase high pressure liquid chromatography column at the positions of myristic acid and methylmyristate. Thus, TCK expressed in transfected mammalian cell lines contains authentic myristic acid, covalently attached through amide linkage. [3H]Myristoyl TCK comigrated on two-dimensional gels with the purified lipophilic isoform TCK II from sea urchins. Furthermore, like TCK II, [3H]myristoyl TCK associated with phospholipid liposomes, suggesting that myristoylation may mediate the observed membrane association of TCK. Myristoylation of sea urchin sperm flagellar creatine kinase may play a role in confining this enzyme to the flagellum during spermatogenesis.     

Cell-specific fatty acylation of proteins in cultured cells of neuronal and glial origin

Distinct sets of cellular proteins were labeled with [³H]myristic and [³H]palmitic acids in primary (rat neurons and astroglia) and continuous (murine N1E-115 neuroblastoma and rat C6 glioma) cell cultures derived from the nervous system. Both soluble and membrane proteins were modified by myristate in a hydroxylamine-stable (amide) linkage, while palmitoylated proteins were esterlinked and almost exclusively membrane bound. Chain elongation of both labeled fatty acids prior to acylation was observed, but no protein amide-liked [³H]myristate originating from [³H]palmitate was detected. Fatty acylation profiles differed considerably among most of the cell lines, except for rat astroglial and glioma cells in which myristoylated proteins appeared to be almost identical based on SDS gel electrophoresis. An unidentified 47 kDa myristoylated protein was labeled to a significantly greater extent in astroglial than in glioma cells; the expression of this protein could be related to transformation or development in cells of glial origin.     

Phorbol ester-stimulated phosphorylation of keratinocyte transglutaminase in the membrane anchorage region.

The membrane-bound transglutaminase of cultured keratinocytes became radioactively labelled upon addition of [32P]Pi to the medium. Transglutaminase phosphorylation was also demonstrable using particulate material isolated from cell homogenates. Compatible with mediation of the labelling by protein kinase C, the degree of phosphorylation in intact cells was stimulated approx. 5-fold in 4 h on treatment with the tumour-promoting phorbol ester phorbol 12-myristate 13-acetate, but not by phorbol. The extent of labelling was virtually unaffected by cycloheximide inhibition of protein synthesis, indicating that it arose primarily through turnover of phosphate in the membrane-bound enzyme. Phosphoamino acid analysis detected labelling only of serine residues. Most of the label was removed by trypsin release of the enzyme from the particulate fraction of cell homogenates, which deletes a membrane anchorage region of approximately 10 kDa. Upon trypsin treatment of the enzyme after immunoprecipitation, the phosphate label was recovered in soluble peptide material with a size of several thousand Da or less. Indicative of fragmentation of the membrane anchorage region, this material was separable by h.p.l.c. into two equally labelled peptides. Moreover, when the enzyme was labelled with [3H]palmitate or [3H]myristate, the fatty-acid-labelled peptide material required non-ionic detergent for solubilization and was separable from the phosphate-labelled material by gel filtration. Phorbol ester treatment of cultured keratinocytes in high- or low- Ca2(+)-containing medium was not accompanied by an appreciable protein-synthesis-independent change in transglutaminase activity. Independent of possible alteration of the intrinsic catalytic activity of the enzyme, phosphorylation may well modulate its interaction with substrate proteins, a potential site for physiological regulation.     

The cytosolic phorboid receptor correlates with hormone dependency in six mammary carcinoma cell lines

Potent, structurally different tumor promoters inhibited growth of 6 human mammary carcinoma cell lines (ROOS et al, PNAS in press). This growth inhibition was investigated by measuring the phorboid receptor binding using [3H] PDBu (4 beta-phorbol 12, 13 dibutyrate). Specific, high affinity receptors were found in all six cell lines. [3H] PDBu binding affinities were higher in the cytosolic fractions than in the corresponding intact cells (K alpha = app. 1nM vs K alpha = app. 15nM). The hormone-independent cell lines (BT-20, HBL-100 and MDA-MB-231) exhibited significantly higher levels of cytosolic [3H] PDBu receptors than the hormone-dependent cells (MCF-7, T-47-D and ZR-75-1). The subcellular distribution of the [3H] PDBu binding correlated well with the distribution of the protein kinase C activity (r = 0.95).     

The lipid binding, regulatory domain of protein kinase C. A 32-kDa fragment contains the calcium- and phosphatidylserine-dependent phorbol diester binding activity

Trypsinization of rat brain protein kinase C (80 kDa) into 50- and 32-kDa fragments occurred without inhibition of [3H]phorbol dibutyrate ([3H]PDBu) binding activity. The 50-kDa fragment, the catalytic domain (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616), was further degraded by trypsin, whereas the 32-kDa fragment was resistant. Protein kinase activity and the [3H]PDBu binding activity were completely separated upon gel filtration of a solution containing Triton X-100/phosphatidylserine mixed micelles and trypsinized protein kinase C. Pooled fractions of the [3H]PDBu binding activity contained a 32-kDa fragment exclusively. The binding of [3H]PDBu to this fragment was dependent on calcium and phosphatidylserine and was of high affinity (Kd = 2.8 nM) and of essentially identical specificity to that of native protein kinase C. It is concluded that the 32-kDa fragment represents a lipid binding, regulatory domain of protein kinase C.     

An Arabidopsis calcium-dependent protein kinase is associated with the endoplasmic reticulum

Arabidopsis contains 34 genes that are predicted to encode calcium-dependent protein kinases (CDPKs). CDPK enzymatic activity previously has been detected in many locations in plant cells, including the cytosol, the cytoskeleton, and the membrane fraction. However, little is known about the subcellular locations of individual CDPKs or the mechanisms involved in targeting them to those locations. We investigated the subcellular location of one Arabidopsis CDPK, AtCPK2, in detail. Membrane-associated AtCPK2 did not partition with the plasma membrane in a two-phase system. Sucrose gradient fractionation of microsomes demonstrated that AtCPK2 was associated with the endoplasmic reticulum (ER). AtCPK2 does not contain transmembrane domains or known ER-targeting signals, but does have predicted amino-terminal acylation sites. AtCPK2 was myristoylated in a cell-free extract and myristoylation was prevented by converting the glycine at the proposed site of myristate attachment to alanine (G2A). In plants, the G2A mutation decreased AtCPK2 membrane association by approximately 50%. A recombinant protein, consisting of the first 10 amino acids of AtCPK2 fused to the amino-terminus of beta-glucuronidase, was also targeted to the ER, indicating that the amino terminus of AtCPK2 can specify ER localization of a soluble protein. These results indicate that AtCPK2 is localized to the ER, that myristoylation is likely to be involved in the membrane association of AtCPK2, and that the amino terminal region of AtCPK2 is sufficient for correct membrane targeting.     

Protein kinase C activation patterns are determined by methodological variations. Studies of insulin action in BC3H-1 myocytes and rat adipose tissue

In BC3H-1 myocytes, insulin has been reported to (a) increase diacyglycerol (DAG) production and provoke increases in protein kinase C enzyme activity of crude or DEAE-Sephacel-purified cytosol and membrane fractions in BC3H-1 myocytes (Cooper et al. (1987) J. Biol. Chem. 262, 3633-3739), but (b) decrease cytosolic, and transiently increase membrane, immunoreactive protein kinase C (Acevedo-Duncan et al. (1989) FEBS Lett. 244, 174-176). Presently, we used a Mono-Q column to purify protein kinase C and found that, similar to immunoblot findings, enzyme activity decreased in the cytosol, and increased in the membrane during insulin treatment. Similar differences in protein kinase C activation patterns were observed in rat adipose tissue: insulin stimulated cytosolic protein kinase C enzyme activity as measured after DEAE-Sephacel chromatography, but decreased cytosolic enzyme activity when measured after Mono-Q chromatography or by immunoblotting. We presently evaluated the possibility that insulin-induced increases in endogenous DAG may influence protein kinase C during assay in vitro. Crude cytosol from BC3H-1 myocytes contained 25-35% of total and [3H]glycerol-labelled DAG and insulin increased this DAG. Considerable amounts of [3H]glycerol-labelled DAG were present in insulin-stimulated protein kinase C-containing column fractions following DEAE-Sephacel chromatography of cytosol fractions, whereas lesser amounts were recovered after Mono-Q column chromatography. This difference in recovery of DAG and activation of the enzyme by this endogenous DAG may explain why we were able to discern insulin-induced (presumably translocation 'provoked') decreases in cytosolic protein kinase C in the present Mono-Q column preparations of both BC3H-1 myocytes and rat adipose tissue.     

Baculovirus expression of mammalian G protein alpha subunits.

Complementary DNAs encoding three subtypes of the alpha subunit (alpha i-1, alpha o and alpha s) of rat guanyl nucleotide regulatory proteins were used to construct recombinant baculoviruses which direct high-level expression of the corresponding proteins in cultured Sf9 insect cells. The expressed proteins were recognized by polyclonal antisera specific for the different alpha chains, and co-migrated with the native proteins from rat brain membranes in immunoblotting analyses. Soluble and particulate forms of all three immunoreactive alpha chains were observed following ultracentrifugation of cell lysates. Biosynthetic radiolabelling of infected cells with [35S]methionine or [3H]myristate showed that both soluble and particulate forms of alpha i-1 and alpha o were myristoylated; in contrast, alpha s did not incorporate myristate. The soluble fractions from cells expressing alpha chains showed high levels of GTP-binding activity over that observed in uninfected cells, or in cells infected with wild-type virus. The peak expression levels observed at 72 h post-infection were highest for alpha o at ca. 400 pmol of GTP-gamma-35S/mg protein, or roughly 2% of the total soluble protein. The results of this work show that the baculovirus system can be employed for high-level production of mammalian G protein alpha chains which retain GTP-binding activity and are appropriately modified by myristoylation.     

Redistribution of phospholipid/Ca2+-dependent protein kinase in mast cells activated by various agonists

Three types of agonists; receptor-mediated concanavalin A), direct (phorbol ester), and membrane-perturbing (compound 48/80), elicit histamine secretion from rat peritoneal mast cells. We tested whether activation of the mast cells by these agents is accompanied by subcellular redistribution of protein kinase C. Phorbol ester treatment predictably caused a profound decrease of phospholipid/Ca2+-dependent histone kinase activity in the cytosol and a concomitant increase of [3H]PMA-binding capacity in the membrane fraction, in a time- and concentration-dependent manner. Similar, but less marked effects were observed with stimulations by concanavalin A and compound 48/80. When mast cells labeled with [32P] and then stimulated with the agents, phosphorylation of a 50,000-Dalton protein was enhanced in the membrane fraction. These results suggest that protein kinase C may play a role in mast cell activation through phosphorylation of the membrane protein.     

A putative myristoylated 2C-type protein phosphatase, PP2C74, interacts with SnRK1 in Arabidopsis

N-myristoylation is a lipid modification of many signaling proteins in which myristate is added to an N-terminal glycine residue. Here we show that PP2C74, a putative myristoylated 2C-type protein phosphatase (PP2C) in Arabidopsis, is transcribed in various tissues and has protein phosphatase activity. GFP-fused PP2C74 localized to the plasma membrane, but not when a glycine residue at position 2, which is the putative myristoylation site, was substituted with an alanine residue. Yeast two-hybrid analysis and GST pull-down analysis showed that PP2C74 interacts with AKIN10, the catalytic α subunit of the SnRK1 protein kinase complex, the β subunits of which are known targets of myristoylation.     

The Pto kinase of tomato, which regulates plant immunity, is repressed by its myristoylated N terminus

Specific recognition of the Pseudomonas syringae effector proteins AvrPto and AvrPtoB in tomato is mediated by Pto kinase resulting in induction of defense responses, including hypersensitive cell death via a signaling pathway requiring the nucleotide-binding leucine-rich repeats protein Prf. Pto is a myristoylated protein, and N-myristoylation is required for signaling. Here we demonstrated a role for N-myristoylation in controlling Pto kinase activity. A myristoylated peptide corresponding to Pto residues 2-10 significantly impaired the kinase activity of N-truncated Pto. We show that kinase inhibition was specific to the myristoylated form of the peptide and that free myristate supplied in trans was a potent suppressor of Pto kinase activity. Thus, myristate, but not Pto residues 2-10, contributes to suppression of kinase activity in vitro. Accordingly, elimination of the in vivo myristoylation potential of Pto de-repressed kinase activity. The increased potency of free myristate relative to the myristoylated N-peptide inhibitor suggested that the peptide moiety is antagonistic to repression by myristate. Suppression of related protein kinases by myristate declined with similarity to Pto, and the inhibitory activity could be attributed to hydrophobicity. We present evidence that inhibition of Pto by the myristoylated N-peptide is mediated through a previously identified surface regulatory patch. The data show a role for negative regulation of Pto by N-myristoylation, in addition to the previously demonstrated positive role, and are consistent with a model in which the acylated N terminus is sequestered in the catalytic cleft prior to release by Pto activation.     

Effect of Brain Ischemia on Protein Kinase C

We examined the influence of brain ischemia on the activity and subcellular distribution of protein kinase C (PKC). Two different models of ischemic brain injury were used: postdecapitative ischemia in rat forebrain and transient (6-min) cerebral ischemia in gerbil hippocampus. In the rat forebrain model, at 5 and 15 min postdecapitation there was a steady decrease of total PKC activity to 60% of control values. This decrease occurred without changes in the proportion of the particulate to the soluble enzyme pools. Isolated rat brain membranes also exhibited a concomitant decrease of [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding with an apparent increase of the ligand affinity to the postischemic membranes. On the other hand, the ischemic gerbil hippocampus model displayed a 40% decrease of total PKC activity, which was accompanied by a relative increase of PKC activity in its membrane-bound form. This resulted in an increase in the membrane/total activity ratio, indicating a possible enzyme translocation from cytosol to the membranes after ischemia. Moreover, after 1 day of recovery, a statistically significant enhancement of membrane-bound PKC activity resulted in a further increase of its relative activity up to 162% of control values. In vitro experiments using a synaptoneurosomal particulate fraction were performed to clarify the mechanism of the rapid PKC inhibition observed in cerebral tissue after ischemia. These experiments showed a progressive, Ca(2+)-dependent, antiprotease-insensitive down-regulation of PKC during incubation. This down-regulation was significantly enhanced by prior phorbol (PDBu) treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Insulin-like effects of epidermal growth factor and insulin-like growth factor-I on [3H]2-deoxyglucose uptake, diacylglycerol generation and protein kinase C activation in BC3H-1 myocytes.

Epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) were found to provoke increases in [3H]2-deoxyglucose uptake, diacylglycerol (DAG) generation and membrane-bound protein kinase C activity in BC3H-1 myocytes. These effects were similar to those provoked by insulin. The increases in DAG did not appear to be derived from hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol, but may have been derived from synthesis of phosphatidic acid de novo, and hydrolysis of phosphatidylcholine, as revealed by studies with [3H]glycerol and [3H]choline respectively. Accordingly, both EGF and IGF-I increased acute [3H]glycerol labelling of DAG (and other lipids) and [3H]choline labelling of phosphocholine. These labelling responses were similar in time course, suggesting that they are closely coupled. Our findings suggest that EGF and IGF-I, like insulin, increase DAG-protein kinase C signalling, apparently by activating co-ordinated lipid-synthesis and -hydrolysis responses, which are distinctly different from the PIP2-hydrolysis response.