Phosphatidylinositol-4,5-bisphosphate may antecede diacylglycerol as activator of protein kinase C

Phosphatidylserine/calcium-dependent protein kinase C (PKC) from rat brain is activated fifty times more efficiently by phosphatidylinositol-4,5-bisphosphate (PIP2) (Kapp = 0.04 mole% in Triton-lipid micelles) than by diacylglycerol (DG) (Kapp = 2 mole%). Both effector lipids appear to bind to the same site but PIP2 may confer a narrower substrate specificity on the kinase. DG, which together with inositol trisphosphate (IP3) is generated by hydrolysis from PIP2 after cell stimulation, has been considered the natural activator of the kinase but it is likely to be anteceded in this function by PIP2; DG may perhaps retain the function of a back-up activator. The lack of PKC-activation by phosphatidylinositol (PI) or phosphatidylinositol-4-phosphate (PIP) opens the possibility that the Inositide Shuttle, PI reversible PIP reversible PIP2, has a role in controlling the activity of the kinase.     

Interaction of protein kinase C with phosphoinositides.

Calcium/phosphatidylserine-dependent protein kinase C (PKC) is activated by phosphatidylinositol 4,5-bisphosphate (PIP2), as well as by diacylglycerol (DG) and phorbol esters. Here we report that PIP2, like DG, increases the affinity of PKC for Ca2+, and causes Ca(2+)-dependent translocation of the enzyme from the soluble to a particulate fraction (liposomes). Phosphatidylinositol 4-phosphate (PIP) also displaces phorbol ester from PKC and causes Ca(2+)-dependent translocation of the enzyme to liposomes, but is much less efficient than PIP2, and a much weaker activator, with a histone phosphorylation v(PIP)/v(PIP2) of approximately 0.15. Scatchard analysis indicates competitive inhibition between PIP and phorbol ester with Ki(PIP) = 0.26 mol% as compared with Ki(PIP2) = 0.043 mol%. No effect of phosphatidylinositol (PI) on phorbol ester binding to PKC, translocation of PKC, or activation of PKC was observed. These results suggest that both PIP and PIP2 can complex with PKC, but full activation of the enzyme takes place only when PIP is converted to PIP2. We suggest that an inositide interconversion shuttle has a role in the regulation of protein phosphorylation.     

Phosphatidylinositol 4,5-bisphosphate competitively inhibits phorbol ester binding to protein kinase C

Calcium phospholipid dependent protein kinase C (PKC) is activated by diacylglycerol (DG) and by phorbol esters and is recognized to be the phorbol ester receptor of cells; DG displaces phorbol ester competitively from PKC. A phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), can also activate PKC in the presence of phosphatidylserine (PS) and Ca2+ with a KPIP2 of 0.04 mol %. Preliminary experiments have suggested a common binding site for PIP2 and DG on PKC. Here, we investigate the effect of PIP2 on phorbol ester binding to PKC in a mixed micellar assay. In the presence of 20 mol % PS, PIP2 inhibited specific binding of [3H]phorbol 12,13-dibutyrate (PDBu) in a dose-dependent fashion up to 85% at 1 mol %. Inhibition of binding was more pronounced with PIP2 than with DG. Scatchard analysis indicated that the decrease in binding of PDBu in the presence of PIP2 is the result of an altered affinity for the phorbol ester rather than of a change in maximal binding. The plot of apparent dissociation constants (Kd') against PIP2 concentration was linear over a range of 0.01-1 mol % with a Ki of 0.043 mol % and confirmed the competitive nature of inhibition between PDBu and PIP2. Competition between PIP2 and phorbol ester could be demonstrated in a liposomal assay system also. These results indicate that PIP2, DG, and phorbol ester all compete for the same activator-receiving region on the regulatory moiety of protein kinase C, and they lend support to the suggestion that PIP2 is a primary activator of the enzyme.     

Guanine nucleotides stimulate hydrolysis of phosphatidylinositol and polyphosphoinositides in permeabilized Swiss 3T3 cells

Hydrolysis-resistant analogues of GTP specifically stimulate the formation of [3H]inositol mono-, bis- and trisphosphates by saponin-permeabilized Swiss 3T3 cells prelabelled with [3H]inositol. Each inositol phosphate is formed largely by hydrolysis of its parent lipid and not by dephosphorylation of inositol 1,4,5-trisphosphate [(1,4,5)IP3]. Although hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is most sensitive to guanine nucleotides, hydrolysis of phosphatidyl-inositol (PI) and phosphatidylinositol 4-phosphate (PIP) is quantitatively more important. These results suggest that a guanine nucleotide-dependent regulatory protein(s) (G-protein) is involved in regulating the hydrolysis of PI and PIP, as well as PIP2, and so may allow formation of diacylglycerol (DG) without simultaneous production of (1,4,5)IP3 and mobilization of intracellular Ca2+.     

Effect of staurosporine on fMet-Leu-Phe-stimulated human neutrophils: dissociated release of inositol 1,4,5-trisphosphate, diacylglycerol and intracellular calcium.

Staurosporine, a microbial alkaloid, enhances inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG) production rapidly and dose-dependently in fMet-Leu-Phe (FMLP)-stimulated human neutrophils showing maximal effects at 1 microM concentration. The IP3 increase was specific for staurosporine as three other putative protein kinase C (PKC) inhibitors, H7, sphingosine and palmitoylcarnitine were unable to enhance the IP3 generation in FMLP-stimulated human neutrophils. Staurosporine, at concentrations 0.3-1.0 microM, did not affect the initial mobilization of FMLP-induced intracellular Ca2+ (Ca2+i), although a sustained elevation of cytosolic Ca2+ level was observed within 5 min. This effect could not be suppressed, even by 1 microM phorbol-myristate 12,13-acetate (PMA). Whereas lower concentrations of staurosporine (less than or equal to 100 nM) were unable to affect FMLP-induced IP3 production, DG accumulation and Ca2+i, the PMA-inhibited initial Ca2+i signal and IP3 formation triggered by FMLP were almost completely restored. At higher concentrations (greater than or equal to 300 nM) staurosporine reversed the inhibitory effect of other protein kinases, distinct from the PMA-inducible one, which may be responsible for the phosphatidyl inositol 4,5-bisphosphate (PIP2) breakdown, thus causing accumulation of IP3 and DG and an elevation of C2+i level. Whereas IP3 declined to basal level within 5 min, the DG level remained elevated during the same period. This phenomenon is attributed to phospholipase D (PLD) stimulation by staurosporine, which augments the DG synthesis, in part through PA degradation via phosphatidic acid (PA) phosphohydrolase.     

Altered signal transduction in erbB-transformed cells. Implication of enhanced inositol phospholipid metabolism in erbB-induced transformation

To clarify the signal transduction mechanism of the erbB gene (virus oncogene) products leading to cell growth and transformation, the alteration of signal transduction induced by enhanced inositol phospholipid metabolism was studied in chick embryo fibroblast cells (CEF cells) transformed by gag-fused erbB gene-carrying virus (GEV cells). The incorporations of 32P into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate were markedly increased in GEV cells. In GEV cells, the activities of lipid kinases such as phosphatidylinositol (PI), PIP, and diacylglycerol (DG) kinases were also increased. The activities of other important enzymes involved in inositol phospholipid metabolism, such as CDP-DG:myo-inositol transferase and phospholipase C, were not changed in GEV cells. Increased inositol phospholipid metabolism might lead to the production of second messengers, such as 1,2-DG and inositol 1,4,5-trisphosphate. Indeed, the 1,2-DG content was also increased in GEV cells. Moreover, the activity of protein kinase C (the Ca2+/phospholipid-dependent enzyme), which should be stimulated by 1,2-DG, was elevated in GEV cells; the protein kinase C activity in the membrane fraction of GEV cells was especially high. When CEF cells were treated with tetradecanoylphorbol acetate, protein kinase C activator, plus Ca2+ ionophore, [3H]thymidine incorporation was markedly stimulated, and maximal stimulation was observed with 1 nM Ca2+ ionophore A23187 plus 100 nM TPA. On the other hand, when GEV cells were treated with TPA plus Ca2+ ionophore A23187, [3H]thymidine incorporation was consistently inhibited. Next, studies were made to determine whether the erbB gene product itself had kinase activity on PI, PIP, and DG after membranes were mildly solubilized with Triton X-100 to prevent inactivation of these kinases. Immunoprecipitates of a GEV cell lysate with antisera that reacted with the erbB gene product had PI kinase activity, whereas no activity was detected in those of lysates of uninfected CEF cells. However, the activity was very weak compared with the total cellular activity. No difference in the PIP and DG kinase activities of immunoprecipitates of cell lysates of uninfected CEF cells and GEV cells was observed. These results suggest that the erbB gene product enhances inositol phospholipid metabolism and subsequent signal transduction, but that the erbB gene product is not involved directly in lipid kinases, although it is closely associated with lipid kinase.     

Interaction of protein kinase C isozymes with phosphatidylinositol 4,5-bisphosphate.

Interaction of protein kinase C (PKC) isozymes with phosphatidylinositol 4,5-bisphosphate (PIP2) was investigated by monitoring the changes in the intrinsic fluorescence of the enzyme, the kinase activity, and phorbol ester binding. Incubation of PKC I, II, and III with PIP2 resulted in different rates of quenching of PKC fluorescence and different degrees of inactivation of these enzymes. Other inositol-containing phospholipids such as phosphatidylinositol and phosphatidylinositol 4-phosphate also caused differential rates of quenching of the intrinsic fluorescence of these enzymes. These latter two phospholipids were, however, less potent in the inactivation of PKCs than PIP2. The IC50 of PIP2 were 2, 4, and 11 microM for PKC I, II, and III, respectively. Inactivation of PKCs by PIP2 cannot be reversed by extensive dilution of PIP2 with Nonidet P-40 nor by digestion of PIP2 with phospholipase C. Interaction of PIP2 with the various PKC isozymes was greatly facilitated in the presence of Mg2+ or Ca2+ as evidenced by the accelerated quenching of the PKC fluorescence, however, these divalent metal ions protected PKC from the PIP2-induced inactivation. Binding of PIP2 to PKC in the absence of divalent metal ion also caused a reduction of [3H]phorbol 12,13-dibutyrate binding as a result of reducing the affinity of the enzyme for phorbol ester. Based on gel filtration chromatography, it was estimated that one molecule of PKC interacted with one PIP2 micelle with an aggregation number of 80-90. The PIP2-bound PKC could further interact with phosphatidylserine in the presence of Ca2+ to form a larger complex. Binding of PKC to both PIP2 and phosphatidylserine in the presence of Ca2+ was also evident by changes in the intrinsic fluorescence of PKC. As the interaction of PKC with PIP2, but not with phosphatidylserine, could be enhanced by millimolar concentrations of Mg2+, we propose that PIP2 may be a component of the membrane anchor for PKC under basal physiological conditions when [Ca2+]i is low and Mg2+ is plentiful. Under the in vitro assay conditions, PIP2 could stimulate PKC activity to a level approximately 10-20% of that by diacylglycerol. The stimulatory effect of PIP2 on PKC apparently is not due to binding to the same site recognized by diacylglycerol or phorbol ester, because PIP2 cannot effectively compete with phorbol 12,13-dibutyrate in the binding assay.     

Interruption of inositol sphingolipid synthesis triggers Stt4p-dependent protein kinase C signaling

The protein kinase C (PKC)-MAPK signaling cascade is activated and is essential for viability when cells are starved for the phospholipid precursor inositol. In this study, we report that inhibiting inositol-containing sphingolipid metabolism, either by inositol starvation or treatment with agents that block sphingolipid synthesis, triggers PKC signaling independent of sphingoid base accumulation. Under these same growth conditions, a fluorescent biosensor that detects the necessary PKC signaling intermediate, phosphatidylinositol (PI)-4-phosphate (PI4P), is enriched on the plasma membrane. The appearance of the PI4P biosensor on the plasma membrane correlates with PKC activation and requires the PI 4-kinase Stt4p. Like other mutations in the PKC-MAPK pathway, mutants defective in Stt4p and the PI4P 5-kinase Mss4p, which generates phosphatidylinositol 4,5-bisphosphate, exhibit inositol auxotrophy, yet fully derepress INO1, encoding inositol-3-phosphate synthase. These observations suggest that inositol-containing sphingolipid metabolism controls PKC signaling by regulating access of the signaling lipids PI4P and phosphatidylinositol 4,5-bisphosphate to effector proteins on the plasma membrane.     

Potentiation of thyrotropin releasing hormone-induced inositol phospholipid metabolism and Ca2+ mobilization by cyclic AMP-increasing agents

Thyrotropin releasing hormone (TRH) caused significant breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) in GH3 cells, but vasoactive intestinal peptide (VIP) did not. However, VIP enhanced the TRH-induced hydrolysis of PIP2, the conversion of phosphatidylinositol 4-phosphate (PIP) to PIP2 and the accumulation of phosphatidic acid (PA). On the other hand, the tumor promoter, tetradecanoyl phorbol acetate (TPA), suppressed the TRH-induced hydrolysis of PIP2. In the membrane fraction, the addition of cAMP inhibited the PI kinase activity in a dose-dependent manner, but stimulated the PIP kinase activity. TPA did not affect the PI and PIP kinase activities at all. VIP enhanced the first spike phase of the TRH-induced increase in the intracellular Ca2+ level, while TPA inhibited such Ca2+ mobilization. These results suggested that cAMP-increasing agents enhanced inositol phospholipid metabolism and Ca2+ mobilization induced by TRH in GH3 cells but that TPA inhibited them.     

Phosphorylation of erythrocyte membrane liberates calcium

Washed and permeabilized human erythrocyte ghosts were found to discharge calcium on treatment with ATP. Concomitantly, there was a decrease in phosphatidylinositol (PI) and an increase in phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2). These results support the hypothesis that an inositide shuttle, PI in equilibrium PIP in equilibrium PIP2, operates to maintain intracellular Ca2+ levels. The cation is thought to be sequestered in a cage formed by the head groups of two acidic phospholipid molecules, e.g., phosphatidylserine and phosphatidylinositol, with participation of both PO and fatty acid ester CO groups. These cages are stabilized by inter-headgroup hydrogen bonding. When the inositol group is phosphorylated in positions 4 and 5, inter-lipid hydrogen bonding is disrupted and the cage opens to release its Ca2+.     

Ca2+ regulation of phosphatidylinositol turnover in the plasma membrane of tobacco suspension culture cells.

The biochemical properties of the enzymes involved in phosphatidylinositol (PI) turnover in higher plants were investigated using the plasma membrane isolated from tobacco suspension culture cells by aqueous two-phase partitioning. Submicromolar concentrations of Ca2+ inhibited PI kinase and phosphatidylinositol 4-phosphate (PIP) kinase and stimulated phospholipase C. Diacylglycerol (DG) kinase was inhibited by Ca2+, but required a higher concentration than the physiological level. From the above results we postulate the following scheme: signal coupled activation of phospholipase C produces IP3 which induces Ca2+ release from the intracellular Ca2+ compartment, the increased cytoplasmic Ca2+ in turn activates phospholipase C and causes a further increase of the cytoplasmic Ca2+ level. This inhibits PI kinase and PIP kinase and brings about a limited supply of PIP2, the substrate of phospholipase C. Consequently, IP3 production decreases and Ca2+ mobilization ceases. Then cytosolic Ca2+ returns to the stationary level by the Ca2+ pump at the plasma membrane and at the endoplasmic reticulum and Ca2+/H+ antiporter at the plasma membrane and at the tonoplast.     

Differences in the metabolism of inositol and phosphoinositides by cultured cells of neuronal and glial origin

Phosphoinositide and inositol metabolism was compared in glioma (C6), neuroblastoma (N1E-115) and neuroblastoma X glioma hybrid (NG 108-15) cells. All cell lines had similar proportions of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2). Neuroblastoma and hybrid cells had almost identical phospholipid and phosphoinositide compositions and similar activities for the enzymes metabolizing polyphosphoinositides (PI kinase, PIP phosphatase, PIP kinase, PIP2 phosphatase, PIP2 phosphodiesterase). Glioma cells differed by having greater proportions of ethanolamine plasmalogen and sphingomyelin, lower PIP kinase, 3-5-fold higher PIP phosphatase activity and 10-15-fold greater PIP2 phosphodiesterase activity. Higher PIP phosphatase and PIP2 diesterase activities appear to be characteristic of cells of glial origin, since similar activities were found in primary cultures of astroglia. Glioma cells also metabolize inositol differently. In pulse and pulse-chase experiments, glioma cells transported inositol into a much larger water-soluble intracellular pool and maintained a concentration gradient 30-times greater than neuroblastoma cells. Label in intracellular inositol was less than in phosphoinositides in neuroblastoma and exchanged rapidly with extracellular inositol. In glioma, labeling of intracellular inositol greatly exceeded that of phosphoinositides. As a consequence, radioactivity in prelabeled phosphoinositides could not be effectively chased from glioma cells by excess unlabeled inositol. Such differences between cells of neuronal and glial origin suggest different and possibly supportive roles for these two cell types in maintaining functions regulated through phosphoinositide-linked signalling systems in the central nervous system.     

Sustained diacylglycerol formation from inositol phospholipids in angiotensin II-stimulated vascular smooth muscle cells

Angiotensin II acts on cultured rat aortic vascular smooth muscle cells to stimulate phospholipase C-mediated hydrolysis of membrane phosphoinositides and subsequent formation of diacylglycerol and inositol phosphates. In intact cells, angiotensin II induces a dose-dependent increase in diglyceride which is detectable after 5 s and sustained for at least 20 min. Angiotensin II (100 nM)-stimulated diglyceride formation is biphasic, peaking at 15 s (227 +/- 19% control) and at 5 min (303 +/- 23% control). Simultaneous analysis of labeled inositol phospholipids shows that at 15 s phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP) decline to 52 +/- 6% control and 63 +/- 5% control, respectively, while phosphatidylinositol (PI) remains unchanged. In contrast, at 5 min, PIP2 and PIP have returned toward control levels (92 +/- 2 and 82 +/- 4% control, respectively), while PI has decreased substantially (81 +/- 2% control). The calcium ionophore ionomycin (15 microM) stimulates diglyceride accumulation but does not cause PI hydrolysis. 4 beta-Phorbol 12-myristate 13-acetate, an activator of protein kinase C, inhibits early PIP and PIP2 breakdown and diglyceride formation, without inhibiting late-phase diglyceride accumulation. Thus, angiotensin II induces rapid transient breakdown of PIP and PIP2 and delayed hydrolysis of PI. The rapid attenuation of polyphosphoinositide breakdown is likely caused by a protein kinase C-mediated inhibition of PIP and PIP2 hydrolysis. While in vascular smooth muscle stimulated with angiotensin II inositol 1,4,5-trisphosphate formation is transient, diglyceride production is biphasic, suggesting that initial and sustained diglyceride formation from the phosphoinositides results from different biochemical and/or cellular processes.     

Response of alkalinization or acidification by phytohemagglutinin is dependent on the activity of protein kinase C in human peripheral T Cells

The increase of intracellular free calcium concentration ([Ca(2+)](i)) and protein kinase C (PKC) activity are two major early mitogenic signals to initiate proliferation of human T cells. However, a rapid change in intracellular pH (pH(i)), acidification or alkalinization during the activation, is also associated after these two signals. The aim of this study was to define whether the change in pH(i) is affected by calcium and protein kinase C (PKC), in phytohemagglutinin (PHA)-stimulated T cells. T cells were isolated from human peripheral blood. The [Ca(2+)](i) and the pH(i) were measured using, respectively, the fluorescent dyes, Fura-2, and BCECF. In addition, down-regulation of PKC activity by PMA (1 microM, 18 h) was confirmed in these cells using a protein kinase assay. The results indicated that, (1) alkalinization was induced by PHA or PMA in T cells; the results of alkalinization was PKC-dependent and Ca(2+)-independent, (2) in PKC down-regulated T cells, PHA induced acidification; this effect was enhanced by pre-treating the cells with the Na(+)/H(+) exchange inhibitor, 5-(N,N-dimethyl)-amiloride, (DMA, 10 microM, 20 min), (3) the acidification was dependent on the Ca(2+) influx and blocked by removal of extracellular calcium or the addition of the inorganic channel blocker, Ni(2+), and (4) Thapsigargin (TG), a Ca(2+)-ATPase inhibitor, confirmed that acidification by the Ca(2+) influx occurred in T cells in which PKC was not down-regulated. These findings indicate two mechanisms, alkalinization by PKC and acidification by Ca(2+) influx, exist in regulating pH(i) in T cells. This is the first report that PHA stimulates the acidification by Ca(2+) influx but not alkalinization in T cells after down-regulation of PKC. In conclusion, the activity of PKC in T cells determines the response in alkalinization or acidification by PHA.     

Putative role of inositol phospholipid metabolism in neurons

Inositol phospholipids play a crucial role in the intracellular signal transduction in most cell types. Activation of an enzyme called phospholipase C or PIP2-phosphodiesterase (PIP2-PDE) leads to the production of two second messenger molecules, diacylglycerol (DG) and inositol 1,4,5-triphosphate (IP3). DG activates a kinase called protein kinase C, whereas IP3 mediates the release of Ca2+ from intracellular storage sites. The measurement of IP3 and its degradation products, inositol diphosphate (IP2) and inositol monophosphate (IP1) provides a way of assessing the extent to which this complex system has been activated. In the central nervous system (CNS) most of the studies on the neurotransmitter stimulated formation of inositol phosphates (IPs) have been performed on brain slices, a mixture of mainly neurons and glial cells. The recent development of pure neuronal cultures provides a means of determining which of these responses were of neuronal origin. The purpose of this review is to summarize the results obtained in neurons in primary culture together with a brief appraisal of the possible function of this second messenger system in neurons.     

Phospholipase C activation and diacylglycerol kinase inactivation lead to an increase in diacylglycerol content in spontaneously hypertensive rat

Activities of three kinases, phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and diacylglycerol (DG) kinases, and phospholipase C were measured in erythrocyte ghosts from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). PI kinase activity was significantly higher in SHR than WKY but there was no significant difference in PIP kinase activity between SHR and WKY. The activity of phospholipase C, which hydrolyzes PIP2, was also increased in SHR. However, DG kinase activity was, on the contrary, decreased in SHR. These results suggest that there is a tendency to accumulate DG in SHR. Indeed, DG content in erythrocytes of SHR increased 1.7-fold compared to that of WKY. Such DG accumulation may cause the sustained activation of protein kinase C in SHR, since DG is a physiological activator for protein kinase C.     

Critical role of PIP5KI{gamma}87 in InsP3-mediated Ca(2+) signaling

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is the obligatory precursor of inositol 1,4,5-trisphosphate (InsP(3) or IP(3)) and is therefore critical to intracellular Ca(2+) signaling. Using RNA interference (RNAi), we identified the short splice variant of type I phosphatidylinositol 4-phosphate 5-kinase gamma (PIP5KIgamma87) as the major contributor of the PIP(2) pool that supports G protein-coupled receptor (GPCR)-mediated IP(3) generation. PIP5KIgamma87 RNAi decreases the histamine-induced IP(3) response and Ca(2+) flux by 70%. Strikingly, RNAi of other PIP5KI isoforms has minimal effect, even though some of these isoforms account for a larger percent of total PIP(2) mass and have previously been implicated in receptor mediated endocytosis or focal adhesion formation. Therefore, PIP5KIgamma87's PIP(2) pool that supports GPCR-mediated Ca(2+) signaling is functionally compartmentalized from those generated by the other PIP5KIs.     

Activation of phosphatidylinositol kinase and phosphatidylinositol-4-phosphate kinase by cAMP in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, cAMP-dependent phosphorylation plays an essential role at the start of the cell cycle. It has also recently been demonstrated that the breakdown of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol is a requisite process for cell proliferation (Uno, I., Fukami, K., Kato, H., Takenawa, T., and Ishikawa, T. (1988) Nature 333, 188-190). To clarify the relationship between the cAMP- and inositol phospholipid-mediated signal transduction systems, alterations in the inositol phospholipid metabolism of cAMP mutants were examined. The incorporation of [32P]Pi into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was markedly reduced in ras2, which produces low levels of cAMP, and increased in bcy1, which produces cAMP-independent protein kinase. The incorporation of [32P]Pi into ATP and phosphatidylinositol (PI) was almost the same in wild type, ras1, ras2, and bcy1 yeast strains. The addition of exogenous cAMP to cyr1-2 caused a tremendous increase in [32P]Pi incorporation into PIP and PIP2 without any effect on incorporation into ATP and PI, suggesting that cAMP plays an important role in polyphosphoinositide synthesis. We therefore examined the activities of PI and PIP kinases, the enzymes that catalyze the sequential steps from PI to PIP2 via PIP. The activities of both kinases were found to be very low in the membranes of cry1-2 and ras2 but very high in the membranes of bcy1 and ras1 ras2 bcy1 strain cells. The addition of cAMP to cyr1-2 cells caused the activation of PI and PIP kinases. Furthermore, the treatment of membranes with cAMP or dibutyryl cAMP caused the activation of PI kinase in wild type, ras1, cry1-2, and ras2 strains, but not in bcy1 strain cells. The effect was most prominent in membranes from cyr1-2 and ras2 cells. These results show that cAMP-dependent phosphorylation enhances polyphosphoinositide synthesis through activation of PI and PIP kinase, an effect which may lead to the enhanced production of inositol 1,4,5-trisphosphate and diacylglycerol.     

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

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

Differential effect on inositol-phospholipid hydrolysis, cytosolic-free Ca2+ concentration, protein kinase C activity and protein phosphorylation of 1-oleoyl-2-acetyl-sn-glycerol growth-stimulated ascites tumor cells

This study shows that the membrane-permeable stereospecific 1-oleoyl-2-acetyl-sn-glycerol (OAG), which is the analog of the natural 1,2-diacylglycerol (DAG), can stimulate the growth of ascites tumor cells. OAG can fully replace high serum concentrations in the culture medium and stimulates DNA synthesis in a dose-dependent manner. Investigation of the protein kinase C (PKC) isolated from a Triton extract of a 100,000g membrane pellet revealed that OAG can directly activate this enzyme. Concomitantly the phosphorylation of several cytosolic proteins with the molecular weights of 26, 33, 49, 55, 64, and 90 kDa is observed which is also found in serum-stimulated cells. Since DAG as a second messenger molecule originates from the hydrolysis of phosphoinositides we have investigated the metabolism of these lipids after labeling the cells with [3H]inositol. In detail, we have measured the amount of radioactive inositol trisphosphate (IP3) and the phosphodiesterase hydrolyzing phosphatidylinositol-4,5-bisphosphate (PIP2). The decreased radioactivity level of IP3 in OAG-stimulated cells as compared to non-growing cells (1-2% serum) indicates a feedback regulation of PIP2 hydrolysis which is substantiated by a profound reduction of PIP2-specific phospholipase C activity. The reduced IP3 formation has apparently no inhibitory effect on the cytoplasmic free Ca2+ concentration of OAG-stimulated cells, suggesting that the Ca2+ release is not directly correlated to the amount of IP3, which is also demonstrated for the non-growing cells. These data indicate that OAG apparently has a duel effect on the inositol phospholipid-mediated signal transfer system.(ABSTRACT TRUNCATED AT 250 WORDS)