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Identification of two cytosolic diacylglycerol kinase isoforms in rat brain, and in NIH-3T3 and ras-transformed fibroblasts.

ABSTRACT: Two major species of diacylglycerol kinase (type I and type II) were separated from brain cytosol and from NIH-3T3 or ras-transformed 3T3 cells by heparin-agarose chromatography. Multiple species of diacylglycerol kinase were also detected by non-denaturing isoelectric focusing. The two peaks of activity were of similar size, both co-eluted at approximately 95 kDa from a Superose f.p.l.c. column. Type II enzyme (pI 8.0) was more active when substrate was presented in a deoxycholate/phosphatidylserine undefined environment, as opposed to an octyl glucoside/phosphatidylserine micellar environment. Type II activity was also enhanced by the presence of phosphatidylcholine as cofactor. Type I enzyme (pI 4.0) was more active in the presence of either phosphatidylserine or phosphatidylinositol. Type I and II enzymes had different ATP affinities. Both enzymes showed a preference for diacylglycerol substrates with saturated acyl chains of 10-12 carbon atoms. The cytosolic enzyme activity was able to bind to diacylglycerol-enriched membranes in NIH-3T3 fibroblasts, and this translocation was unaffected in ras-transformed 3T3 cells. These results demonstrate the presence of multiple diacylglycerol kinases in brain cytosol and NIH-3T3 and ras-transformed 3T3 cells. The enzymes differ in cofactor, ATP and substrate requirements. These results can explain some of the contradictions between previous studies of cytosolic diacylglycerol kinase activity, and suggest the presence of a family of such kinases that are differentially regulated by phospholipid cofactors.

SUBMITTER: Stathopoulos VM

PROVIDER: S-EPMC1149746 | biostudies-other | 1990 Dec

REPOSITORIES: biostudies-other

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Overexpression of kinase suppressor of Ras upregulates the high-molecular-weight tropomyosin isoforms in ras-transformed NIH 3T3 fibroblasts.

Project description:The down-regulation of the high-molecular-weight isoforms of tropomyosin (TM) is considered to be an essential event in cellular transformation. In ras-transformed fibroblasts, the suppression of TM is dependent on the activity of the Raf-1 kinase; however, the requirement for other downstream effectors of Ras, such as MEK and ERK, is less clear. In this study, we have utilized the mitogen-activated protein kinase scaffolding protein Kinase Suppressor of Ras (KSR) to further investigate the regulation of TM and to clarify the importance of MEK/ERK signaling in this process. Here, we report that overexpression of wild-type KSR1 in ras-transformed fibroblasts restores TM expression and induces cell flattening and stress fiber formation. Moreover, we find that the transcriptional activity of a TM-alpha promoter is decreased in ras-transformed cells and that the restoration of TM by KSR1 coincides with increased transcription from this promoter. Although ERK activity was suppressed in cells overexpressing KSR1, ERK inhibition alone was insufficient to upregulate TM expression. The KSR1-mediated effects on stress fiber formation and TM transcription required the activity of the ROCK kinase, because these effects could be suppressed by the ROCK inhibitor, Y27632. Overexpression of KSR1 did not directly regulate ROCK activity, but did permit the recoupling of ROCK to the actin polymerization machinery. Finally, all of the KSR1-induced effects were mediated by the C-terminal domain of KSR1 and were dependent on the KSR-MEK interaction.

| S-EPMC151698 | biostudies-literature

Differential pathways (phospholipase C and phospholipase D) of bradykinin-induced biphasic 1,2-diacylglycerol formation in non-transformed and K-ras-transformed NIH-3T3 fibroblasts. Involvement of intracellular Ca2+ oscillations in phosphatidylcholine breakdown.

Project description:Bradykinin (BK) induced a biphasic increase in 1,2-diacylglycerol (DAG) in both K-ras-transformed fibroblasts (DT) and the parent NIH-3T3 cells. The first phase was coincident with the increase in Ins(1,4,5)P3 resulting from PtdIns(4,5)P2 hydrolysis, and the second, sustained, phase was derived from phosphatidylcholine (PtdCho) hydrolysis. In NIH-3T3 cells, stimulation by BK induced greater production of choline than phosphocholine in [3H]choline-labelled cells and appreciable phosphatidylethanol (PtdEtOH) formation in [3H]myristic acid-labelled cells, suggesting that PtdCho was hydrolysed mainly by a phospholipase D (PLD) activity. Pretreatment with propranolol, an inhibitor of phosphatidate phosphohydrolase, markedly diminished the second DAG accumulation, supporting the above notion. In DT cells, BK induced predominantly phosphocholine generation and little PtdEtOH formation, indicating that the PtdCho hydrolysis was due to a phospholipase C (PLC) activity. The BK-induced oscillations in intracellular Ca2+ concentration ([Ca2+]i) observed in single DT cells [Fu, Sugimoto, Oki, Murakami, Okano & Nozawa (1991) FEBS Lett. 281, 263-266] were detected as a sustained [Ca2+]i elevation when assayed in a cell suspension. A receptor-operated Ca2+ channel blocker, SK&F 96365, suppressed both the BK-induced phosphocholine generation and the sustained [Ca2+]i elevation in a similar dose-dependent manner. These results thus suggested that oscillations in [Ca2+]i are involved in the activation of PtdCho-specific PLC in DT cells.

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Purification and characterization of biologically active scatter factor from ras-transformed NIH 3T3 conditioned medium.

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Desensitization of prostaglandin F2 alpha-stimulated inositol phosphate generation in NIH-3T3 fibroblasts transformed by overexpression of normal c-Ha-ras-1, c-Ki-ras-2 and c-N-ras genes.

Project description:The stimulation of inositol phosphate generation in control and ras-gene-transformed NIH-3T3 cells by prostaglandin F2 alpha (PGF2 alpha) was investigated. Compared with the control cells, a desensitization of the response was observed in cells transformed by the overexpression of N-, Ha-, or Ki-ras genes. This desensitization was without effect upon the concentration causing half-maximal effect (EC50), dissociation constant (Kd) or number of PGF2 alpha receptors. Inhibition of PG synthesis was without effect upon desensitization, demonstrating that the effect was not agonist-induced. Desensitization could be induced in NIH-3T3 cells by culturing under conditions where the cells were all in the exponential growth phase, or by a 12 h exposure to a C-kinase-activating phorbol ester. These results suggest that desensitization of certain agonist-induced inositol phospholipid responses in ras-transformed cells is a consequence of increased cell proliferation and associated amplification in C-kinase activity and is an indirect consequence of transformation by ras.

| S-EPMC1131370 | biostudies-other

Bradykinin B2 receptor-induced and inositol tetrakisphosphate-evoked Ca2+ entry is sensitive to a protein tyrosine phosphorylation inhibitor in ras-transformed NIH/3T3 fibroblasts.

Project description:Signal transduction from mouse bradykinin B2 receptors to calcium influx was studied in ras-transformed NIH/3T3 (DT) fibroblasts. DT cells were preloaded with fura-2 and whole-cell voltage-clamped. Activation of B2 receptors resulted in a decrease of cellular fluorescence at the excitation wavelength of 340, or 360 nm after MnCl2 application, in both the presence and absence of external Ca2+ in DT cells, at a holding potential of -40 mV. This Mn2+ entry through the Ca2+ influx pathway increased with membrane hyperpolarization. Internal application of inositol 1,3,4,5-tetrakisphosphate (InsP4), but not of inositol 1,4,5-trisphosphate, mimicked membrane potential-dependent Mn2+ entry. Bradykinin- and InsP4-induced Ca2+ influx was blocked by 10-100 microM genistein, a tyrosine kinase inhibitor. B2 receptor activation induced time-dependent tyrosine phosphorylation of mitogen-activated protein kinase and 120 kDa protein, which was dose-dependently inhibited by genistein. Bradykinin was unable to induce Ca2+ oscillations in genistein-treated DT cells. Our results show that bradykinin-induced Ca2+ influx and oscillations depend upon protein tyrosine phosphorylation. The results suggest that two bradykinin B2 receptor-activated signal pathways, protein tyrosine phosphorylation and formation of InsP4, merge at the Ca2+ influx process in ras-transformed NIH/3T3 fibroblasts.

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The gene expression profile induced by Wnt 3a in NIH 3T3 fibroblasts.

Project description:Wnt proteins play important roles in regulating cell differentiation, proliferation and polarity. Wnts have been proposed to play roles in tissue repair and fibrosis, yet the gene expression profile of fibroblasts exposed to Wnts has not been examined. We use Affymetrix genome-wide expression profiling to show that a 6-h treatment of fibroblasts of Wnt3a results in the induction of mRNAs encoding known Wnt targets such as the fibrogenic pro-adhesive molecule connective tissue growth factor (CTGF, CCN2). Wnt3a also induces mRNAs encoding potent pro-fibrotic proteins such as TGFbeta and endothelin-1 (ET-1). Moreover, Wnt3a promotes genes associated with cell adhesion and migration, vasculature development, cell proliferation and Wnt signaling. Conversely, Wnt3a suppresses gene associated with skeletal development, matrix degradation and cell death. Results were confirmed using real-time polymerase chain reaction of cells exposed to Wnt3a and Wnt10b. These results suggest that Wnts induce genes promoting fibroblast differentiation towards angiogenesis and matrix remodeling, at the expense of skeletal development.

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BCR-ABL oncogenic transformation of NIH 3T3 fibroblasts requires the IL-3 receptor.

Project description:Oncogenic transformation of hematopoietic cells by the Bcr-Abl oncoprotein directly involves the activation Jak2 tyrosine kinase and the Stat5 transcription factor. Both proteins are normally linked to the interleukin (IL)-3/granulocyte-macrophage colony-stimulating factor receptors for growth and survival. Since fibroblastic cells are not targets of BCR-ABL-induced oncogenesis, we determined whether forced expression of the IL-3 receptor would allow oncogenic transformation of NIH 3T3 fibroblasts known to be resistant to transformation by BCR-ABL. NIH 3T3 cells transduced with the human IL-3 receptor alpha and beta chains were highly susceptible to oncogenic transformation by expression of BCR-ABL. Forced expression of both receptor chains but not either one alone allowed efficient foci formation of NIH 3T3 cells expressing BCR-ABL (triple positive cells), and these cells formed colonies in soft agar, whereas BCR-ABL+ NIH 3T3 cells lacking IL-3 receptor expression did not. Signaling studies indicate that the BCR-ABL/IL-3 receptor+ NIH 3T3 cells utilize the Gab2/PI-3 kinase pathway activated by Jak2, and the Stat5 pathway activated separately by Bcr-Abl, whereas BCR-ABL+ NIH 3T3 cells lacking the IL-3 receptor do not utilize the Jak2 pathway, but still maintain activation of Stat5. The Bcr-Abl kinase inhibitor imatinib mesylate (1 microM) and two Jak2 kinase inhibitors strongly inhibited agar colony formation and the activation of Gab2 caused by Jak2. All of these findings indicate that Bcr-Abl oncoprotein requires the IL-3 receptor/Jak2/Stat5 pathways for oncogenic transformation of NIH 3T3 fibroblasts.

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The nucleus is an intracellular propagator of tensile forces in NIH 3T3 fibroblasts.

Project description:Nuclear positioning is a crucial cell function, but how a migrating cell positions its nucleus is not understood. Using traction-force microscopy, we found that the position of the nucleus in migrating fibroblasts closely coincided with the center point of the traction-force balance, called the point of maximum tension (PMT). Positioning of the nucleus close to the PMT required nucleus-cytoskeleton connections through linker of nucleoskeleton-to-cytoskeleton (LINC) complexes. Although the nucleus briefly lagged behind the PMT following spontaneous detachment of the uropod during migration, the nucleus quickly repositioned to the PMT within a few minutes. Moreover, traction-generating spontaneous protrusions deformed the nearby nucleus surface to pull the nuclear centroid toward the new PMT, and subsequent retraction of these protrusions relaxed the nuclear deformation and restored the nucleus to its original position. We propose that the protruding or retracting cell boundary transmits a force to the surface of the nucleus through the intervening cytoskeletal network connected by the LINC complexes, and that these forces help to position the nucleus centrally and allow the nucleus to efficiently propagate traction forces across the length of the cell during migration.

| S-EPMC4457156 | biostudies-literature

Characterization of sample preparation methods of NIH/3T3 fibroblasts for ToF-SIMS analysis.

Project description:The information that is obtained from single cells during time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis is influenced by the method that was used to prepare the cells. The removal of extracellular media before analysis is necessary, but the rinsing technique should not damage the plasma membrane of the cell. The presence of intracellular salts reduced the secondary ion yield an average of 2.6-fold during Bi3 (+)/C60 (++) depth profiles. Chemical fixation followed by rinsing removed a majority of the intracellular salts, "recovering" the positive secondary ion yields. The formaldehyde-fixation process removed a majority of the intracellular Cl(-), but other key anions were not removed in significant amounts. The data presented here is consistent the anion neutralization mechanism largely responsible for the lower ion yields. All of the organic secondary ions that were detected in the freeze-dried cells were also detected in the formaldehyde-fixed cells, suggesting that the fixation process did not remove any molecular species to an extent that is detectable by ToF-SIMS. Compared to freeze dried cells, well preserved, frozen-hydrated cells showed little increase, or a decreased yield, for most low mass ions, but an increased yield for larger mass fragments. This is consistent with a reduced damage cross section at cryogenic analysis temperatures, although proton donation from water and reduction the salt effects in the presence of water likely also play roles. Numerous ions detected from the frozen-hydrated cells were not detected from the freeze dried cells, however many of these ions were attributed to chemical combinations of water, salts and the ammonium acetate rinsing solution.

| S-EPMC4000548 | biostudies-literature

NIH-3T3 cells transformed with a ras oncogene exhibit a protein kinase C-mediated inhibition of agonist-stimulated Ca2+ inflow.

Project description:1. The ability of bombesin or platelet-derived growth factor (PDGF) to stimulate Ca2+ inflow (assessed by measuring changes in the intracellular free Ca2+ concentration in cells loaded with fura-2) in NIH-3T3 cells transformed with the EJ/T24-Ha-ras-1 oncogene is inhibited when compared with the action of the agonists on wild-type cells. 2. The effects of transformation with the ras oncogene are associated with complete inhibition of the ability of bombesin to release Ca2+ from intracellular stores, a substantial decrease in the number of bombesin receptors, no change in the ability of foetal calf serum or ionomycin to release Ca2+ from intracellular stores and the activation of protein kinase C. 3. The effects of transformation with the H-ras oncogene on the ability of bombesin or PDGF to stimulate Ca2+ inflow were mimicked by a 30 min exposure of wild-type cells to phorbol dibutyrate. This action of phorbol dibutyrate was completely blocked by prior treatment of wild-type cells for 24 h with the phorbol ester. 4. It is concluded that one of the actions of the H-ras oncogene in fibroblasts is to inhibit agonist-stimulated Ca2+ inflow by a mechanism which involves the activation of protein kinase C.

| S-EPMC1149555 | biostudies-other

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