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D A Foster

Publications and source records attributed to D A Foster.

At least 55 records · Page 3Linked to original sources
PubMed1994

Epidermal growth factor induces the production of biologically distinguishable diglyceride species from phosphatidylinositol and phosphatidylcholine via the independent activation of type C and type D phospholipases.

An early response to epidermal growth factor in A431 cells is the generation of diglyceride, a physiological activator of protein kinase C. By differentially prelabeling cellular phospholipids with [3H]arachidonate and [3H]myristate, which are incorporated primarily into phosphatidylinositol and phosphatidylcholine, respectively, we have found that epidermal growth factor induces an increase in diglyceride levels from both phosphatidylinositol and phosphatidylcholine via distinct mechanisms and kinetics. The epidermal growth factor-induced increase in phosphatidylinositol-derived diglyceride was transient and peaked at 5 min. As diglyceride levels dropped, there was a corresponding increase in phosphatidic acid, suggesting that the diglyceride is efficiently converted to phosphatidic acid by a diglyceride kinase. In contrast, epidermal growth factor-induced increases in phosphatidylcholine-derived diglyceride peaked at 30 min and remained elevated for greater than 2 h. The epidermal growth factor-induced increases in phosphatidic acid detected in [3H]myristate-prelabeled cells paralleled the increase in diglyceride, suggesting that the phosphatidylcholine-derived diglyceride is produced from phosphatidic acid via a phosphatidic acid phosphatase. Consistent with this hypothesis, epidermal growth factor also induced a protein kinase C-independent phospholipase D activity that was specific for phosphatidylcholine. These data suggest that epidermal growth factor induces diglyceride production from phosphatidylinositol and phosphatidylcholine via two distinct mechanisms: a rapid and transient induction of diglyceride that likely involves phospholipase c-gamma-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate and a slower, more sustained induction of diglyceride via a phospholipase D-mediated hydrolysis of phosphatidylcholine to produce phosphatidic acid, which is then converted to diglyceride by a phosphatidic acid phosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1993

v-Src-induced transformation is inhibited by okadaic acid.

The tumor promoter okadaic acid is a potent inhibitor of the serine/threonine protein phosphatases 1 and 2A. Addition of okadaic acid to v-Src-transformed BALB/c 3T3 cells reverted them to a flat morphology, increased fibronectin levels in the extracellular matrix, reduced saturation density, and inhibited the formation of colonies in soft agar. The ability of v-Src-transformed cells to proliferate in low serum was also inhibited by okadaic acid. These data implicate serine/threonine phosphatases in v-Src-induced transformation.

3T3 Cells↗
PubMed1993

v-Src activates a unique phospholipase D activity that can be distinguished from the phospholipase D activity activated by phorbol esters.

Phospholipase D (PLD) activity, as measured by the transphosphatidylation of cellular phospholipids, is elevated in BALB/c 3T3 cells transformed by v-Src. Phorbol esters that activate protein kinase C (PKC) also increase PLC activity in BALB/c 3T3 cells. v-Src-induced PLD activity could be distinguished from phorbol ester-induced PLD activity by differential radiolabelling of phospholipids, which are the substrates of PLD. Both v-Src- and phorbol ester-induced PLD activity could be detected when phospholipids were prelabelled with either radiolabelled myristate or palmitate; however, only phorbol ester-induced PLD activity could be detected when either arachidonate or 1-O-alkyl-sn-glyceryl-3-phosphorylcholine (alkyl-lysoPC) was used to prelabel the phospholipids. The increased PLD activity in v-Src-transformed cells was not detected when the cells were prelabelled with either arachidonic acid or alkyl-lysoPC, which contains an ether linkage at sn-1 of the glycerol backbone. As both arachidonic acid and alkyl-lysoPC are incorporated into phosphatidylcholine (PC), the substrate for v-Src-induced PLD activity, these data suggest that the PLD activated by v-Src can distinguish PCs lacking arachidonic acid and ether linkages. Consistent with v-Src activating a PLD activity that is distinct from that activated by phorbol esters that activate PKC directly, neither depleting cells of PKC nor treatment with the protein kinase inhibitor, staurosporine, had any effect on v-Src-induced PLD activity, whereas both PKC depletion and staurosporine inhibited phorbol ester-induced PLD activity. Taken together, these data suggest that v-Src activates a PKC-independent PLD activity that is specific for a subpopulation of PC and distinct from the PLD activity induced by PKC activity induced by phorbol esters. The diacylglycerol produced from PC by the action of the v-Src-induced PLD may therefore be responsible for the activation of PKC by v-Src.

3T3 Cells↗
PubMed1993

A dominant negative Raf-1 mutant prevents v-Src-induced transformation.

A vector expressing a dominant negative mutant of Raf-1 was stably introduced into BALB/c 3T3 cells expressing a temperature-sensitive derivative of v-Src. High levels of the Raf-mutant were detected in these cells. Expression of the Raf-1 mutant blocked v-Src-induced transformation, as determined by reversion to a flat non-transformed morphology and the inability to form colonies in soft agar. Cells transfected with the parental vector lacking the mutant Raf-1 could be transformed by v-Src. These data suggest that intracellular signals activated by v-Src that are mediated by Raf-1 are required for transformation by v-Src.

3T3 Cells↗
PubMed1993

Intracellular signalling mediated by protein-tyrosine kinases: networking through phospholipid metabolism.

In recent years, it has become apparent that receptor-mediated intracellular signals are not linear cascades beginning at the plasma membrane and terminating with the production of a needed metabolite or the induction of gene expression. Instead, complex networks of interactive intracellular signals are activated in response to extracellular stimuli. Many responses to extracellular stimuli are mediated by protein-tyrosine kinases (PTKs). Activating PTKs leads to the recruitment of a variety of intracellular signalling molecules that execute a complex set of instructions. The response to PTK activity is dependent upon which PTK is activated and the cellular context in which the PTK exists. Several signalling molecules recruited by PTKs are involved in the metabolism of phospholipids. In this Mini Review, intracellular signalling networks activated by PTKs are discussed with an emphasis on the potential for generating highly specific and sophisticated responses to PTK activity through phospholipid metabolism.

Animals↗
PubMed1993

Ha-Ras functions downstream from protein kinase C in v-Fps-induced gene expression mediated by TPA response elements.

v-Fps activates promoters under the control of the 12-O-tetradecanoyl phorbol 13-acetate (TPA) response element (TRE). The induction of TRE-mediated transcription by v-Fps was sensitive to a dominant-negative mutant of Ha-Ras. An activated derivative of Ha-Ras, v-Ha-Ras, also activated TRE-mediated transcription. v-Fps-induced TRE-mediated gene expression was sensitive to depleting cells of protein kinase C (PKC), whereas v-Ha-Ras-induced TRE-mediated transcription was insensitive to PKC depletion, suggesting that Ha-Ras functions downstream from PKC in v-Fps-induced TRE-mediated gene expression. Consistent with this hypothesis, the induction of TRE-mediated gene expression by phorbol esters that activate PKC directly was blocked by the dominant-negative Ha-Ras mutant. Thus, v-Fps-induced activation of TRE-mediated gene expression is via an intracellular signaling mechanism that is dependent upon both PKC and Ha-Ras and Ha-Ras functions downstream from PKC.

3T3 Cells↗
PubMed1992

Evidence that Ha-Ras mediates two distinguishable intracellular signals activated by v-Src.

v-Src activates promoters under the control of 12-O-tetradecanoylphorbol-13-acetate (TPA) response elements (TREs) and serum response elements (SREs) via two distinguishable intracellular signaling mechanisms. The induction of TRE- and SRE-mediated gene expression by v-Src could be distinguished by a differential sensitivity to depleting cells of protein kinase C (PKC) and to a dominant negative Raf-1 mutant. Thus, PKC depletion and the dominant negative Raf-1 mutant were able to distinguish two intracellular signaling mechanisms activated by v-Src. Both of these v-Src-induced intracellular signals were sensitive to a dominant negative mutant of Ha-Ras. These data suggest that Ha-Ras functions to coordinately regulate multiple intracellular signaling mechanisms activated by v-Src.

3T3 Cells↗
PubMed1992

Influenza vaccine effectiveness in preventing hospitalization for pneumonia in the elderly.

During the winter of 1989-1990, influenza type A(H3N2) circulated widely, causing excess morbidity and mortality nationwide. From November through April, 1989-1990, hospitalized cases of pneumonia and influenza occurring among noninstitutionalized individuals 65 or more years of age were identified by 20 acute care hospitals in southern lower Michigan. These cases were group matched on age, sex, race, and zip code to randomly sampled, community-based controls from a comprehensive listing of Medicare beneficiaries residing in the study area. Self-reported data were collected from cases and controls on influenza vaccine status for the 1989-1990 season and on a number of other factors which could have influenced vaccination status or outcome. Questionnaires were completed by 1,907 individuals, 449 of whom were cases, resulting in an overall response rate of 76%. A community-based influenza surveillance system was implemented to determine the timing and intensity of viral activity and influenza-like illness. Vaccine effectiveness in preventing overall pneumonia and influenza hospitalizations was estimated by logistic regression. During the 3-month period of surveillance-confirmed peak influenza type A(H3N2) circulation, vaccine effectiveness was 45% (95% confidence interval 14-64, p = 0.009). However, during the 3-month period of low or absent virus activity, identical methodology and model specification resulted in an effectiveness estimate of 21% that was not statistically different from zero (p = 0.36). The effectiveness determined during the peak period of virus circulation is felt to be a conservative estimate, since agents other than influenza are responsible for pneumonia and influenza hospitalizations, even during times of peak influenza activity.

Aged↗
PubMed1992

v-Fps-responsiveness in the Egr-1 promoter is mediated by serum response elements.

Egr-1, a mitogen-responsive transcription factor, is rapidly induced by v-Fps in the absence of protein synthesis. Thus, Egr-1 is a primary response to the protein-tyrosine kinase activity of v-Fps. To determine the v-Fps-responsive elements in the Egr-1 promoter, deletion mutants of the Egr-1 promoter were used in transient expression assays. A v-Fps expression vector was contransfected into NIH 3T3 cells with chloramphenicol acetyl transferase (CAT) gene expression vectors under the control of the Egr-1 promoter or the Egr-1 promoter containing various deletions. Responsiveness to v-Fps was restricted to a region that contained repeated CC(A/T)6GG sequences, known as CArG boxes. CArG boxes form the core of serum response element (SREs). v-Fps-induced Egr-1 promoter activation was lost by sequential removal of four tandemly repeated SREs. This region, containing four SREs, was found to be sufficient for maximal Egr-1 induction by v-Fps when placed upstream from a heterologous promoter. Individual SREs from this region were able to respond to v-Fps, however, the activation of the individual SREs was lower than that observed for the clustered SREs. These data suggest that v-Fps-responsiveness in the Egr-1 promoter is mediated by SREs.

3T3 Cells↗
PubMed1992

MARCKS protein is transcriptionally down-regulated in v-Src-transformed BALB/c 3T3 cells.

Activation of protein kinase C (PKC) by tumor-promoting phorbol esters leads to the phosphorylation of an 80-kilodalton PKC substrate (known as MARCKS) in murine fibroblasts. In BALB/c 3T3 cells stably transformed by v-Src, phorbol esters were unable to induce phosphorylation of MARCKS. Western blot analysis and in vitro kinase assays showed that both PKC protein levels and kinase activity were unchanged in v-Src-transformed relative to the parental nontransformed BALB/c 3T3 cells. However, MARCKS protein levels were reduced in v-Src-transformed cells relative to nontransformed cells. MARCKS RNA levels were also correspondingly reduced in v-Src-transformed cells. Nuclear "run-on" assays showed decreased transcription of MARCKS in v-Src-transformed cells. Thus, the absence of MARCKS in v-Src-transformed cells could be explained by a down-regulation of MARCKS transcription. Inhibiting the protein tyrosine kinase activity of v-Src with herbimycin A restored MARCKS RNA levels, MARCKS transcription, and MARCKS protein, suggesting that down-regulation of MARCKS in v-Src-transformed BALB/c 3T3 cells is a direct effect of v-Src.

3T3 Cells↗
PubMed1992

The induction of Egr-1 expression by v-Fps is via a protein kinase C-independent intracellular signal that is sequentially dependent upon HaRas and Raf-1.

Activating the protein-tyrosine kinase activity of v-Fps leads to the rapid transcriptional activation of the Egr-1 gene, which encodes a mitogen-responsive transcription factor. Activation of Egr-1 by v-Fps was insensitive to protein kinase C depletion, suggesting that a protein kinase C-independent signal activated by v-Fps leads to the induction of Egr-1. Expression of v-Fps in transient expression assays induced Egr-1 promoter activation. v-HaRas and v-Raf also activated the Egr-1 promoter. To characterize HaRas and Raf-1 involvement in v-Fps-induced Egr-1 expression, we used recently characterized dominant negative mutants of HaRas and Raf-1. v-Fps-induced Egr-1 promoter activation was inhibited by the dominant negative mutants of both HaRas and Raf-1. v-HaRas-induced Egr-1 promoter activation was blocked by the negative Raf-1 mutant; however, v-Raf-1-induced Egr-1 promoter activation was unaffected by the inhibitory HaRas mutant. These data suggest that v-Fps activates a protein kinase C-independent intracellular signaling pathway that is dependent on both HaRas and Raf-1, where Raf-1 functions downstream of HaRas.

Animals↗
PubMed1992

Cloning and characterization of a thermolabile v-src gene for use in reversible transformation of mammalian cells.

The use of temperature-sensitive (ts) src mutants for studies of cell transformation and differentiation has been limited by the availability of cloned ts-src genes that are inactivated at temperatures compatible with growth of mammalian cells. In this report, we describe the cloning and characterization of the tsLA90src gene, which displays tight thermal sensitivity at 39.5 degrees C. Nucleotide sequence comparison of tsLA90 and wild-type src genes from the Schmidt-Ruppin subgroup A and D strains of Rous sarcoma virus (RSV) revealed four amino acid differences in tsLA90src. Substitution of one of these residues (Lys-280) from tsLA90src with its wild-type homolog (Glu-280) caused a reversion to a wild-type src phenotype. The cloned tsLA90 gene, designated tsUP1, was introduced into avian and mammalian retroviral vectors. Chicken embryo fibroblasts and immortalized mouse 3T3 cells infected with these viral vectors displayed a temperature-dependent transformed phenotype as assessed by cell morphology, secretion of plasminogen activator, transcriptional activation of the primary response genes, Egr-1 and TIS 10, and stimulation of tyrosine phosphorylation. In addition, chicken myoblasts (infected with RSVtsUP1) showed a temperature-dependent differentiation into myotubes. Thus, this cloned src gene should be ideally suited for inducing reversible transformation and differentiation of mammalian cells in culture.

3T3 Cells↗
PubMed1992

Evidence that activation of the Egr-1 promoter by v-Raf involves serum response elements.

The constitutively active serine/threonine kinase encoded by the v-raf oncogene, v-Raf, activates the Egr-1 promoter in transient expression assays. To characterize the v-Raf-responsive transcriptional control elements, deletion mutants of the Egr-1 promoter were used in transient expression assays. A v-Raf expression vector was co-transfected into NIH3T3 cells with reporter chloramphenicol acetyl transferase (CAT) expression vectors under the control of the Egr-1 promoter or the Egr-1 promoter containing various deletions. Responsiveness to v-Raf was restricted to a region that contained repeated CC(A/T)6GG sequences, known as CArG boxes. CArG boxes form the core of serum response elements (SREs). v-Raf-induced Egr-1 promoter activation was lost by removal of the four tandemly repeated SREs. This region, between -425 and -250, which was necessary for v-Raf responsiveness, was also found to be sufficient for maximal Egr-1 induction by v-Raf when placed upstream from a minimal heterologous promoter. Three out of four SREs from this region were able to respond to v-Raf, however the activation of the individual SREs was lower than the clustered SREs. This cluster of SREs has previously been shown to be responsive to several mitogenic stimuli and the oncogene v-src. Thus, the SREs contained in this cluster may be an important target for cell division signals.

3T3 Cells↗
PubMed1992

Sustained induction of egr-1 by v-src correlates with a lack of fos-mediated repression of the egr-1 promoter.

Serum stimulation of quiescent fibroblasts leads to a transient induction of the transcription factor egr-1. However, the induction of egr-1 by v-src was found to be sustained rather than transient. The proto-oncogene fos has been reported to be co-regulated with egr-1 and to repress serum-induced egr-1 expression. We found that c-fos prevents v-src-induced gene expression regulated by the egr-1 promoter. Thus, the sustained induction of egr-1 by v-src could be explained by a lack of c-fos induction by v-src. Consistent with this hypothesis, egr-1 and c-fos were co-induced by serum, but not by v-src, in Balb/c 3T3 cells; v-src did not induce c-fos expression in these cells. We propose that sustained expression of egr-1 induced by v-src in Balb/c 3T3 cells is due to a lack of c-fos down-regulation of egr-1.

3T3 Cells↗
PubMed1991

An inhibitory mutant of c-Raf-1 blocks v-Src-induced activation of the Egr-1 promoter.

The protein-tyrosine kinase activity of v-Src leads to the transcriptional activation of the mitogen-responsive transcription factor Egr-1. c-Raf-1 is a serine/threonine protein kinase that has been implicated in the transduction of signals induced by mitogens. The involvement of c-Raf-1 in v-Src-induced Egr-1 expression was investigated using an inhibitory mutant of c-Raf-1. We report here that expression of a kinase-defective mutant of c-Raf-1 inhibits v-Src-induced activation of the Egr-1 promoter. This inhibition is reversed by overexpression of wild type c-Raf-1. Consistent with an involvement of c-Raf-1 in a signaling pathway leading to Egr-1 expression, we find that v-Raf induces Egr-1 promoter activation. These data suggest that c-Raf-1 is a component in an intracellular signaling pathway initiated by v-Src leading to the induction of the mitogen-responsive transcription factor Egr-1.

3T3 Cells↗
PubMed1991

Evidence that a G-protein transduces signals initiated by the protein-tyrosine kinase v-Fps.

The protein-tyrosine kinase (PTK) v-Fps induces protein kinase C (PKC)-dependent expression of the transformation-related 9E3 gene in chicken embryo fibroblasts (Spangler, R., Joseph, C., Qureshi, S.A., Berg, K., and Foster, D.A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7017-7021). We present evidence here that a GTP-binding protein (G-protein) is a component of this PKC-dependent signaling pathway. 1) A GTP analogue that stimulates G-protein-mediated signals induced 9E3 gene expression. 2) A GDP analogue that inhibits signaling through G-proteins inhibited expression of 9E3 and phosphorylation of a 67-kDa PKC substrate induced by v-Fps. The GDP analogue had no effect on phosphorylation of the PKC substrate or the expression of 9E3 induced by direct activation of PKC with phorbol ester. 3) Increased v-Fps PTK activity led to increased GTP binding to a 50-kDa protein. The molecular weight of this GTP-binding protein is consistent with the molecular weight of alpha-subunits of G-proteins of the heterotrimeric class. The data suggest that a G-protein functions upstream from PKC in a signaling pathway that connects v-Fps PTK activity to increased 9E3 gene expression.

Animals↗
PubMed1991

v-Src activates mitogen-responsive transcription factor Egr-1 via serum response elements.

Activating the protein-tyrosine kinase activity of v-Src in murine fibroblasts leads to increased expression of Egr-1, a mitogen-responsive transcription factor. v-Src-induced expression of Egr-1 is independent of protein synthesis and is controlled at the level of transcription. Target sequences responsive to v-Src-induced signals were investigated using deletion mutant and analysis of the Egr-1 promoter. Upstream Egr-1 promoter sequences linked to a reporter gene were cotransfected with a v-Src expression vector into NIH 3T3 cells. v-Src-enhanced gene expression from the Egr-1 promoter was dependent upon the presence of CC(A/T)6GG elements. The CC(A/T)6GG motif forms the core element of serum response elements (SREs) and is the binding site for serum response factor. The Egr-1 promoter sequences responsive to v-Src contained four SREs. Sequential deletion of these SREs reduced v-Src responsiveness to basal transcription levels. A single SRE from this region was able to confer v-Src responsiveness to a heterologous promoter, and a mutation to the CC(A/T)6GG box of this SRE abolished v-Src-enhanced gene expression. Thus, an early response of v-Src-induced intracellular signaling is the transcriptional activation of a growth factor-responsive transcription factor via an SRE.

Animals↗