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Biomedical subjects

A R Olivier

Publications and source records attributed to A R Olivier.

13 recordsLinked to original sources
PubMed1996

The co-mitogenic combination of transforming growth factor beta 1 and bombesin protects protein kinase C-delta from late-phase down-regulation, despite synergy in diacylglycerol accumulation.

Bombesin induces the down-regulation of protein kinase C-delta (PKC-delta) and PKC-epsilon in Swiss 3T3 cells. Simultaneous addition of transforming growth factor beta 1 (TGF beta 1) selectively blocks PKC-delta down-regulation at mid-S-phase, whereas PKC-epsilon levels continue to decline. Northern blot analysis shows that PKC-epsilon levels could be controlled in part at the level of transcription; PKC-delta mRNA levels remained constant at these later times. Bombesin induces a sustained elevation of some species of diacylglycerol (DAG), consistent with the observed loss of PKC-delta and PKC-epsilon. Interestingly, the combination of bombesin and TGF-beta 1 produces an even greater DAG response. Flow cytometric analysis demonstrates that bombesin induces only 15% of the cells to enter the cell cycle, in contrast to the combination of TGF beta 1 plus bombesin which induces 75-80% of the cells to progress through the cycle. The protection of PKC-delta from down-regulation under conditions of sustained DAG elevation correlates with the mitogenic response and implies that the down-regulation process itself is regulated. Consistent with this, it is demonstrated that bombesin plus TGF beta 1 protects PKC-delta from phorbol ester-induced down-regulation.

3T3 Cells↗
PubMed1994

Bombesin, platelet-derived growth factor, and diacylglycerol induce selective membrane association and down-regulation of protein kinase C isotypes in Swiss 3T3 cells.

Swiss 3T3 cells contain protein kinase C (PKC) isotypes alpha, delta, epsilon and zeta (Olivier, A. R., and Parker, P. J. (1992) J. Cell. Physiol. 152, 240-244). Acute stimulation of quiescent cells with the neuropeptide bombesin decreases the mobility of PKC-delta and PKC-epsilon on SDS-polyacrylamide gels. These slower migrating forms of PKC-delta and PKC-epsilon rapidly (within 1 s) and selectively are found associated with the Triton X-100-soluble membrane fraction. No change in the mobility or distribution of PKC-alpha or PKC-zeta is detected. Long-term treatment of cells with bombesin induces selective membrane association and down-regulation of PKC-delta and PKC-epsilon (decreasing 70 and 65%, respectively). No change in the long-term distribution of PKC-alpha and PKC-zeta was detected. Bombesin did, however, increase PKC-alpha protein levels by 60% compared to control cells. PKC-zeta levels remained unchanged. Both the shift in mobility and down-regulation of PKC-delta and PKC-epsilon were only induced by mitogenic doses of bombesin. The potent mitogen platelet-derived growth factor induced similar effects on the PKC isotypes delta and epsilon. PKC-alpha and PKC-zeta levels were unaffected. Repeated doses of the synthetic diglyceride 1-oleoyl-2-acetyl-sn-glycerol induced PKC-delta and PKC-epsilon down-regulation and stimulated the cells to divide. Again PKC-alpha and PKC-zeta levels were unaffected. These results show a correlation between the membrane association and down-regulation of PKC-delta and PKC-epsilon and the entry of cells into S phase.

3T3 Cells↗
PubMed1994

12-Deoxyphorbol-13-O-phenylacetate-20-acetate is not protein kinase C-beta isozyme-selective in vivo.

The phorbol ester, 12-deoxyphorbol-13-O-phenylacetate-20-acetate (DOPPA) has been shown to activate specifically the protein kinase C (PKC)-beta 1 isozyme in vitro (1). We have investigated the potential of DOPPA as a PKC-beta 1/2 isozyme-specific agonist in intact cells, employing U937 cells, which express beta 1/2, epsilon and zeta PKC and in Swiss 3T3 cells which lack PKC-beta 1/2 but express alpha, delta, epsilon and zeta PKC. Immunoblot analysis with isozyme-specific antibodies indicated that DOPPA can mediate the subcellular redistribution and down-modulation of all endogenous PKC isozymes (except PKC-zeta) in both U937 and Swiss 3T3 cells. Prolonged treatment (> 6 h) of cultures in down-modulation studies is complicated by the metabolism of DOPPA to 12-deoxyphorbol-13-phenylacetate (DOPP), a compound which activates all PKC isozymes tested in vitro (Ryves, W. J., et al. (1991) FEBS Lett., 288, 5-9). Nevertheless, because DOPPA induced rapid and dose-dependent phosphorylation of p80 in cells which do not express PCK-beta, p80 phosphorylation in Swiss 3T3 cells indicates that DOPPA can activate a non-beta PKC in vivo. The data suggest that DOPPA cannot be used as a PKC-beta-selective agonist in intact cell studies.

3T3 Cells↗
PubMed1992

Identification of multiple PKC isoforms in Swiss 3T3 cells: differential down-regulation by phorbol ester.

The expression of members of the Ca2+ and phospholipid-dependent protein kinase (PKC) family were studied in murine Swiss 3T3 cells. In addition to PKC-alpha, the presence of immunoreactive PKC-delta, -epsilon, and zeta was detected. Treatment with 500 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) led to the down-regulation of alpha, delta, and epsilon isoforms, but not that of zeta. Higher concentrations of TPA similarly had no effect on the level of PKC-zeta. In contrast to PKC-alpha, the membrane localization of PKC-delta, -epsilon, and -zeta was not enhanced by extraction in Ca(2+)-containing buffers, whereas acute TPA treatment increased membrane association of PKC-alpha, -delta, and -epsilon but not that of PKC-zeta.

3T3 Cells↗
PubMed1991

Expression and characterization of protein kinase C-delta.

A cDNA encoding protein kinase C-delta (PKC-delta) was isolated from a rat brain library. The coding region was subcloned into the expression vector pmt2 and transfected into COS-1 cells. Expression of the protein led to an 11-fold increase in activity as determined with a synthetic peptide based on the PKC-delta pseudosubstrate site. The Mr of PKC-delta as determined by SDS/PAGE and immunoblot analysis using anti-(PKC-delta C-terminal) antibodies was 77,000. The enzyme was purified to near homogeneity and showed total dependency on phospholipid and diacylglycerol (or phorbol esters) for activity. Like PKC-epsilon, PKC-delta displays no Ca2+ dependence for activation. The substrate specificity of PCK-delta is similar to that of PKC-epsilon but quite different from other PKCs.

Amino Acid Sequence↗
PubMed1991

Activation of the PKC-isotypes alpha, beta 1, gamma, delta and epsilon by phorbol esters of different biological activities.

Phorbol esters, tetradecanoylphorbolacetate, sapintoxin-A, 12-deoxyphorbol-phenylacetate, 12-deoxyphorbol-phenylacetate-20-acetate, thymeleatoxin and resiniferatoxin were investigated for their abilities to activate the PKC-isotypes alpha, beta 1, gamma, delta and epsilon. PKC-isotypes were grouped into two classes on the basis of Ca2+ requirements for activation by phorbol esters; alpha, beta 1, and gamma being Ca(2+)-dependent forms and delta and epsilon being Ca(2+)-independent. PKC-isotype selective activation by phorbol esters was observed in that SAPA failed to activate PKC-delta up to a concentration of 1000 ng.ml-1 and DOPPA only activated PKC-beta 1 over the same range of concentrations.

Animals↗
PubMed1990

Three forms of phosphatase type 1 in Swiss 3T3 fibroblasts. Free catalytic subunit appears to mediate s6 dephosphorylation.

The major 40 S ribosomal protein S6 phosphatase in Swiss mouse 3T3 fibroblasts is a type 1 enzyme (Olivier, A. R., Ballou, L. M., and Thomas, G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4720-4724). Polyclonal antibodies were raised against a synthetic peptide containing the carboxyl-terminal 14 amino acids of the catalytic subunit of phosphatase 1 (PP-1C). Results from Western blot analysis and immunoprecipitation show that the peptide antiserum specifically recognizes PP-1C in cell extracts. Anion-exchange chromatography of cell extracts and Western blot analysis revealed three peaks of PP-1C termed A, B, and C. Peaks A and C are associated with the major type 1 S6 phosphatase activities, but peak B exhibits little activity. The phosphatase in peak A (Mr 39,000) appears to represent the free catalytic subunit, whereas the enzymes in peaks B and C display sizes of 68,000-140,000. Peak B contains two additional proteins of Mr 26,000 and 48,000 that co-immunoprecipitate with PP-1C, while peak C has a single additional protein of Mr 100,000. Fifteen min after serum withdrawal there is a 2-fold stimulation of S6 phosphatase activity in peak A that can be accounted for by an increase in the amount of PP-1C. The amount of PP-1C in the inactive peak B fraction also increases during this time and this increase is associated with changes in the phosphorylation state of the Mr 26,000 and 48,000 proteins. The results are discussed in relation to regulatory mechanisms which are thought to modulate the activity of type 1 phosphatase.

Animals↗
PubMed1989

EGF induces biphasic S6 kinase activation: late phase is protein kinase C-dependent and contributes to mitogenicity.

Detailed kinetics reveal that EGF-induced S6 kinase activation is biphasic: an early phase appears at 10-15 min, followed by a late phase between 30 and 60 min. Both activities exhibit the same chromatographic behavior and sensitivity to phosphatase 2A. Direct activation of protein kinase C by TPA induces only late phase activity. Down-regulation of protein kinase C leads to loss of both TPA- and EGF-induced late phase activity, while the early phase is unaffected. The loss of late phase kinase activity results in decreased EGF-induced S6 phosphorylation, protein synthesis, and cell growth. The results indicate that EGF differentially regulates S6 kinase activation by two distinct signaling pathways and that loss of the late or protein kinase C-dependent phase leads to a diminished mitogenic response.

Animals↗
PubMed1988

Differential regulation of S6 phosphorylation by insulin and epidermal growth factor in Swiss mouse 3T3 cells: insulin activation of type 1 phosphatase.

Insulin and epidermal growth factor (EGF) induce distinct kinetics of S6 kinase activation and S6 phosphorylation in Swiss 3T3 cells. Both events are differentially regulated by specific phosphatases. The major S6 phosphatase in cell extracts was identified as a type 1 enzyme by its chromatographic properties, its sensitivity to inhibitor 2, and its substrate specificity. This enzyme is different from the major S6 kinase phosphatase, which is a type 2A enzyme. Insulin at physiological concentrations causes up to a 2-fold activation of a type 1 S6 phosphatase, whereas at higher concentrations this effect is significantly diminished. EGF alone has little effect on this enzyme, and with both agents together the total phosphatase activity remains basal. The results are consistent with the phosphorylation state of S6 observed in vivo and suggest a role of phosphatase type 1 in the regulation of protein synthesis.

Animals↗