Functional interactions of the ras oncogene with protein kinase C and RasGTPase activating protein
Abstract
To identify the molecular mechanism by which Ras regulates cellular proliferation, we have analyzed the role of biochemical changes introduced by activated ras. Analysis of temporal aspects of the regulation of Protein kinase C (PKC) by an activated c-Ha-ras gene (T24ras), in C3H 10T1/2 fibroblasts with exogenously inducible expression of T24ras (MTras cell lines) indicated that down-regulation of PKC in the MTras cells occurred late following Ras expression and concurrent with morphological alterations. Earlier studies had showed that Protein kinase C (PKC) levels were partially lowered ('down-regulated') by constitutive expression of the T24ras oncogene (ras9B and ras 11A cell lines) in C3H 10T1/2 cells. Ras9B and ras11A cell lines expressed lower levels of PKC$\alpha$ and PKC$\epsilon$ mRNA than C3H 10T1/2 fibroblasts. Down-regulation of PKC$\alpha$ mRNA in the inducible cell lines occurred at doses of inducer where protein and activity decreases also occurred. This implies that a decrease in PKC$\alpha$ mRNA is causal in T24ras-induced down-regulation of PKC. Expression of Krev-l, the ras-related, ras-revertant gene, in normal C3H 10T1/2 fibroblasts (10Trev cells) inhibited proliferation and antagonized serum- and phorbol ester-induced of the extra-cellular signal regulated kinases (ERKs). It was hypothesized that comparative studies of ras-transformed cells and cells reverted from ras transformation, by Krev-1, would permit identification of specific biochemical effects of ras necessary for transformation since these may be blocked in revertant cell lines. Levels of PKC-specific kinase activity, immunoreactive PKC, and PKC$\alpha$ mRNA were significantly higher in Krev-1-reverted cells (9Brev cells) than those in the parent ras9B cells in apparent reversion of ras-induced down-regulation of PKC. Levels of PKC$\epsilon$ mRNA were not up-regulated in the 9Brev cell lines indicating that this effect of T24ras was blocked by the expression of Krev-1 and that reversion of morphology did not require a reversion in PKC$\epsilon$ mRNA levels. Higher levels of RasGTPase activating protein (RasGAP), a cytosolic candidate as a Ras-effector, fractionated with the membrane (where p21$\sp{ras}$ is also present) and the cytoskeleton in the ras9B and ras 11A cells than in C3H 10T1/2 or 9Brev cells. Temporal analysis with the MTras cells indicated that RasGAP localization to the particulate fractions occurs early following expression of T24Ras and prior to marked morphological alteration. These results suggest that T24ras-programmed, transcriptionally-mediated, down-regulation of PKC, and alterations in sub-cellular localization of RasGAP may be involved in T24Ras-regulated pathways for morphological alteration. Krev-1 can block these biochemical effects of oncogenic Ras and such interference may be involved in its ras-reverting actions. Krev-l also can interfere with functional signaling along Ras-dependent mitogenic pathways and this is may be its normal cellular function.
Degree
Ph.D.
Advisors
Ashendel, Purdue University.
Subject Area
Biochemistry|Molecular biology
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