Abstract
Ras-related proteins are small GTPases that are post-translationally modified with mevalonate-derived isoprenoids. Although the effects of inhibition of isoprenylation on protein function have been examined, the consequences of depletion of isoprenoid pools on regulation of expression of isoprenylated proteins have yet to be investigated. In these studies we have shown that depletion of mevalonate results in increased total levels of Ras, Rap1a, RhoA, and RhoB in K562 cells. Cycloheximide and [(35)S]methionine pulse/pulse-chase experiments reveal that mevalonate depletion increases the de novo synthesis of Ras and RhoA and decreases the degradation of existing Ras and RhoA protein. Pretreatment with actinomycin D completely prevents the induced up-regulation of RhoB and only partially prevents the up-regulation of Ras, Rap1a, and RhoA. Although depletion of mevalonate does not alter steady state levels of Ras mRNA, there is an increase in RhoB mRNA. Our results are the first to demonstrate that mevalonate depletion induces up-regulation of Ras and Ras-related proteins by discrete mechanisms that include modulation of transcriptional, translational, and post-translational processes.
Highlights
Members of the Ras protein superfamily, including Ras, Rap1a, and the Rho proteins, are membrane-bound small GTPases that cycle between an active GTP-bound state and an inactive GDP-bound state
K562 cells were incubated with 10 M lovastatin for 0 –24 h with cells collected for Western blot analysis at 2-h intervals
For Ras this effect could be observed after only 2 h of incubation with lovastatin, as indicated by the appearance of the more slowly migrating band in the Ras immunoblot signifying unmodified Ras protein
Summary
DIFFERENTIAL TRANSCRIPTIONAL, TRANSLATIONAL, AND POST-TRANSLATIONAL UP-REGULATION OF Ras, Rap1a, RhoA, AND RhoB*. The effects of inhibition of isoprenylation on protein function have been examined, the consequences of depletion of isoprenoid pools on regulation of expression of isoprenylated proteins have yet to be investigated. In these studies we have shown that depletion of mevalonate results in increased total levels of Ras, Rap1a, RhoA, and RhoB in K562 cells. To further understand the consequences of mevalonate depletion on expression of the small GTPases that are normally isoprenylated we have investigated the effects of mevalonate depletion on Ras, Rap1a, RhoA, and RhoB expression
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