Newly synthesized Rho A, not Ras, is isoprenylated and translocated to membranes coincident with progression of the G1 to S phase of growth-stimulated rat FRTL-5 cells

Ras and Rho are involved in the regulation of signal transduction events governing cell growth and cell proliferation. Protein prenylation is essential for the activation and/or the translocation of these small GTPases; however, protein geranylgeranylation rather than farnesylation is required for G(1)/S transition. We studied prenylation and translocation of Ras and Rho A during G(1)/S progression in growth-stimulated rat thyroid FRTL-5 cells. Immunoblot analysis revealed that both Ras and Rho A were detected in membrane fractions at G(0). Rho A was eliminated from the membrane fraction during G(1) and was not detected on the membrane at mid-G(1). Translocation of Rho A from the cytoplasm back to the membranes was observed during late G(1) phase. In contrast, Ras remains in the membrane fraction through the cell cycle progression from G(1) to S phase. The immunoprecipitation of Rho A from the membrane fraction demonstrated that newly synthesized Rho A, labeled by pulsing cells with [S-35]methionine and [S-35]cysteine, was geranylgeranylated and associated with the membrane in late G(1). These results indicate that Rho A, not Ras, was eliminated from membrane fraction during G(1) progression and that newly synthesized Rho A is geranylgeranylated and translocated to membranes during G(1)/S progression in growth-stimulated FRTL-5 cells.