Abstract
Ras proteins serve as critical relays in signal transduction pathways that control growth and differentiation and must undergo posttranslational modifications before they become functional. While it is established that farnesylation is necessary for membrane binding and cellular functions of all Ras proteins, the significance of palmitoylation is unclear. We have studied the contribution of Ha-Ras palmitoylation for biological activity in Xenopus oocytes. In contrast to wild-type Ha-Ras, which binds to membranes and induces meiosis when microinjected into oocytes, a nonpalmitoylated but farnesylated and methylated mutant mislocalizes to the cytosol and fails to promote maturation. This lack of responsiveness correlates with the inability of the mutant to induce phosphorylation and activation of mitogen-activated protein kinase and maturation promoting factor, which are both strongly activated by wild-type Ha-Ras. Costimulation of oocytes with insulin increases their responsiveness to Ras and partially rescues the biological activity of the palmitoylation-resistant mutant. However, 25-50 times higher doses of mutant were required to elicit responses equivalent to wild-type Ha-Ras. These results suggest that palmitoylation and membrane association of Ha-Ras is necessary for efficient activation of the mitogen-activated protein kinase cascade in vivo and are consistent with a biochemical function for Ras as a membrane targeting signal for downstream effectors in this pathway.
Highlights
§ To whom correspondence should be addressed: Depts. of Chemistry and Biochemistry, Box 351700, University of Washington, Seattle, WA 98195-1700
We have analyzed the functional significance of Ha-Ras palmitoylation for signal transduction in vivo using the Xenopus oocyte system
Our results show that while WT Ha-Ras is predominantly membrane-bound and promotes activation of mitogenactivated protein kinase (MAPK) and meiotic maturation when microinjected into stage VI oocytes, a nonpalmitoylated but farnesylated and methylated mutant remains cytosolic and is inactive under these conditions (Fig. 2)
Summary
§ To whom correspondence should be addressed: Depts. of Chemistry and Biochemistry, Box 351700, University of Washington, Seattle, WA 98195-1700. In order to clarify the functional significance of Ha-Ras palmitoylation and membrane targeting for the activation of signal transduction cascades and biological responses in vivo, we have used Xenopus laevis oocytes as an experimental system.
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