Anchors away: An alternative role for farnesylation of Ras

Misty D Handley, Purdue University

Abstract

Ras, a 21-kDa membrane-bound GTPase, is a major regulator of cell differentiation, growth, and survival. The ras gene is mutated in 30% of all cancers and in 90% of human pancreatic tumors. Although all three isoforms of Ras, H-, N-, and K-Ras, are farnesylated, they are differentiated by their unique membrane-targeting motifs. Whereas N- and H-Ras are singly and dually palmitoylated, respectively, K-Ras relies on a polybasic domain to help target it to the membrane. These unique membrane-targeting motifs were shown to be responsible for submembrane localization of Ras. Our goal was to determine how the localization of Ras directs its ability to transmit signals from the T cell antigen receptor. Initially, we investigated the presence and membrane microdomain localization of the Ras isoforms using human Jurkat T cells as a model system. Whereas N- and K-Ras isoforms were present in Jurkat E6.1 T cells, H-Ras was undetectable. Using isoform specific antibodies in immunoblotting analyses of detergent soluble and insoluble fractions, we determined that N-Ras localized to non-raft regions of the membrane, whereas K-Ras was located in the detergent insoluble fractions commonly known as lipid rafts. These results were surprising as the non-palmitoylated K-Ras is localized in the non-raft regions of the membrane in fibroblasts. In an effort to alter the localization of Ras, we optimized conditions for modifying cellular Ras with a structurally diverse library of farnesylpyrophosphate analogs synthesized by the Gibbs laboratory. Although localization studies utilizing GFP-N- and K-Ras revealed that incorporation of the farnesylpyrophosphate analogs do not alter the location of the Ras isoforms, incorporation of the farnesylpyrophosphate analogs modulate T cell receptor dependent phosphorylation of Erk. These findings indicate that although the identity of the farnesyl moiety may not play a dominant role in the localization of Ras, the structure of farnesyl plays a role in Ras-mediated signaling.

Degree

Ph.D.

Advisors

Harrison, Purdue University.

Subject Area

Biochemistry

COinS