Mutational and Biochemical Analysis of Plasma Membrane Targeting Mediated by the Farnesylated, Polybasic Carboxy Terminus of K-ras4B

Abstract

Mutational analysis and in vitro assays of membrane association have been combined to investigate the mechanism of plasma membrane targeting mediated by the farnesylated, polybasic carboxy-terminal sequence of K-ras4B in mammalian cells. Fluorescence-microscopic localization of chimeric proteins linking the enhanced green fluorescent protein (EGFP) to the K-ras4B carboxy-terminal sequence, or to variant forms of this sequence, reveals that the normal structure of this targeting motif can be greatly altered without compromising plasma membrane-targeting activity so long as an overall strongly polybasic/amphiphilic character is retained. An EGFP/K-ras4B(171-188) chimeric protein was readily abstracted from isolated cell membranes by negatively charged lipid vesicles, and this abstraction was markedly enhanced by the anionic lipid-binding agent neomycin. Our results strongly favor a mechanism in which at the plasma membrane the carboxy-terminal sequence of K-ras4B associates not with a classical specific proteinaceous receptor but rather with nonspecific but highly anionic 'sites' formed at least in part by the membrane lipid bilayer. Our findings also suggest that the recently demonstrated prenylation-dependent trafficking of immature forms of K-ras4B through the endoplasmic reticulum [Choy et al. (1999) Cell 98, 69-80], while required for maturation of the protein, beyond this stage may not be essential to allow the ultimate delivery of the mature protein to the plasma membrane.

Phosphorylated K-Ras limits cell survival by blocking Bcl-xL sensitization of inositol trisphosphate receptors

“…A large body of evidence supports a model whereby the polybasic region of KRas4B contributes to membrane affinity through an electrostatic interaction with the inner leaflet of the plasma membrane (4,5,20,21). Phosphorylation of serine 181 sufficiently weakens this interaction to cause K-Ras4B to dissociate from the plasma membrane.…”

High Affinity for Farnesyltransferase and Alternative Prenylation Contribute Individually to K-Ras4B Resistance to Farnesyltransferase Inhibitors

“…Our observation that anchorage-independent growth of NIH 3T3 cells stably expressing K-Ras4B is partially sensitive to FTI although this Ras isoform contains both a methioninecontaining CAAX motif and a polybasic domain, becomes alternatively prenylated, and signals efficiently through Elk-1 in the presence of FTI suggests that H-Ras and K-Ras4B are functionally distinct, a hypothesis for which considerable evi- NARAERRREK CRCV ND dence has accumulated (31)(32)(33)(34)51). In our studies, K-Ras and H-(K 6 )-CVIM appear to induce anchorage-independent growth via incompletely overlapping mechanisms that are not equally affected by FTI.…”

“…Several physical and biological characteristics of K-Ras4B have been shown to be dependent on the polybasic domain. The polybasic domain has been shown to be required for 1) K-Ras4B to transform NIH 3T3 and Rat-1 cells (53), 2) K-Ras4B membrane localization (33), 3) formation of ionic interactions between the polybasic domain and membrane anionic lipids to stabilize membrane association (33,54), and 4) K-Ras4B association with microtubules (55), an interaction that has implications for differential subcellular transport and localization of Ras isoforms. Our work adds a new function to this list by demonstrating the role of the polybasic domain in increasing Ras affinity for FTase, thereby resulting in enhanced functional Ras FTI resistance and suggesting that FTase may distinguish among Ras proteins in part through the polybasic domain.…”

“…These differences may result from functional differences between H-Ras and K-Ras4B that are unrelated to prenylation. Several lines of evidence have suggested that H-Ras and K-Ras4B are functionally distinct, possibly related to their different microlocalization (31)(32)(33)(34). If this is the case, K-Ras4B-induced soft agar colony growth may depend more strongly on the function of other farnesylated proteins (35,36) that are themselves sensitive to FTI, whereas H-Ras-induced colony growth may rely less on other farnesylated proteins.…”

H-Ras Does Not Need COP I- or COP II-dependent Vesicular Transport to Reach the Plasma Membrane

“…There, farnesylated Ras proteins are further modified by an ER 2 -resident prenyl-dependent protease (Rce1), and by a second ER-resident methyltransferase enzyme (Icmt) (6,7). At this point the K-Ras4B protein is released from the surface of the ER and rapidly appears at the plasma membrane (3,8,9). No internal membrane or vesicular intermediates have been observed for this step, and the transport mechanism is currently unknown (10).…”