K-Ras associates with the plasma membrane (PM) through farnesylation that functions in conjunction with an adjacent polybasic sequence. We show that phosphorylation by protein kinase C (PKC) of S181 within the polybasic region promotes rapid dissociation of K-Ras from the PM and association with intracellular membranes, including the outer membrane of mitochondria where phospho-K-Ras interacts with Bcl-XL. PKC agonists promote apoptosis of cells transformed with oncogenic K-Ras in a S181-dependent manner. K-Ras with a phosphomimetic residue at position 181 induces apoptosis via a pathway that requires Bcl-XL. The PKC agonist bryostatin-1 inhibited the growth in vitro and in vivo of cells transformed with oncogenic K-Ras in a S181-dependent fashion. These data demonstrate that the location and function of K-Ras are regulated directly by PKC and suggest an approach to therapy of K-Ras-dependent tumors with agents that stimulate phosphorylation of S181.

Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes.

Proteins that terminate with a consensus sequence known as CAAX undergo a series of posttranslational modifications that include polyisoprenylation, endoproteolysis, and carboxyl methylation. These modifications render otherwise hydrophilic proteins hydrophobic at their C termini such that they associate with membranes. Whereas prenylation occurs in the cytosol, postprenylation processing is accomplished on the cytoplasmic surface of the endoplasmic reticulum and Golgi apparatus. Among the numerous CAAX proteins encoded in mammalian genomes are many signaling molecules such as monomeric GTPases, including the Ras proteins that play an important role in cancer. In the course of their processing, nascent Ras proteins traffic from their site of synthesis in the cytosol to the endomembrane and then out to the plasma membrane (PM) by at least two pathways. Recently, retrograde pathways have been discovered that deliver mature Ras from the PM back to the Golgi. The Golgi has been identified as a platform upon which Ras can signal. Thus, the subcellular trafficking of Ras proteins has the potential to increase the complexity of Ras signaling by adding a spatial dimension. The complexity of Ras trafficking also affords a wider array of potential targets for the discovery of drugs that might inhibit tumors by interfering with Ras trafficking.-Wright, L. P., and M. R. Philips. CAAX modification and membrane targeting of Ras. J. Lipid Res. 2006. 47: 883-891. Supplementary key words farnesylation . trafficking . carboxyl methylation Ras proteins have been studied intensively for more than a quarter century because of their role in human cancer. Indeed, Ras was one of the first oncogenes to be identified. At the biochemical level, Ras is the founding member of a large superfamily of monomeric GTPases that function as molecular switches. Ras is also the founding member of a class of peripheral membrane proteins known as CAAX proteins, where C stands for cysteine, A for an aliphatic amino acid, and X for any amino acid. The primary translation product of CAAX protein genes end with a CAAX sequence that serves as a substrate for three enzymes that sequentially modify the sequence to create a lipidated, hydrophobic domain that mediates the association with cellular membranes. These enzymes include two prenyltransferases, farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase), Ras-converting enzyme 1 (Rce1), and isoprenylcysteine carboxyl methyltransferase (Icmt). Plasma membrane (PM) association of Ras was originally understood as the simple and direct consequence of CAAX processing by these enzymes. We now understand that CAAX modification is a highly orchestrated process that takes proteins on a journey through various subcellular compartments and can terminate on compartments other than the PM. An in-depth look at prenyltransferases, the first and best understood of the CAAX processing enzymes, was the focus of the review by Beese in this series (1), and Basso, Kirschmeier, and Bishop (2) described ef...

The CAAX motif at the C terminus of most monomeric GTPases is required for membrane targeting because it signals for a series of three posttranslational modifications that include isoprenylation, endoproteolytic release of the C-terminal-AAX amino acids, and carboxyl methylation of the newly exposed isoprenylcysteine. The individual contributions of these modifications to protein trafficking and function are unknown. To address this issue, we performed a series of experiments with mouse embryonic fibroblasts (MEFs) lacking Rce1 (responsible for removal of the -AAX sequence) or Icmt (responsible for carboxyl methylation of the isoprenylcysteine). In MEFs lacking Rce1 or Icmt, farnesylated Ras proteins were mislocalized. In contrast, the intracellular localizations of geranylgeranylated Rho GTPases were not perturbed. Consistent with the latter finding, RhoGDI binding and actin remodeling were normal in Rce1-and Icmtdeficient cells. Swapping geranylgeranylation for farnesylation on Ras proteins or vice versa on Rho proteins reversed the differential sensitivities to Rce1 and Icmt deficiency. These results suggest that postprenylation CAAX processing is required for proper localization of farnesylated Ras but not geranygeranylated Rho proteins.

Myelin-associated glycoprotein (MAG), which mediates certain myelin-neuron cell-cell interactions, is a lectin that binds to sialylated glycoconjugates. Gangliosides, the most abundant sialylated glycoconjugates in the brain, may be the functional neuronal ligands for MAG. Cells engineered to express MAG on their surface adhered specifically to gangliosides bearing an alpha 2,3-linked N-acetylneuraminic acid on a terminal galactose, with the following relative potency: GQ1b alpha >> GD1a, GT1b >> GM3, GM4 (GM1, GD1b, GD3, and GQ1b did not support adhesion). MAG binding was abrogated by modification of the carboxylic acid, any hydroxyl, or the N-acetyl group of the ganglioside's N-acetylneuraminic acid moiety. Related immunoglobulin (Ig) superfamily members either failed to bind gangliosides (CD22) or bound with less stringent specificity (sialoadhesin), whereas a modified form of MAG (bearing three of its five extra-cellular Ig-like domains) bound only GQ1b alpha. Enzymatic removal of sialic acids from the surface of intact nerve cells altered their functional interaction with myelin. These data are consistent with a role for gangliosides in MAG-neuron interactions.