Ras proteins are signal transducers that regulate cellular growth, differentiation and apoptosis in response to different stimulus (1). Ras proteins act as binary molecular switches, cycling between inactive GDP-bound and active GTP-bound forms which are regulated by the concerted action of different proteins such as guanine nucleotide exchange factors, which stimulate the GDP release from GDP-loaded Ras allowing the binding of the most abundant GTP and GTPase activating proteins, which enhances the intrinsic GTPase activity of Ras (2).
There are three ubiquitous isoforms of Ras proteins, namely H-Ras, N-Ras and K-Ras4B, referred as K-Ras for the rest of this summary. These proteins are more than 90% homologues and their functions are not redundant (3). Knockout mice of the K-ras gene are not viable while deletions of the N-Ras and H-Ras genes have no observable side effects (4). In addition, it has been identified distinct signal outputs from different Ras isoforms (5) and different potencies of their corresponding activated alleles for oncogenic transformation (6).
It is widely assumed that the association of Ras proteins with membranes, particularly with the inner leaflet of the plasma membrane, is necessary and sufficient for the proper function of these proteins (7). The association of these proteins with membranes is a consequence of their post-translational lipid modification. The polypeptide sequence of all Ras isoforms contain a C-terminal CAAX motif (C, cysteine; A, aliphatic and X, any other amino acid), which is first modified in the cytosol with a farnesyl anchor to the cysteine residue, then the AAX sequence is cleaved by an endopeptidase associated to the cytosolic surface of the endoplasmic reticulum and, finally, the farnesylated cysteine is carboximetylated. Depending on the Ras isoform, a second signal of membrane anchor is present immediately to the farnesylated cysteine. H-Ras is dually palmitoylated at cysteine residues 181 and 184 while N-Ras is palmitoylated at cysteine residue 184 (3). In opposite, K-Ras is not further lipid modified, but contains a polybasic domain (six contiguous lysine residues) with binding capacity to phospholipid headgroups of lipid bilayers (8). The CAAX motif and the second signal (palmitoylation or the polybasic domain) are essential for efficient attachment of Ras proteins to membranes, in particular plasma membrane (7).
As described above, different functions have been attributed to Ras proteins. It has been recognized that Ras isoforms are capable of activate different effectors, thus affecting different signaling pathways (1,9,10). Because transmembrane receptors and Ras proteins are mainly localized in plasma membrane, one plausible explanation for these results is that they are localized in different plasma membrane microdomains (11), which are formed by the lateral segregation of lipids based in their dissimilar biophysical properties (12). The differential activities of Ras proteins can be also explained if they are localized in different membrane subcompartments (13). In this sense, it has been demonstrated that intracellular pools of H-Ras localized in membranes of endoplasmic reticulum and Golgi apparatus become active when cells are stimulated with epidermal growth factor (14).
In spite of becoming apparent that subcellular distribution of Ras isoforms is important for proper function, the underlying mechanisms of intracellular transport and distribution of these proteins are poorly understood. K-Ras, after synthesized in the cytosol and post-translationally modified in the endoplasmic reticulum, can reach the plasma membrane by a desorption-absortion mechanism. This mechanism is driven by the negative electrostatic potential of the plasma membrane, which is enriched in anionic lipids such as phophatidylserine (inner leaflet) and sialic acid containing glicolipids and glicoproteins (outer leaflet) (15). Conversely, the current data suggest that H-Ras, and probably N-Ras, are transported to different subcompartments by vesicular traffic (16) or by a nonvesicular pathway involving a constitutive de/reacylation cycle (17,18).
In our laboratory, we have investigated the membrane association, subcellular distribution and intracellular trafficking of H- and K-Ras proteins in Chinese hamster ovary (CHO)-K1 cells. By using confocal microscopy and biochemical analysis we demonstrate that H-Ras, at steady state, localized at the recycling endosome in addition to the cytoplasmic leaflet of the plasma membrane. In contrast, K-Ras mainly localized at the plasma membrane. Interestingly, we found that sorting signals of H- and K-Ras are contained within the C-terminal domain of these proteins, and that palmitoylation on this region of H-Ras might operates as a dominant sorting signal for proper subcellular localization of this protein. Using selective photobleaching techniques and time-lapse fluorescence microscopy, we demonstrate the dynamic association of H-Ras to the recycling endosome. We also found that Rab5 and Rab11 activities are required for delivery of H-Ras to this organelle. Using a chimera containing the Ras binding domain of c-Raf-1 fused to a fluorescent protein, we demonstrate that H-Ras can be activated (GTP-bound state) at the Rab11-positive recycling endosome after proper stimulation. These evidences support roles for novel signaling pathways regulated by H-Ras in membranes from recycling endosome.
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