The differential palmitoylation states of N-Ras and H-Ras determine their distinct Golgi subcompartment localizations.

Despite a high degree of structural homology and shared exchange factors, effectors and GTPase activating proteins, a large body of evidence suggests functional heterogeneity among Ras isoforms. One aspect of Ras biology that may explain this heterogeneity is the differential subcellular localizations driven by the C-terminal hypervariable regions of Ras proteins. Spatial heterogeneity has been documented at the level of organelles: palmitoylated Ras isoforms (H-Ras and N-Ras) localize on the Golgi apparatus whereas K-Ras4B does not. We tested the hypothesis that spatial heterogeneity also exists at the sub-organelle level by studying the localization of differentially palmitoylated Ras isoforms within the Golgi apparatus. Using confocal, live-cell fluorescent imaging and immunogold electron microscopy we found that, whereas the doubly palmitoylated H-Ras is distributed throughout the Golgi stacks, the singly palmitoylated N-Ras is polarized with a relative paucity of expression on the trans Golgi. Using palmitoylation mutants, we show that the different sub-Golgi distributions of the Ras proteins are a consequence of their differential degree of palmitoylation. Thus, the acylation state of Ras proteins controls not only their distribution between the Golgi apparatus and the plasma membrane, but also their distribution within the Golgi stacks.

In TCR-stimulated T-cells, N-ras regulates specific genes and signal transduction pathways.

It has been recently shown that N-ras plays a preferential role in immune cell development and function; specifically: N-ras, but not H-ras or K-ras, could be activated at and signal from the Golgi membrane of immune cells following a low level T-cell receptor stimulus. The goal of our studies was to test the hypothesis that N-ras and H-ras played distinct roles in immune cells at the level of the transcriptome. First, we showed via mRNA expression profiling that there were over four hundred genes that were uniquely differentially regulated either by N-ras or H-ras, which provided strong evidence in favor of the hypothesis that N-ras and H-ras have distinct functions in immune cells. We next characterized the genes that were differentially regulated by N-ras in T cells following a low-level T-cell receptor stimulus. Of the large pool of candidate genes that were differentially regulated by N-ras downstream of TCR ligation, four genes were verified in qRT-PCR-based validation experiments (Dntt, Slc9a6, Chst1, and Lars2). Finally, although there was little overlap between individual genes that were regulated by N-ras in unstimulated thymocytes and stimulated CD4(+) T-cells, there was a nearly complete correspondence between the signaling pathways that were regulated by N-ras in these two immune cell types.

Kaposi's Sarcoma-associated herpesvirus lytic switch protein stimulates DNA binding of RBP-Jk/CSL to activate the Notch pathway.

Kaposi's sarcoma-associated herpesvirus (KSHV) lytic switch protein, Rta, is a ligand-independent inducer of the Notch signal transduction pathway, and KSHV cannot reactivate from latency in cells null for the Notch target protein RBP-Jk. Here we show that Rta promotes DNA binding of RBP-Jk, a mechanism that is fundamentally different from that established for the RBP-Jk-activating proteins, Notch intracellular domain (NICD) and Epstein-Barr virus EBNA2. Although constitutively active RBP-Jk and NICD do not transactivate KSHV promoters independently, cotransfection of an Rta mutant lacking its transactivation domain robustly restores transcriptional activation. Cooperation requires intact DNA binding sites for Rta and RBP-Jk and trimeric complex formation between the three molecules in vitro. In infected cells, RBP-Jk is virtually undetectable on a series of viral and cellular promoters during KSHV latency but is significantly enriched following Rta expression during viral reactivation. Accordingly, Rta, but not EBNA2 and NICD, reactivates the complete viral lytic cycle.

The lytic switch protein of KSHV activates gene expression via functional interaction with RBP-Jkappa (CSL), the target of the Notch signaling pathway.

The RTA protein of the Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is responsible for the switch from latency to lytic replication, a reaction essential for viral spread and KS pathogenesis. RTA is a sequence-specific transcriptional activator, but the diversity of its target sites suggests it may act via interaction with host DNA-binding proteins as well. Here we show that KSHV RTA interacts with the RBP-Jkappa protein, the primary target of the Notch signaling pathway. This interaction targets RTA to RBP-Jkappa recognition sites on DNA and results in the replacement of RBP-Jkappa's intrinsic repressive action with activation mediated by the C-terminal domain of RTA. Mutation of such sites in target promoters strongly impairs RTA responsiveness. Similarly, such target genes are induced poorly or not at all by RTA in fibroblasts derived from RBP-Jkappa(-/-) mice, a defect that can be reversed by expression of RBP-Jkappa. In vitro, RTA binds to two adjacent regions of RBP-Jkappa, one of which is identical to the central repression domain that binds the Notch effector fragment. These results indicate that KSHV has evolved a ligand-independent mechanism for constitutive activation of the Notch pathway as a part of its strategy for reactivation from latency.