Ras proteins are prototypical GTPases that control signaling pathways by switching between a GDP-bound inactive and a GTP-bound active state. Mutations in the genes encoding Ras proteins are found in more than 30% of human cancers. Thus, a more comprehensive knowledge of Ras signaling will not only be important for understanding fundamental issues of cell signaling but will also have a direct impact on human health. Until recently the study of Ras and other signaling pathways has been confined to biochemical assays that report no spatial information. We discovered that Ras transits endomembrane compartments en route to the plasma membrane (PM). To determine if intracellular Ras was capable of signaling we developed fluorescent probes that report when and where Ras becomes activated in living cells. Using this technology, in the last cycle of GM55779 we discovered that N-Ras and H-Ras become activated in fibroblasts on both the PM and Golgi apparatus. We mapped the pathway to activation on Golgi as one involving EGFR/PLC(/DAG + Ca++/RasGRP1. More recently we have discovered that in T lymphocytes, signaling through the antigen receptor (TCR) and the (2 integrin LFA-1 combine to modulate the spatial distribution of activated Ras. We now seek to more completely understand what determines the spatial distribution of activated Ras and what are the biological consequences of Ras signaling from various subcellular compartments. To accomplish this we propose the following aims.
Aim 1. Compartment Specific Ras Signaling I: upstream regulation by DAG/RasGRP1. We will study the regulation of DAG on the Golgi apparatus as it relates to Ras activation. The roles of sphingomyelin synthase and PLD1 will be determined and PLD1 regulation on the Golgi by PITB( and by Arf, Rho, and Ral GTPases will be explored. We will identify the sequences of the C1 domain of RasGRP1 that gives it affinity for the Golgi. We will also determine the role of PLD2 in Ras activation on the PM of T cells downstream of LFA-1 signaling.
Aim 2. Compartment-specific Ras Signaling II: downstream pathways. We will study the differential signaling output from Ras proteins or Ras GEFs and GAPs that are stringently targeted to various subcellular compartments using systems inducible at the level of gene expression or at the level of acute targeting by rapamycin-induced heterodimerization. The signaling outputs from T cells stimulated through the TCR?LFA-1 will also be studied and correlated with the distribution of Ras activation.
Aim 3. Compartment-specific Ras Signaling III: in vivo models. We will study the effect on T cell development of reconstituting RasGRP1-/- animals with lymphocytes that express targeted RasGRP1. We will study the mechanism of compartment specific Ras signaling in Schizosaccharomyces pombe. We will follow up an intriguing discovery that Ras in Drosophila is not membrane associated by studying the effect of membrane association of Ras on eye development in flies. Together, these studies will give a fuller picture of the spatial aspect of the regulation of Ras signaling and will help explain how a one signaling molecule can regulate, in an independent fashion, multiple signaling pathways in a single cell.
Showing the most recent 10 out of 15 publications
