Compartmentalized signaling by Ras family GTPases

Ras proteins regulate cellular growth and differentiation and are mutated in 30% of cancers. The plasma membrane (PM) has been considered to be a unique platform from which Ras signaling emanates. Using a novel in vivo probe for activated Ras, here we show that Ras signals from the Golgi apparatus as well as the PM. In response to Src-dependent activation of phospholipase Cγ1, the Ras guanine nucleotide exchange factor RasGRP1 translocated to the Golgi where it activated Ras. Whereas Ca++ positively regulated Ras on the Golgi through RasGRP1, the same second messenger negatively regulated Ras on the PM via the Ras GTPase activating protein CAPRI. Ras activation following T cell receptor stimulation in Jurkat cells, rich in RasGRP1, was limited to the Golgi through the action of CAPRI, demonstrating unambiguously a physiologic role for Ras on Golgi. Activation of Ras on Golgi also induced differentiation of PC12 cells, transformed fibroblasts and mediated radio resistance. Thus, activation of Ras on Golgi has important biological consequences and proceeds via a pathway distinct from the one that activates Ras on PM.; Rap1a and Ras are closely related monomeric GTPases that share some effectors but have distinct functions. To gain insight into the regulation of Rapt, we have studied its subcellular localization and its sites of activation in living cells. Rap1a was localized on both PM and endosomes. Using a novel, in vivo fluorescent probe for GTP-bound Rap1a we show that Rap1 was upregulated on and activated on PM during mitogen stimulation. Moreover, inhibition of PM upregulation abolished the ability of Rap1 to promote integrin-mediated adhesion of T cells. Thus, unlike Ras, the membrane localizations of Rap1a are dynamically regulated and the PM is the exclusive platform from which Rap1 signaling emanates.; Compartmentalization of signaling events allows for a greater diversity of signals than can be generated from a limited number of signaling molecules functioning in only one location. These studies will facilitate an understanding of the where and when of signaling and may offer new targets for therapeutics designed to block one but not all outputs from a particular signaling molecule.