We are currently investigating the function and regulation of two small molecular weight GTPases: Ras, which is mutated in many human neoplasms and plays a role in the molecular pathology of neurofibromatosis type 1 (NF1), and Rap1, a closely related protein that interacts with the protein product encoded by one of the genes altered in patients with tuberous sclerosis (TSC). Ras proteins are activated via their interactions with guanine nucleotide exchange factors such as the guanine nucleotide release factor (GRF). We investigated the interaction of Ras with GRF, and identified a novel region of Ras (amino acids 102 to 105) that is critical for stimulation of guanine nucleotide release. Following the activation of Ras, it binds to and stimulates the activity of the serine/threonine kinase Raf-1, leading to the transmission of further downstream signals. Raf-1 undergoes a series of modifications associated with its activation and subsequent inactivation. We have found that alterations within the Raf-1 C-terminus resulted in an altered electrophoretic mobility; these changes were shown to result from the modification of a previously characterized phosphorylation site (serine 621) as well as an additional nearby residue (serine 624). Modification of Raf-1 at these sites is likely to play a critical role in mitogenic signal transduction. Tuberous sclerosis (TSC) is a genetic syndrome characterized by the development of benign tumors in a variety of tissues, as well as rare malignancies. We have previously shown that tuberin, the protein product of the tuberous sclerosis-2 gene (TSC2) can interact in vitro with Rap1, a small Ras-related GTPase. We have now obtained direct experimental evidence that TSC2 functions as a tumor suppressor gene, as expression of TSC2 or its C-terminal domain that interacts with Rap1 in two different tuberin-negative cell lines suppressed their anchorage-independent growth and tumorigenicity. We have also found by immunofluorescence analysis that the majority of tuberin is localized to the cis/medial Golgi stacks in several different human cell lines. The tuberin staining pattern also showed significant overlap with Rap1, consistent with a possible interaction between these proteins in vivo. Immunohistochemical analyses with anti-tuberin antibodies showed that tuberin is expressed in the arterioles of the kidney, skin, and heart, and the pyramidal neurons and Purkinje cells of the brain and cerebellum. These represent the major target organs for the manifestations of tuberous sclerosis in TSC patients. In many of the same cells, abundant staining of Rap1 was also observed. We conclude that the loss of functional tuberin in these cells may lead to growth defects, and play a role in the development of lesions of patients with TSC.
