Many tumor suppressor proteins act to blunt the effects of mitogenic signaling pathways. Loss of function mutations in the Merlin tumor suppressor underlie Neurofibromatosis type 2 (NF2) a familial autosomal dominant cancer syndrome. Studies of Drosophila suggest that Hippo ( Hpo) is required for inhibition of cell proliferation mediated by dMer, the orthologue of human Merlin. Mammalian sterile 20-like kinase-2 (Mst2) is a mammalian Hpo orthologue; and numerous studies implicate Mst2 as a tumor suppressor. Mst2 is negatively regulated by the proto oncoprotein Raf-1 in a manner independent of Raf-1's kinase activity. We sought to determine if, in mammalian cells, Merlin could positively regulate Mst2. We also sought to determine if Mst2, in addition to being negatively regulated by Raf-1, itself might reciprocally regulate Raf-1. In contrast to findings from Drosophila, we find no evidence that mammalian Merlin positively regulates mammalian Mst2. Instead, surprisingly, RNAi silencing of Mst2 leads to elevated inhibitory phosphorylation of Raf-1 at Ser259 and impaired Raf-1 kinase activity. Consequent to this, ERK pathway activation and cell proliferation are attenuated. Phosphatase 2A (PP2A) dephosphorylates Raf-1 Ser259 in response to mitogens. Interestingly RNAi silencing of Mst2 triggers a striking proteasome-dependent decrease in the levels of the catalytic subunit of PP2A (PP2A-C). A similar effect is achieved upon silencing of large tumor suppressor (Lats1/2) direct downstream substrates of Mst2. Our studies reveal a more complex role for Mst2 than previously thought. The Mst2-Lats1/2 pathway, by maintaining PP2A-C levels, may, in some situations, positively affect mitogenic signaling.

Chapter II of this thesis presents unpublished and preliminary data indicating that Merlin may function as a tumor suppressor through negative regulation of mTORC1 complex and promotion of autophagy.