American Association for Cancer Research, Cancer Research, 3(70), p. 1195-1203, 2010
DOI: 10.1158/0008-5472.can-09-3147
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Mammalian sterile20-like kinases (MST1/2) are involved in stress-induced apoptosis signalling. MST2 is inhibited by Raf-1 binding, and its activation requires dissociation from Raf-1 and binding to the RASSF1A tumour suppressor protein. Here, we have investigated the regulation of MST2 by the pro-survival phosphoinositide-3 kinase (PI3K)-Akt pathway. Akt phosphorylates MST2 in response to mitogens, oncogenic Ras expression or depletion of the tumour suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN). We identified two Akt phosphorylation sites (T117 and T384) in MST2. Mutation of these sites individually reduced phosphorylation, while the double mutation abolished it. These mutations, especially the double mutation, inhibited MST2 interactions with Raf-1, but enhanced binding to RASSF1A resulting in higher activation of downstream stress signalling pathways (JNK and p38 MAPK) and apoptosis. Biochemical and in situ FLIM experiments revealed a dual mechanism of inhibition. Akt phosphorylation of MST2 (i) blocks binding to RASSF1A and promotes sequestration into the inhibitory complex with Raf-1; and (ii) prevents MST2 homo-dimerisation which is essential for MST2 activation. Our results further show that the dissociation of the Raf-1-MST2 complex is part of mitogenic signalling, thereby linking induction of proliferation with the risk of apoptosis. Results with Ras effector domain mutants that selectively couple to either PI3K or Raf-1 show that Akt activation is necessary to abrogate MST2 activation in response to mitogenic stimulation. Thus, MST2 serves as a hub to integrate the biological outputs of the Raf-1 and Akt pathways.