Molecular mechanisms regulating MEK-MAP kinase cell survival: A critical role for RSK and PDK1

This thesis extends our understanding of the molecular mechanisms whereby the classical Mitogen-Activated Protein Kinase (MAPK) cascade promotes cell survival. The MAPK-activated 90 kiolodalton Ribosomal S6 Kinase (RSK) is shown to negatively regulate the cell death machinery in part through phosphorylation of serine 112 of BAD, a pro-death member of the Bcl-2 family. A novel activated allele of RSK mediated constitutive S112 phosphorylation of BAD and promoted cell survival in IL-3 dependent hematopoietic cell lines. Conversely, a dominant-interfering mutant of RSK eliminated IL-3 dependent survival afforded by MAPK signaling, and reduced serine 112 phosphorylation of BAD. The activated RSK allele contains an aminoterminal myristoylation signature targeting it to the plasma membrane, placing it in proximity to its essential activator 3-Phosphoinositide dependent Kinase 1 (PDK1), and permitting activated RSK activity when MAPK is inactive.; Cooperation between PDK1 and MAPK was further explored by an analysis of the anti-tumorigenic and anti proliferative agent N-α-tosyl-L-phenylalanyl Chloromethyl Ketone (TPCK). Although the biological effects of TPCK had been known for decades, the cellular targets of TPCK had not been defined. TPCK was shown to negatively regulate signaling downstream of PDK1, preventing PDK1-dependent activation of RSK, the 70/85 kilodalton Ribosomal S6 Kinase 1 and Akt, without inhibiting acute activation of Protein Kinase A, Phosphoinositide 3-Kinase and MAPK. Consistent with the role of RSK and Akt in promoting cell survival, in part through phosphorylation of BAD, TPCK prevented IL-3 mediated survival and BAD phosphorylation. The molecular mechanism whereby TPCK prevents PDK1-dependent RSK activation remains to be fully elucidated. However, TPCK prevented PDK1 from interacting with RSK, prevented the generation of a phospho-serine PDK1 docking site in RSK and covalently modified PDK1 at cysteine 181. Interestingly, the RSK family members, Mitogen and Stress-activated Kinase-1 and -2 (MSK1/2), can be activated independent of PDK1 but remain sensitive to TPCK. This implies that TPCK disrupts not only PDK1-dependent signaling, but also signaling of PDK1-related kinases. Defining the molecular basis for cell survival and the characterization of small molecules that disrupt it elucidates basic cellular processes and permits the development of therapeutic approaches to address excessive or insufficient cell survival manifest in neoplasia, autoimmunity and tissue degeneration.