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Saha, Madhurima

Biology

2012

PhD

Cells employ different signaling mechanisms to interpret externaJ stimuli and thereby govern their cellular fates. This dissertation extends our understanding of cooperative signaling mechanism between two very important proteins, RSK and 14-3-3. RSK., the p90 ribosomal S6 kinase and 14-3-3 play key roles in regulating the biological processes in a cell. Mediated by the RAS-MAPK pathway, RSK phosphorylates various substrates ranging from cytosolic to nuclear proteins. These phosphorylation events regulate cell growth, cell motility, cell proliferation and cell survival. RSK prefers a target motif on its substrate for phosphorylation which is RXXS. where S is the phosphorylated serine. 14-3-3 interacts with proteins phosphorylated in motifs that overlap with RSK target sequences as it prefers to bind to (RXXpS/TXP), a phosphorylated serine or a threonine target motif within its binding partners.
Interaction with 14-3-3 leads to the regulation of these substrates commonly by masking subcellular localization motifs and! or protein-protein interaction domains. Therefore our work highlights that RSK substrates are potential targets for 14-3-3 interaction and explores their cooperativity in signaling mechanisms. Our fIrst study focuses on a novel RSK substrate, SOS1 (Son of Sevenless). The regulation of SOS1 by 14-3-3 after RSK phosphorylation following PMA and EGF stimulation describes a newfound path to regulate RAS-MAPK signaling. Cooperation between RSK and 14-3-3 was further explored by using a mass-spectrometry based approach called SILAC (Stable Isotopic Labeling of Amino acid in Cell Culture). Histone (H2B) was identifIed as a major MEKIRSK-dependent 14-3-3 binding partner using this approach. These substrates are novel targets for RSK and 14-3-3 co-operativity. Together these data pave a new way to understand the extent of RSK and 14-3-3 co-operativity in cell signaling mechanisms.