Association of Protein Phosphatase 2A with S6 Kinase Is Regulated In an MTOR-Dependent Manner

Date

2007-08-08

Authors

Mahmood, Nadir Ahmeduddin

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The mammalian target of rapamycin (mTOR) pathway senses nutrient and growth factors to regulate protein synthesis and cell growth. Strict control of the components of this pathway is essential for cells to reach an appropriate size. Aberrant mTOR signaling has been implicated in multiple diseases such as cancer, diabetes, and heart failure. Activation of mTOR by nutrients and growth factors leads to the phosphorylation of its two substrates, S6 kinase 1 (S6K1) and 4E binding protein 1 (4EBP1) to promote translation initiation. Many reports have demonstrated the rapid dephosphorylation of S6K1 following treatment with the mTOR inhibitor, rapamycin, as well as by the depletion of amino acids. These data suggest a role for a serine/threonine protein phosphatase in mediating this dephosphorylation. This was supported by studies showing the co-immunoprecipitation of the catalytic subunit of protein phosphatase 2A (PP2A) with S6K1. PP2A is a ubiquitously expressed phosphatase that has been implicated in many intracellular signaling pathways. In this dissertation, an association between S6K1 and PP2A was investigated to elucidate the role of this phosphatase in regulating nutrient signaling and cell growth. Initial studies utilized an inhibitor of PP2A and related phosphatases to block the dephosphorylation of S6K1 under conditions that inactivate mTOR. Immunoprecipitation studies identified an interaction between the PP2A catalytic and scaffolding subunits and S6K1 that was enhanced under conditions that lead to S6K1 dephosphorylation and decreased upon stimulation of the pathway by insulin. An siRNA screen targeting PP2A regulatory subunits was used to identify specific subunits that were mediating this association. Depletion of the B56gamma and B56delta regulatory subunits reduced co-immunoprecipitation of PP2A with S6K1. However, functional studies found no effect of knockdown of these subunits on the dephosphorylation of S6K at T389 or on S6K1 kinase activity. There was no effect on cell size in cells depleted of B56gamma or B56delta , but cell proliferation was reduced in these cells. It is likely that cell proliferation was affected due to the roles of these subunits in other pathways. Additionally, knockdown of PP2A-like phosphatases did not definitively identify the phosphatase that dephosphorylates S6K1 at T389.

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