Figure 4.

Possible mechanisms of PP2A regulation by FRAP. (A) FRAP phosphorylates PP2A in vitro. Kinase-active (WT) and kinase-dead (DA) FRAP expressed in insect cells by baculovirus infection were immunoprecipitated and incubated with GST–4E-BP1 and/or PP2A as substrates in the presence of [γ-³²P]ATP. The products of the kinase reactions were separated by using SDS/PAGE before autoradiography (Upper). Relative quantities of kinase substrates were determined by silver staining (Lower). (B) A model of FRAP intervention in the p70^s6k signaling pathway. Mitogenic stimuli promote phosphorylation of p70^s6k which in turn promotes translation of a subset of cellular mRNAs. Inhibition of FRAP by amino acid deprivation or by rapamycin treatment interferes with p70^s6k function by activating PP2A. PP2A dephosphorylates and inactivates p70^s6k through its interaction with the N terminus of p70^s6k.