Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycinassociated protein

Abstract

The FKBP12-rapamycin-associated protein (FRAP; also called RAFT1/mTOR) regulates translation initiation and entry into the cell cycle. Depriving cells of amino acids or treating them with the small molecule rapamycin inhibits FRAP and results in rapid dephosphorylation and inactivation of the translational regulators 4E-BP1(eukaryotic initiation factor 4E-binding protein 1) and p70(s6k) (the 70-kDa S6 kinase). Data published recently have led to the view that FRAP acts as a traditional mitogen-activated kinase, directly phosphorylating 4E-BP1 and p70(s6k) in response to mitogenic stimuli. We present evidence that FRAP controls 4E-BP1 and p70(s6k) phosphorylation indirectly by restraining a phosphatase. A calyculin A-sensitive phosphatase is required for the rapamycin- or amino acid deprivation-induced dephosphorylation of p70(s6k), and treatment of Jurkat I cells with rapamycin increases the activity of the protein phosphatase 2A (PP2A) toward 4E-BP1. PP2A is shown to associate with p70(s6k) but not with a mutated p70(s6k) that is resistant to rapamycin- and amino acid deprivation-mediated dephosphorylation. FRAP also is shown to phosphorylate PP2A in vitro, consistent with a model in which phosphorylation of PP2A by FRAP prevents the dephosphorylation of 4E-BP1 and p70(s6k), whereas amino acid deprivation or rapamycin treatment inhibits FRAP's ability to restrain the phosphatase.

Figure 1

Phosphatase inhibition interferes with FRAP-mediated dephosphorylation of 4E-BP1 and p70^s6k. (A) The activity of endogenous p70^s6k immunoprecipitated from TAg Jurkat cells after treatment for 45 min with or without amino acids, calyculin A, and rapamycin. Kinase activity is shown as a percentage of activity from cells grown in the presence of amino acids without calyculin A or rapamycin. Three experiments were performed for each condition; means and SD are shown. (B) α-4E-BP1 Western blot showing the relative quantities of hyper- (Upper) and hypophosphorylated (Lower) 4E-BP1 after various rapamycin and calyculin A treatments. Quiescent NIH 3T3 cells were either untreated, treated with 30 nM rapamycin or calyculin A for 60 min, or treated with 30 nM rapamycin for 30 min followed by addition of 30 nM calyculin A for an additional 30 min. Cell lysates were boiled and centrifuged to remove heat-unstable proteins before separation of heat-stable proteins by SDS/PAGE.

Figure 2

Rapamycin increases serine-threonine phosphatase activity. Removal of ³²P from exogenously phosphorylated and purified GST–4E-BP1 was measured. Cell lysates (A) or PP2A immune complexes (B) from untreated (open symbols) and rapamycin-treated (filled symbols) TAg Jurkat cells were incubated with ³²P-labeled GST–4E-BP1. Aliquots of the reaction mix were removed at the times indicated, and the amount of ³²P label remaining on the GST–4E-BP1 was determined by SDS/PAGE followed by phosphoimager quantification. Each point represents the percent decrease in GST–4E-BP1-bound ³²P relative to ³²P present at t = 0. Three experiments were performed; error bars reflect the SEM for each point.

Figure 3

PP2A interacts with full-length p70^s6k but not with a rapamycin-resistant p70^s6k mutant. (A) Western blot of PP2A in cell lysates and p70^s6k immune complexes. TAg Jurkat cells were treated with or without 25 nM rapamycin before lysis in buffer containing phosphatase inhibitors. They were then incubated with an α-p70^s6k polyclonal antibody or a control polyclonal antibody. The immunoprecipitated proteins were separated by using SDS/PAGE, and PP2A was visualized by Western blotting. (B) Western blot of HA-tagged p70^s6k in PP2A immune complexes. TAg Jurkat cells were mock-transfected or transfected with plasmids that express HA-tagged p70^s6k or a rapamycin-resistant, HA-tagged p70^s6k mutant that has been truncated at the N and C termini (NT/CT). PP2A was immunoprecipitated from the lysed cells, and proteins were separated by using SDS/PAGE. HA-tagged proteins were visualized by Western blotting.

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.