Rapamycin, Wortmannin, and the Methylxanthine SQ20006 Inactivate p70s6k by Inducing Dephosphorylation of the Same Subset of Sites

Abstract

, Ser 404 , and Ser 411 is rescued by FK506, providing further evidence that the inhibitory effect is exerted through a complex of rapamycin-FKBP12. Wortmannin treatment pre-or post-serum stimulation inhibits phosphorylation of the same set of sites as rapamycin, supporting the argument that both agents act on the same pathway. Likewise, methylxanthine phosphodiesterase inhibitors block p70 s6k activation and phosphorylation of the same set of sites as wortmannin and rapamycin. However, other agents that raise intracellular cAMP levels have no inhibitory effect, leading to the hypothesis that the inhibitory actions of methylxanthines on p70 s6k activity are not through activating protein kinase A but through inhibition of an upstream kinase. Together the results indicate that there are two kinase signaling pathways that must converge to activate p70 s6k and that only one of these pathways is sensitive to rapamycin, wortmannin, and methylxanthine inhibition.

The Insulin-Induced Signalling Pathway Leading to S6 and Initiation Factor 4E Binding Protein 1 Phosphorylation Bifurcates at a Rapamycin-Sensitive Point Immediately Upstream of p70s6k

“…To test this possibility, two-dimensional tryptic-chymotryptic phosphopeptide maps of either myc-p70 s6k -GST or HA-4E-BP1 were analyzed. In agreement with earlier observations with endogenous and overexpressed p70 s6k (13,24,54), addition of rapamycin selectively blocked insulin-induced phosphorylation of T 229 , T 389 , and S 404 and suppressed phosphorylation of S 411 (compare Fig. 5A and B).…”

“…Total ribosomes were pelleted by centrifugation at 2°C for 17 h at 75,000 rpm in a TLA 100.3 rotor (Beckman). Ribosomal proteins were prepared as described earlier (64), except that after acetone precipitation, the protein pellet was dissolved in 50 l of 1ϫ sodium dodecyl sulfate (SDS) sample buffer and boiled for 10 min (24). Equal amounts of each sample were resolved by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) and blotted on Immobilon-P membranes (Millipore).…”

“…After 1 h of incubation at 4°C, unbound proteins were removed by three 1-ml washes with LCB. Bound proteins were released by boiling in 20 l of 1ϫ SDS sample buffer (24) and analyzed by Western blotting with the anti-4E-BP1 antibody 11208 (22) or the anti-eIF4E antibody 10C6 (22) and a swine anti-rabbit horseradish peroxidase-coupled secondary antibody (DAKO). The horseradish peroxidase signal was detected with Super Signal TM Substrate (Pierce) and visualized with Biomax film (Kodak).…”

Phorbol 12-myristate 13-acetate and serum synergize to promote rapamycin-insensitive cell proliferation via protein kinase C-eta

“…The kinase activity of mTOR and the phosphorylation of p70S6K and 4EBP-1 are inhibited by rapamycin (Jefferies et al, 1997). Rapamycin, a bacterial macrolide with high specificity for mTOR, has been used clinically to prevent organ transplant rejection and restenosis (Chung et al, 1992;Han et al, 1995;Aspeslet and Yatscoff, 2000;Rodriguez et al, 2003;Levy et al, 2004). Rapamycin is currently in clinical trials as a chemotherapeutic agent in several cancers including recurrent brain tumors (Newton, 2004;Xu et al, 2004).…”

Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase

“…In mammalian cells, the phosphorylation and activation of S6K by PDK1 promotes phosphorylation of the 40S ribosomal protein S6, which in turn controls cell growth and proliferation via the consequential regulation of 5´-terminal oligopyrimidine tract mRNA translation [2]. Interestingly, one of the A. thaliana S6K homologues, AtS6k2, has been shown to phosphorylate a plant ribosomal S6 protein in vivo, a phosphorylation sensitive to wortmannin and LY294002, PtdIns 3-kinase inhibitors that suppress mammalian S6K activity [8][9][10]. Also in maize (Zea mays), an S6K homologue, ZmS6K, phosphorylated a ribosomal S6 protein in vivo [11].…”