Hydrophobic motif phosphorylation is not required for activation loop phosphorylation of p70 ribosomal protein S6 kinase 1 (S6K1)

Abstract

p70 ribosomal protein S6 kinase 1 (S6K1) is regulated by multiple phosphorylation events. Three of these sites are highly conserved among AGC kinases (cAMP dependent Protein Kinase, cGMP dependent Protein Kinase, and Protein Kinase C subfamily): the activation loop in the kinase domain, and two C-terminal sites, the turn motif and the hydrophobic motif. The common dogma has been that phosphorylation of the hydrophobic motif primes S6K1 for the phosphorylation at the activation loop by phosphoinositide-dependent protein kinase 1 (PDK1). Here, we show that the turn motif is, in fact, phosphorylated first, the activation loop second, and the hydrophobic motif is third. Specifically, biochemical analyses of a construct of S6K1 lacking the C-terminal autoinhibitory domain as well as full-length S6K1, reveals that S6K1 is constitutively phosphorylated at the turn motif when expressed in insect cells and becomes phosphorylated in vitro by purified PDK1 at the activation loop. Only the species phosphorylated at the activation loop by PDK1 gets phosphorylated at the hydrophobic motif by mammalian target of rapamycin (mTOR) in vitro. These data are consistent with a previous model in which constitutive phosphorylation of the turn motif provides the key priming step in the phosphorylation of S6K1. The data provide evidence for regulation of S6K1, where hydrophobic motif phosphorylation is not required for PDK1 to phosphorylate S6K1 at the activation loop, but instead activation loop phosphorylation of S6K1 is required for mTOR to phosphorylate the hydrophobic motif of S6K1.

FIGURE 1.

Domain organization, S6K1 constructs used in study, and purification. a, sequence alignment of various representative AGC kinases showing three conserved phosphorylation sites (underlined) in three separate sites, namely activation loop, turn motif, and hydrophobic motif. Also shown is conservation of these sites in S6K1 across various orthologs. b, schematic representation of S6K1αI and S6K1αII isoforms showing seven phosphorylation sites. c, the two baculovirus S6K1αII isoform constructs with truncation of AID (1–398) used in the study. When expressed in insect cells, both have phosphorylation at turn motif site Ser-371. The lower panel shows construct with HM residue Thr mutated to a phosphomimetic Glu.

FIGURE 2.

Phosphorylation status of S6K1 overexpression in insect cells. a, Coomassie gel shows purification of S6K1 constructs as well as PDK1ΔPH, all generated from insect cells. b, S6K1ΔAID and S6K1ΔAIDE, when overexpressed solely in insect cells for 52 h, get phosphorylated only at turn motif Ser-371(upper and lower panel, respectively) as probed by Ser(P)-371 antibody (in duplicate). c, transfection time course (72 h) of S6K1ΔAID expression using His₆ tag antibody in parallel with Ser(P)-371 antibody shows that phosphorylation of S6K1 at turn motif is constitutive. d, specificity check for Ser(P)-371 antibody shows that the S371A mutant was not detected by the antibody.

FIGURE 3.

In vitro and in vivo phosphorylation of S6K1ΔAID with PDK1. a, 4-h time course of PDK1 phosphorylation of S6K1ΔAID using Mg-ATP. As shown in the figure only activation loop site (Thr-229) gets phosphorylated (upper panel), but the HM site (Thr-389) is never phosphorylated in vitro (lower panel). b, co-transfection of S6K1ΔAID with PDK1ΔPH (upper panel) resulting in phosphorylation of all three sites, namely activation loop (Thr-229), turn motif (Ser-371), and the HM (Thr-389). Co-transfection of S6K1ΔAIDE with PDK1ΔPH (lower panel) results in phosphorylation of two sites, namely activation loop (Thr-229) and turn motif (Ser-371), and the HM (Thr-389) site is mutated to Glu. All blots are representative of three different experiments.

FIGURE 4.

In vitro phosphorylation of S6K1ΔAID with mTOR and mobility shift of phospho-isoforms of S6K1ΔAID. a, S6K1ΔAID, when incubated with mTOR (myc-mTOR expressed in HEK293 cells and immunoprecipitated), and Mg-ATP showed no phosphorylation at HM site Thr-389, but when S6K1ΔAID was preincubated with Mg-ATP and PDK1 followed by mTOR incubation, showed Thr-389 phosphorylation. Turn motif phosphorylation was used as a loading control. b, 10% SDS-PAGE is shown. Lane 1, marker; lane 2, Lambda protein phosphatase 1-treated (1 h) S6K1ΔAID (no phosphate); lane 3, S6K1ΔAID (Ser(P)-371 turn motif phosphorylated); lane 4, S6K1ΔAID incubated with PDK1 and Mg-ATP for 1 h (turn motif and activation loop phosphorylated); lane 5, S6K1ΔAID and PDK1ΔPH co-transfected (turn motif, activation loop and HM site phosphorylated).

FIGURE 5.

HM site phosphorylation is not required for phosphorylation of activation loop Thr-229 in full-length S6K1. a, EGF stimulation of HA-S6K1 pretreated with PI3K inhibitor, shows increase in AID site Ser-421/Ser-424 but has no effect on basal levels of Thr-229 and Thr-389. Ser-371 is constitutively phosphorylated and also serves as loading control. b, EGF stimulated and immunopurified HA-S6K1, when incubated with mTOR alone shows no increase in basal Thr-389 phosphorylation but when pretreated with PDK1 followed by mTOR, shows increase in basal Thr-389 and Thr-229 phosphorylation. Ser(P)-421/Ser(P)-424 and Ser(P)-371 show no enhancement due to mTOR and PDK1 treatment and serve as control.

FIGURE 6.

Model of S6K1ΔAID activation via sequential phosphorylation. The turn motif Ser-371 phosphorylation is constitutive and results in ordering of the C-terminal tail onto the N-lobe of S6K1ΔAID (the conserved C-terminal tail with Ade motif, the HM segment, and two phosphorylation sites are pictured in detail at the bottom). This allows for phosphorylation of activation loop by PDK1 followed by the HM site by mTOR kinase. The HM site may not get phosphorylated after the turn motif because the unphosphorylated activation loop presumably causes steric hindrance for the accessibility of the HM segment by mTOR kinase. PDK1 phosphorylation of activation loop is a prerequisite for mTOR phosphorylation of HM site in S6K1ΔAID.