Mechanism for activation of the growth factor-activated AGC kinases by turn motif phosphorylation

Abstract

The growth factor/insulin-stimulated AGC kinases share an activation mechanism based on three phosphorylation sites. Of these, only the role of the activation loop phosphate in the kinase domain and the hydrophobic motif (HM) phosphate in a C-terminal tail region are well characterized. We investigated the role of the third, so-called turn motif phosphate, also located in the tail, in the AGC kinases PKB, S6K, RSK, MSK, PRK and PKC. We report cooperative action of the HM phosphate and the turn motif phosphate, because it binds a phosphoSer/Thr-binding site above the glycine-rich loop within the kinase domain, promoting zipper-like association of the tail with the kinase domain, serving to stabilize the HM in its kinase-activating binding site. We present a molecular model for allosteric activation of AGC kinases by the turn motif phosphate via HM-mediated stabilization of the alphaC helix. In S6K and MSK, the turn motif phosphate thereby also protects the HM from dephosphorylation. Our results suggest that the mechanism described is a key feature in activation of upto 26 human AGC kinases.

Figure 1

Molecular modelling suggests a widely conserved binding site for the tail/linker phosphoSer/Thr within the catalytic domain of AGC kinases. (A) Model of active PKBβ shown as a ribbon representation with side chains of selected residues. The kinase domain is shown in green and the tail/linker in red. Phosphate groups are shown in yellow. K/R residues predicted to bind the phosphate of phosphoT451 are shown in blue. Other phosphate-binding residues are in cyan. The HM, glycine-rich loop and ATP analogue are shown in magenta, orange and white, respectively. Partial sequence alignment of the tail/linker (B) and predicted binding site for the tail/linker phosphate (C) of AGC kinases. (B, C) Phosphorylation sites/phosphate-mimicking residues are shown in red. The aromatic residues that define the HM are underlined. Basic residues predicted to bind the tail/linker phosphoSer/Thr are shown in blue and labelled 1–4. Sequences are human except S6K1 (rat, p70 isoform) and RSK2 (mouse).

Figure 2

Role of the tail phosphorylation site in activation and phosphorylation of AGC kinases in vivo. COS7 cells were transfected with plasmid expressing haemagglutinin (HA)- or glutathione S-transferase (GST)-tagged wt or mutant kinase. After 16 h and a final 4 h serum-starvation period, cells were exposed to 1 μM insulin for 10 min (PKBα), to 20 nM EGF for 30 min (S6K1) or 15 min (RSK2), to 10 μg/ml anisomycin for 40 min (MSK1) or left in serum-containing medium (PRK2_Δ1−500) and then lysed. The kinases were precipitated from aliquots of the cell lysates with antibody to the HA tag or with glutathione beads. The precipitates were subjected to kinase assay, to immunoblotting with the indicated phosphorylation site-specific Ab or anti-HA Ab or stained for protein. Experiments were repeated at least three times and activity data (expressed as percent) are means±s.d.

Figure 3

The tail phosphate-binding site is essential for normal activation and phosphorylation of AGC kinases in vivo. The activity and phosphorylation state of wt and mutant AGC kinases expressed in COS7 cells (A, C) or S2 cells (B) were analysed as described in Figure 2, except that Drosophila S6K was activated by exposure of cells to 1 μM insulin and 10 μM pervanadate for 15 min. Experiments were repeated at least three times and activity data (expressed as percentage) are means±s.d.

Figure 4

Further evidence of the tail phosphate-binding site. The activity of wt and mutant PRK2_Δ1−500 (A) or MSK1 (B) expressed in COS7 cells were analysed as described in the legend of Figure 2. However, in (A) kinase activity was also determined in the presence of the indicated concentration of Zn²⁺ and expressed as percentage of activity in the absence of Zn²⁺. Experiments were repeated at least three times and activity data are means±s.d.

Figure 5

The tail phosphate synergistically enhances the ability of the HM phosphate to activate S6K, dependent on the tail phosphate-binding site. (A) The kinase activity of purified kinase domain of S6K1 (S6K1₁₋₃₆₄), pre-phosphorylated by PDK1 in the activation loop, was determined in the presence of increasing concentrations of synthetic S6K1 tail peptide (residues 366–395) that was either nonphosphorylated (S371/T389), phosphorylated at Ser371 (pS371/T389) or Thr389 (S371/pT389) or phosphorylated at both sites (pS371/pT389). (B) The kinase activity of S6K1₁₋₃₆₄ and S6K1₁₋₃₆₄ K144N, either pre-phosphorylated or not by PDK1, was determined in the absence or presence of 90 μM S371/pT389 or pS371/pT389. (C) pS371/pT389 S6K1 tail peptide was biotinylated and used to coat streptavidin Sensor Chips. The chips were thereafter analysed for binding to purified GST-S6K1₁₋₃₆₅ or GST-S6K1₁₋₃₆₅ K144N. (A–C) Experiments were repeated at least three times and activity data (expressed as percent) are means ±s.d.

Figure 6

The tail phosphate promotes a compact global conformation of the AGC kinase domain and protects the tail phosphate-binding site and αC helix from solvent exposure. (A) Effect of S6K1 tail peptides, described in the legend of Figure 5, on global deuteron uptake by purified GST-S6K1₁₋₃₆₅. (B) Kinase activity of wt and mutant GST-PKCζ. (C) Global deuteron uptake by wt and mutant GST-PKCζ. (D) Local deuteron uptake by wt and mutant GST-PKCζ. In the PKCζ model, peptides showing strong and no-or-slight protection by the tail phosphate are shown in pink and grey, respectively. The panels show HX curves of the peptides. Experiments were repeated two times (A, C, D) or three times (B) and data are means ±range (A, C) or ±s.d. (B)

Figure 7

The tail phosphate may correspond to E333 of PKA. (A) Analogous positions and interactions of E333 of PKA and the tail phosphate. Left and right panels show a structure of PKA (1ATP) and our model of RSK2, respectively. Colour codes are described in the legend of Figure 1A, except that residues known/thought to interact with the turn motif phosphate of PKA and phosphoS375 of RSK2 are shown in grey. (B) Effect of mutation of E332/E333 and R56 on autophosphorylation and activity of PKAα expressed in Escherichia coli. (C) Effect of mutation of S375 in RSK2 expressed and analysed as described in the legend of Figure 2. (B, C) Data are means ±s.d. of three independent experiments. (D) Proposed alignment and naming of phosphorylation sites in the tail of AGC kinases.