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, socalled 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 aC 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.

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…10 of the enzyme [28,29,50]. While phosphorylation at the TM influences enzyme stability, differences have been reported as to the importance of this site for catalytic activity (Table 1).…”

“…While phosphorylation at the TM influences enzyme stability, differences have been reported as to the importance of this site for catalytic activity (Table 1). For example, phosphorylation at the TM is required for PKC I and PKC II activity [51,52], while it is dispensable for PKC , PKC and PKC activity [53,31,50]. In contrast, conflicting results have been reported as to importance of TM phosphorylation for PKC activity [54,55] and PKC activity [56,50].…”

“…For example, phosphorylation at the TM is required for PKC I and PKC II activity [51,52], while it is dispensable for PKC , PKC and PKC activity [53,31,50]. In contrast, conflicting results have been reported as to importance of TM phosphorylation for PKC activity [54,55] and PKC activity [56,50]. The simplest explanation is that individual PKCs may differ in their requirement for TM phosphorylation for catalytic activity, perhaps due to structural differences between the individual isoforms, but this does not explain the differences reported for TM phosphorylation for PKC and PKC .…”

“…Required for activity [33,34] Required for activity [50] Not required for activity [56] Not present in PKC PKC Required for activity [131] Not reported Not present in PKC …”

Turn motif phosphorylation negatively regulates activation loop phosphorylation in Akt

“…This Thr residue is constitutively phosphorylated in the absence of extracellular stimuli (Alessi et al, 1996), depending on mTORC2 (Facchinetti et al, 2008;Ikenoue et al, 2008). Structural studies have suggested that this modification contributes to the correct conformation of Akt (Hauge et al, 2007). Early Akt activation studies showed that TM phosphorylation is not essential for phosphorylation of the other sites (Toker and Newton, 2000); however, considering the emerging complexity of the Akt activation mechanism, such as the discrepancy over the mutual dependency of A-loop and HM phosphorylation, the involvement of TM phosphorylation in the phosphorylation of the A-loop and/or the HM needs to be re-evaluated.…”