A 3-Phosphoinositide-dependent Protein Kinase-1 (PDK1) Docking Site Is Required for the Phosphorylation of Protein Kinase Cζ (PKCζ) and PKC-related Kinase 2 by PDK1

Anudharan Balendran,Ricardo M Biondi,Peter C.F Cheung,Antonio Casamayor,Maria Deak,Dario R Alessi

doi:10.1074/jbc.m000421200

Abstract

Members of the AGC subfamily of protein kinases including protein kinase B, p70 S6 kinase, and protein kinase C (PKC) isoforms are activated and/or stabilized by phosphorylation of two residues, one that resides in the T-loop of the kinase domain and the other that is located C-terminal to the kinase domain in a region known as the hydrophobic motif. Atypical PKC isoforms, such as PKCzeta, and the PKC-related kinases, like PRK2, are also activated by phosphorylation of their T-loop site but, instead of possessing a phosphorylatable Ser/Thr in their hydrophobic motif, contain an acidic residue. The 3-phosphoinositide-dependent protein kinase (PDK1) activates many members of the AGC subfamily of kinases in vitro, including PKCzeta and PRK2 by phosphorylating the T-loop residue. In the present study we demonstrate that the hydrophobic motifs of PKCzeta and PKCiota, as well as PRK1 and PRK2, interact with the kinase domain of PDK1. Mutation of the conserved residues of the hydrophobic motif of full-length PKCzeta, full-length PRK2, or PRK2 lacking its N-terminal regulatory domain abolishes or significantly reduces the ability of these kinases to interact with PDK1 and to become phosphorylated at their T-loop sites in vivo. Furthermore, overexpression of the hydrophobic motif of PRK2 in cells prevents the T-loop phosphorylation and thus inhibits the activation of PRK2 and PKCzeta. These findings indicate that the hydrophobic motif of PRK2 and PKCzeta acts as a "docking site" enabling the recruitment of PDK1 to these substrates. This is essential for their phosphorylation by PDK1 in cells.

Highlights

Stimulation of cells with growth factors, phorbol esters, and insulin induces the activation of certain members of the AGC subfamily of protein kinases that include protein kinase B (PKB)1 [1, 2], p70 S6 kinase (p70 S6K) [3, 4], serum and glucocorticoid-induced kinase (SGK), [5,6,7] and many protein kinase C (PKC) isoforms [8, 9]
PDK1 Interacts with the Hydrophobic Motif of Atypical and Related PKC Isoforms—A yeast two-hybrid screen was carried out to identify proteins expressed in human brain that interact with PDK1
From this screen we identified a large number of positive clones that interacted with full-length PDK1 but not with the pleckstrin homology domain of PDK1, which corresponded to the C-terminal fragments of various PKC isoforms (Fig. 1A)

Summary

Introduction

Stimulation of cells with growth factors, phorbol esters, and insulin induces the activation of certain members of the AGC subfamily of protein kinases that include protein kinase B (PKB)1 [1, 2], p70 S6 kinase (p70 S6K) [3, 4], serum and glucocorticoid-induced kinase (SGK), [5,6,7] and many protein kinase C (PKC) isoforms [8, 9]. PDK1 phosphorylated a synthetic dodecapeptide, corresponding to the sequences surrounding the T-loop site in PKB at a low rate, but a peptide comprising the dodecapeptide fused to the C-terminal 24 residues of PIF (containing the PDK1 binding motif) was a vastly superior substrate [31]. These findings raised the possibility that the PIF-binding pocket on the kinase domain of PDK1 acts as a “docking site,” enabling it to interact with and enhance the phosphorylation of some substrates. We show that PDK1 interacts with these kinases through their hydrophobic motif and that this interaction is required to enable these kinases to become phosphorylated at their T-loop motif in cells

Methods

Results

Conclusion