Further evidence that 3‐phosphoinositide‐dependent protein kinase‐1 (PDK1) is required for the stability and phosphorylation of protein kinase C (PKC) isoforms

Open Access
Publisher Website
Google Scholar
Abstract
The multi‐site phosphorylation of the protein kinase C (PKC) superfamily plays an important role in the regulation of these enzymes. One of the key phosphorylation sites required for the activation of all PKC isoforms lies in the T‐loop of the kinase domain. Recent in vitro and transfection experiments indicate that phosphorylation of this residue can be mediated by the 3‐phosphoinositide‐dependent protein kinase‐1 (PDK1). In this study, we demonstrate that in embryonic stem (ES) cells lacking PDK1 (PDK1−/− cells), the intracellular levels of endogenously expressed PKCα, PKCβI, PKCγ, PKCδ, PKCϵ, and PKC‐related kinase‐1 (PRK1) are vastly reduced compared to control ES cells (PDK1+/+ cells). The levels of PKCζ and PRK2 protein are only moderately reduced in the PDK1−/− ES cells. We demonstrate that in contrast to PKCζ expressed PDK1+/+ ES cells, PKCζ in ES cells lacking PDK1 is not phosphorylated at its T‐loop residue. This provides the first genetic evidence that PKCζ is a physiological substrate for PDK1. In contrast, PRK2 is still partially phosphorylated at its T‐loop in PDK1−/− cells, indicating the existence of a PDK1‐independent mechanism for the phosphorylation of PRK2 at this residue.