Protein Kinase C Phosphorylates Protein Kinase D Activation Loop Ser744 and Ser748 and Releases Autoinhibition by the Pleckstrin Homology Domain*

Persistent activation of protein kinase D (PKD) via protein kinase C (PKC)-mediated signal transduction is accompanied by phosphorylation at Ser744 and Ser748 located in the catalytic domain activation loop, but whether PKC isoforms directly phosphorylate these residues, induce PKD autophosphorylation, or recruit intermediate upstream kinase(s) is unclear. Here, we explore the mechanism whereby PKC activates PKD in response to cellular stimuli. We first assessed in vitroPKC-PKD transphosphorylation and PKD activation. A PKD738–753 activation loop peptide was well phosphorylated by immunoprecipitated PKC isoforms, consistent with similarities between the loop and their known substrate specificities. A similar peptide with glutamic acid replacing Ser748 was preferentially phosphorylated by PKCε, suggesting that PKD containing phosphate at Ser748is rapidly targeted by this isoform at Ser744. When incubated in the presence of phosphatidylserine, phorbol 12,13-dibutyrate and ATP, intact PKD slowly autophosphorylated in the activation loop but only at Ser748. In contrast, addition of purified PKCε to the incubation mixture induced rapid Ser744 and Ser748 phosphorylation, concomitant with persistent 2–3-fold increases in PKD activity, measured using reimmunoprecipitated PKD to phosphorylate an exogenous peptide, syntide-2. We also further examined pleckstrin homology domain-mediated PKD regulation to determine its relationship with activation loop phosphorylation. The high constitutive activity of the pleckstrin homology (PH) domain deletion mutant PKD-ΔPH was not abrogated by mutation of Ser744 and Ser748 to alanines, suggesting that one function of activation loop phosphorylation in the PKD activation mechanism is to relieve autoinhibition by the PH domain. These studies provide evidence of a direct PKCε-PKD phosphorylation cascade and provide additional insight into the activation mechanism.