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

Richard T Waldron,Enrique Rozengurt

doi:10.1074/jbc.m208075200

Abstract

Persistent activation of protein kinase D (PKD) via protein kinase C (PKC)-mediated signal transduction is accompanied by phosphorylation at Ser(744) and Ser(748) 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 vitro PKC-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 Ser(748) was preferentially phosphorylated by PKCepsilon, suggesting that PKD containing phosphate at Ser(748) is rapidly targeted by this isoform at Ser(744). When incubated in the presence of phosphatidylserine, phorbol 12,13-dibutyrate and ATP, intact PKD slowly autophosphorylated in the activation loop but only at Ser(748). In contrast, addition of purified PKCepsilon to the incubation mixture induced rapid Ser(744) and Ser(748) 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-deltaPH was not abrogated by mutation of Ser(744) and Ser(748) 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 PKCepsilon-PKD phosphorylation cascade and provide additional insight into the activation mechanism.

Highlights

Protein kinase C (PKC)1 is an enzyme family of central importance in signal transduction [1, 2]
We have shown that Ser744 and Ser748 are phosphorylated in vivo during Protein kinase D (PKD) activation in response to phorbol ester stimulation, in a manner blocked by preincubation with GF 109203X [50, 51]
Comparison of the PKD Activation Loop Segment with the Known Substrate Recognition Sequences of PKC Isoforms—The initial molecular cloning and expression of PKD and its human homologue PKC␮ was followed by the molecular cloning and expression of another related protein, termed PKD2, and two other clones far identified only at the cDNA level

Summary

Introduction

Protein kinase C (PKC) is an enzyme family of central importance in signal transduction [1, 2]. PKC isoforms exhibit distinct expression patterns, and in response to signaling events, become dynamically targeted to discrete subcellular locations and anchored in positions adjacent to substrates [10, 11]. This differential dynamic localization, as well as intrinsic substrate selectivity [12], may confer separate signaling roles to the individual PKC isoforms. Despite their recognized importance in signal transduction, few links have been established between individual PKC isozymes and the direct targets that specify their individual biological outcomes. PKD Activation Loop Phosphorylation by PKC⑀ Relieves Autoinhibition [21,22,23,24] or lysophosphatidic acid [23, 25], activation of receptor tyrosine kinases such as the platelet-derived growth factor receptor [21], signaling via heterotrimeric and monomeric G proteins [26, 27], and oxidative stress [28] were demonstrated to induce PKD activation in a wide variety of cell types including Swiss 3T3 fibroblasts, small cell lung cancer and pancreatic cancer, normal epithelial and smooth muscle cell lines, cardiocytes, and lymphocytes (23, 25, 29 –34)

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Conclusion