Sequential Protein Kinase C (PKC)-dependent and PKC-independent Protein Kinase D Catalytic Activation via Gq-coupled Receptors: DIFFERENTIAL REGULATION OF ACTIVATION LOOP SER744 AND SER748 PHOSPHORYLATION

Rodrigo Jacamo,James Sinnett-Smith,Osvaldo Rey,Richard T Waldron,Enrique Rozengurt

doi:10.1074/jbc.m800442200

Rodrigo Jacamo, James Sinnett-Smith + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m800442200

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Abstract

Protein kinase D (PKD) is a serine/threonine protein kinase rapidly activated by G protein-coupled receptor (GPCR) agonists via a protein kinase C (PKC)-dependent pathway. Recently, PKD has been implicated in the regulation of long term cellular activities, but little is known about the mechanism(s) of sustained PKD activation. Here, we show that cell treatment with the preferential PKC inhibitors GF 109203X or Gö 6983 blocked rapid (1-5-min) PKD activation induced by bombesin stimulation, but this inhibition was greatly diminished at later times of bombesin stimulation (e.g. 45 min). These results imply that GPCR-induced PKD activation is mediated by early PKC-dependent and late PKC-independent mechanisms. Western blot analysis with site-specific antibodies that detect the phosphorylated state of the activation loop residues Ser(744) and Ser(748) revealed striking PKC-independent phosphorylation of Ser(748) as well as Ser(744) phosphorylation that remained predominantly but not completely PKC-dependent at later times of bombesin or vasopressin stimulation (20-90 min). To determine the mechanisms involved, we examined activation loop phosphorylation in a set of PKD mutants, including kinase-deficient, constitutively activated, and PKD forms in which the activation loop residues were substituted for alanine. Our results show that PKC-dependent phosphorylation of the activation loop Ser(744) and Ser(748) is the primary mechanism involved in early phase PKD activation, whereas PKD autophosphorylation on Ser(748) is a major mechanism contributing to the late phase of PKD activation occurring in cells stimulated by GPCR agonists. The present studies identify a novel mechanism induced by GPCR activation that leads to late, PKC-independent PKD activation.

Highlights

A rapid increase in the synthesis of lipid-derived second messengers with subsequent activation of protein phosphorylation cascades has emerged as a fundamental signal transduction mechanism triggered by multiple extracellular stimuli, including hormones, neurotransmitters, chemokines, and growth factors [1]
In order to determine the role of protein kinase C (PKC) in Protein kinase D (PKD) catalytic activation within cells stimulated for various times with a Gq-coupled receptor agonist, COS-7 cells were transiently co-transfected with expression plasmids encoding wild type green fluorescent protein (GFP)-PKD and the bombesin-preferring G protein-coupled receptor (GPCR)
We have previously demonstrated that the fusion of GFP to the N terminus of PKD did not produce any detectable effect on PKD basal catalytic activity, GPCR-induced activation, and intracellular distribution [38, 57, 58, 62]

Summary

Introduction

A rapid increase in the synthesis of lipid-derived second messengers with subsequent activation of protein phosphorylation cascades has emerged as a fundamental signal transduction mechanism triggered by multiple extracellular stimuli, including hormones, neurotransmitters, chemokines, and growth factors [1]. Requirement of PKD Catalytic Activity for Late-phase Ser748 Phosphorylation—At least two mechanisms involving autophosphorylation or transphosphorylation may contribute to PKC-independent PKD phosphorylation on Ser748 in response to prolonged stimulation with GPCR agonists.

Results

Conclusion