Activation of Protein Kinase D by Signaling through Rho and the α Subunit of the Heterotrimeric G Protein G13

Jingzhen Yuan,Lee W Slice,Enrique Rozengurt

doi:10.1074/jbc.m105530200

Abstract

Protein kinase D (PKD/PKCmu) immunoprecipitated from COS-7 cells transiently transfected with either a constitutively active mutant of Rho (RhoQ63L) or the Rho-specific guanine nucleotide exchange factor pOnco-Lbc (Lbc) exhibited a marked increase in basal activity. Addition of aluminum fluoride to cells co-transfected with PKD and wild type Galpha(13) also induced PKD activation. Co-transfection of Clostridium botulinum C3 toxin blocked activation of PKD by RhoQ63L, Lbc, or aluminum fluoride-stimulated Galpha(13). Treatment with the protein kinase C inhibitors GF I or Ro 31-8220 prevented the increase in PKD activity induced by RhoQ63L, Lbc, or aluminum fluoride-stimulated Galpha(13). PKD activation in response to Galpha(13) signaling was also completely prevented by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. Co-expression of C. botulinum C3 toxin and a COOH-terminal fragment of Galpha(q) that acts in a dominant-negative fashion blocked PKD activation in response to agonist stimulation of bombesin receptor. Expression of the COOH-terminal region of Galpha(13) also attenuated PKD activation in response to bombesin receptor stimulation. Our results show that Galpha(13) contributes to PKD activation through a Rho- and protein kinase C-dependent signaling pathway and indicate that PKD activation is mediated by both Galpha(q) and Galpha(13) in response to bombesin receptor stimulation.

Highlights

PKD/Protein kinase C (PKC)␮ is a serine/threonine protein kinase [6, 7] with distinct structural, enzymological, and regulatory properties [8]
Our results show that G␣13 contributes to PKD activation through a Rho- and protein kinase C-dependent signaling pathway and indicate that PKD activation is mediated by both G␣q and G␣13 in response to bombesin receptor stimulation
Expression of Constitutively Activated Rho Induces PKD Activation—To test whether PKD activation can be induced by signaling pathway(s) initiated by the low molecular weight G proteins of the Rho subfamily, COS-7 cells were co-transfected with PKD and expression vectors encoding wild type Rho, Ras, Rac or constitutively active forms of the three proteins, RhoQ63L, RasVal12, or RacVal12

Summary

Introduction

PKD/PKC␮ is a serine/threonine protein kinase [6, 7] with distinct structural, enzymological, and regulatory properties [8]. Our results show that G␣13 contributes to PKD activation through a Rho- and protein kinase C-dependent signaling pathway and indicate that PKD activation is mediated by both G␣q and G␣13 in response to bombesin receptor stimulation.

Results

Conclusion