cAMP-dependent activation of the Rac guanine exchange factor P-REX1 by type I protein kinase A (PKA) regulatory subunits

J Biol Chem. 2019 Feb 15;294(7):2232-2246. doi: 10.1074/jbc.RA118.006691. Epub 2018 Dec 10.

Abstract

Regulatory subunits of protein kinase A (PKA) inhibit its kinase subunits. Intriguingly, their potential as cAMP-dependent signal transducers remains uncharacterized. We recently reported that type I PKA regulatory subunits (RIα) interact with phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-REX1), a chemotactic Rac guanine exchange factor (RacGEF). Because P-REX1 is known to be phosphorylated and inhibited by PKA, its interaction with RIα suggests that PKA regulatory and catalytic subunits may fine-tune P-REX1 activity or those of its target pools. Here, we tested whether RIα acts as a cAMP-dependent factor promoting P-REX1-mediated Rac activation and cell migration. We observed that Gs-coupled EP2 receptors indeed promote endothelial cell migration via RIα-activated P-REX1. Expression of the P-REX1-PDZ1 domain prevented RIα/P-REX1 interaction, P-REX1 activation, and EP2-dependent cell migration, and P-REX1 silencing abrogated RIα-dependent Rac activation. RIα-specific cAMP analogs activated P-REX1, but lost this activity in RIα-knockdown cells, and cAMP pulldown assays revealed that P-REX1 preferentially interacts with free RIα. Moreover, purified RIα directly activated P-REX1 in vitro We also found that the RIα CNB-B domain is critical for the interaction with P-REX1, which was increased in RIα mutants, such as the acrodysostosis-associated mutant, that activate P-REX1 at basal cAMP levels. RIα and Cα PKA subunits targeted distinct P-REX1 molecules, indicated by an absence of phosphorylation in the active fraction of P-REX1. This was in contrast to the inactive fraction in which phosphorylated P-REX1 was present, suggesting co-existence of dual stimulatory and inhibitory effects. We conclude that PKA's regulatory subunits are cAMP-dependent signal transducers.

Keywords: EP2 receptors; G protein–coupled receptor (GPCR); P-REX1; Rac (Rac GTPase); Rho guanine nucleotide exchange factor (RhoGEF); cell migration; cell signaling; chemotaxis; cyclic AMP (cAMP); endothelial cell; guanine nucleotide exchange factor (GEF); heterotrimeric G protein; protein kinase A (PKA); regulatory subunit alpha (RIα); wound healing.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • AMP-Activated Protein Kinases / genetics
  • AMP-Activated Protein Kinases / metabolism*
  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Cyclic AMP / genetics
  • Cyclic AMP / metabolism*
  • Gene Knockdown Techniques
  • Guanine Nucleotide Exchange Factors / genetics
  • Guanine Nucleotide Exchange Factors / metabolism*
  • HEK293 Cells
  • Humans
  • MCF-7 Cells
  • PDZ Domains
  • Proto-Oncogene Proteins c-akt / genetics
  • Proto-Oncogene Proteins c-akt / metabolism
  • Receptors, Prostaglandin E, EP2 Subtype / genetics
  • Receptors, Prostaglandin E, EP2 Subtype / metabolism
  • Second Messenger Systems*

Substances

  • Guanine Nucleotide Exchange Factors
  • PREX1 protein, human
  • Receptors, Prostaglandin E, EP2 Subtype
  • Cyclic AMP
  • Proto-Oncogene Proteins c-akt
  • AMP-Activated Protein Kinases
  • PRKAA1 protein, human