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

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 (RI alpha) 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 RI alpha suggests that PKA regulatory and catalytic subunits may fine-tune P-REX1 activity or those of its target pools. Here, we tested whether RI alpha acts as a cAMP-dependent factor promoting P-REX1-mediated Rac activation and cell migration. We observed that G(s)-coupled EP2 receptors indeed promote endothelial cell migration via RI alpha-activated P-REX1. Expression of the P-REX1-PDZ1 domain prevented RI alpha/P-REX1 interaction, P-REX1 activation, and EP2-dependent cell migration, and P-REX1 silencing abrogated RI alpha-dependent Rac activation. RI alpha-specific cAMP analogs activated P-REX1, but lost this activity in RI alpha-knockdown cells, and cAMP pulldown assays revealed that P-REX1 preferentially interacts with free RI alpha. Moreover, purified RI alpha directly activated P-REX1 in vitro. We also found that the RI alpha CNB-B domain is critical for the interaction with P-REX1, which was increased in RI alpha mutants, such as the acrodysostosis-associated mutant, that activate P-REX1 at basal cAMP levels. RI alpha and C alpha 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.