Protein Kinase A (PKA) Type I Interacts with P-Rex1, a Rac Guanine Nucleotide Exchange Factor

Abstract

Morphology of migrating cells is regulated by Rho GTPases and fine-tuned by protein interactions and phosphorylation.PKA affects cell migration potentially through spatiotemporal interactions with regulators of Rho GTPases. Here we show that the endogenous regulatory (R) subunit of type I PKA interacts with P-Rex1, a Rac guanine nucleotide exchange factor that integrates chemotactic signals. Type I PKA holoenzyme interacts with P-Rex1 PDZ domains via the CNB B domain of RI␣, which when expressed by itself facilitates endothelial cell migration. P-Rex1 activation localizes PKA to the cell periphery, whereas stimulation of PKA phosphorylates P-Rex1 and prevents its activation in cells responding to SDF-1 (stromal cellderived factor 1). The P-Rex1 DEP 1 domain is phosphorylated at Ser-436, which inhibits the DH-PH catalytic cassette by direct interaction. In addition, the P-Rex1 C terminus is indirectly targeted by PKA, promoting inhibitory interactions independently of the DEP 1 -PDZ 2 region. A P-Rex1 S436A mutant construct shows increased RacGEF activity and prevents the inhibitory effect of forskolin on sphingosine 1-phosphate-dependent endothelial cell migration. Altogether, these results support the idea that P-Rex1 contributes to the spatiotemporal localization of type I PKA, which tightly regulates this guanine exchange factor by a multistep mechanism, initiated by interaction with the PDZ domains of P-Rex1 followed by direct phosphorylation at the first DEP domain and putatively indirect regulation of the C terminus, thus promoting inhibitory intramolecular interactions. This reciprocal regulation between PKA and P-Rex1 might represent a key node of integration by which chemotactic signaling is fine-tuned by PKA.Rho guanine exchange factors (RhoGEFs) 4 are mechanistically linked to fundamental cellular processes, such as migration, adhesion, and morphogenesis. Based on their ability to integrate signaling inputs that result in the activation of Rho GTPases, RhoGEFs indirectly contribute to establish nucleation sites for actin polymerization, thus exerting a tight control on cytoskeleton dynamics (1, 2). RhoGEFs can also play a role as scaffolds of different signaling cascades, thus representing regulatory nodes for spatial and temporal control of signaling (3). These fundamental processes are fine-tuned by phosphorylation of RhoGEFs. However, the complex interplay between RhoGEFs and kinases is not completely understood; relevant examples include RhoGEFs known to be activated or inhibited by phosphorylation, some of them by PKA (4, 5).The ability of PKA to modulate the activity of RhoGEFs might be further facilitated by direct interactions that would also influence the subcellular localization of PKA and its accessibility to specific substrates. The paradigmatic example of this situation is AKAP-Lbc, which is a RhoGEF activated by G 12 -coupled receptors that promotes polymerization of actin filaments downstream of Rho and also interacts with multiple kinases regulating mitogenic inputs (6, 7). AKAP-Lbc ...

PREX1 Protein Function Is Negatively Regulated Downstream of Receptor Tyrosine Kinase Activation by p21-activated Kinases (PAKs)

“…It is known that PREX1 is phosphorylated in the cell, demonstrated by the fact that after gel electrophoresis, it is detected as multiple bands that are collapsed by both -phosphatase and protein phosphatase 1␣ (PP1␣) (30,31). -Phosphatase and PP1␣ dephosphorylation of PREX1 increase GEF activity, which is consistent with reports showing that PKA phosphorylation of PREX1 leads to a reduction in its in vitro GEF activity both in vitro and in cells (31,32,49). Importantly, PREX1 is phosphorylated after the activation of specific RTKs and GPCRs, indicating that the regulation of PREX1 by phosphorylation has a role in these signaling pathways.…”

“…It is known that PREX1 can be phosphorylated downstream of isoproterenol and sphingosine 1-phosphate (31,49), and in this study we identified additional phosphorylation events on PREX1 after treatment with another GPCR agonist, PGE2. PGE2 is produced through the processing of the fatty acid arachidonic acid and has wide-ranging and diverse roles in physiology in addition to being implicated in cancer (42).…”

“…The stimulation of ␤-adrenergic GPCRs with isoproterenol results in the phosphorylation of PREX1, which is likely due to the activation of PKA downstream of the ␤-adrenergic receptor (31). Similarly, downstream of sphingosine 1-phosphate, PKA phosphorylates PREX1 to regulate an intramolecular interaction that reduces PREX1 GEF activity (49). Additionally, PREX1 is phosphorylated after the stimulation of RTKs with neuregulin, insulin-like growth factor 1 (IGF1), platelet-derived growth factor, and fibroblast growth factor, but the kinases that are responsible for these phosphorylation events are not known (18,19).…”

“A-kinase” regulator runs amok to provide a paradigm shift in cAMP signaling

“…Although P-REX1 GEF activity is known to be inhibited by PKAcatalyzed phosphorylation (2), Adame-García et al now report (1) that PKA-RI␣ interacts directly with P-REX1 to increase its GEF activity, an effect that is independent of PKA catalytic subunit activity. This surprising finding is an outgrowth of the same laboratory's prior study, which used a yeast 2-hybrid screen to demonstrate that a fragment of P-REX1 containing two PDZ domains (P-REX1-PDZ1-PDZ2) acted as "bait" to capture its "prey," which corresponded to a C-terminal fragment of PKA-RI␣ (3). At that time, the P-REX1 interaction site on PKA-RI␣ was established to be cyclic nucleotide-binding domain B (CNBD-B), and this site recognized the P-REX1-PDZ1-PDZ2 domains ( Fig.…”

P-Rex1 and P-Rex2 RacGEFs and cancer

“…P-Rex1 PDZ domains are also implicated in the interaction between P-Rex1 and ephrin-B1, a transmembrane ligand for the Eph receptor tyrosine kinases involved in neuronal development, as P-Rex1 mutant lacking PDZ domains could not be coimmunoprecipitated with ephrin-B1 in mouse embryonic cortex lysates [44]. Of note, the P-Rex1 PDZ domains also interact with protein kinase A (PKA) [42,45]. PKA potently inhibits P-Rex1 RacGEF activity by phosphorylating the P-Rex1 DEP1 domain and promoting an autoinhibitory conformation, in which the DEP domain and the C-terminal tail obstruct the DH domain catalytic activity [45].…”