Reciprocal regulation of PKA and Rac signaling

Abstract

Activated G protein-coupled receptors (GPCRs) and receptor tyrosine kinases relay extracellular signals through spatial and temporal controlled kinase and GTPase entities. These enzymes are coordinated by multifunctional scaffolding proteins for precise intracellular signal processing. The cAMP-dependent protein kinase A (PKA) is the prime example for compartmentalized signal transmission downstream of distinct GPCRs. A-kinase anchoring proteins tether PKA to specific intracellular sites to ensure precision and directionality of PKA phosphorylation events. Here, we show that the Rho-GTPase Rac contains A-kinase anchoring protein properties and forms a dynamic cellular protein complex with PKA. The formation of this transient core complex depends on binary interactions with PKA subunits, cAMP levels and cellular GTP-loading accounting for bidirectional consequences on PKA and Rac downstream signaling. We show that GTP-Rac stabilizes the inactive PKA holoenzyme. However, β-adrenergic receptor-mediated activation of GTP-Rac-bound PKA routes signals to the Raf-Mek-Erk cascade, which is critically implicated in cell proliferation. We describe a further mechanism of how cAMP enhances nuclear Erk1/2 signaling: It emanates from transphosphorylation of p21-activated kinases in their evolutionary conserved kinase-activation loop through GTPRac compartmentalized PKA activities. Sole transphosphorylation of p21-activated kinases is not sufficient to activate Erk1/2. It requires complex formation of both kinases with GTP-Rac1 to unleash cAMP-PKA-boosted activation of Raf-Mek-Erk. Consequently GTPRac functions as a dual kinase-tuning scaffold that favors the PKA holoenzyme and contributes to potentiate Erk1/2 signaling. Our findings offer additional mechanistic insights how β-adrenergic receptor-controlled PKA activities enhance GTP-Rac-mediated activation of nuclear Erk1/2 signaling. signal transduction | cross-talk S ignal transduction cascades coordinate the plethora of extracellular stimuli into biological responses within cells. The specificity of receptor-initiated signaling responses is encoded by spatial and temporal dynamics of downstream signaling networks (1). These networks, initiating from e.g., the G protein-coupled receptor (GPCR) superfamily and receptor tyrosine kinases (RTK), tightly regulate signaling pathways at several critical points via feedback loops and cross-talk among other pathways (2-5). A large number of GPCR signaling cascades uses cAMP as an intracellular second messenger (3, 6). In response to hormone binding to distinct GPCRs, cAMP is produced and binds to its canonical effector, the cAMP-dependent protein kinase A (PKA). cAMP binding to the PKA regulatory subunits (R) induces dissociation of the tetrameric PKA holoenzyme, resulting in active PKA catalytic subunits (PKAc; Fig. 1C) (7, 8). To ensure substrate specificity, PKA is tethered to distinct subcellular compartments through physical interaction with A-kinase anchoring proteins (AKAPs; refs. 9 and 10). It has been long regarded that the...

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

“…PKA activation resulted in PAK-dependent phosphorylation of PREX1, suggesting that PKA is upstream of PAKs. A recent study showed that Rac binds to a PKA regulatory subunit and that PKA phosphorylates and activates PAK, setting up a model where PKA binds Rac-GTP to help in the stimulation of PAK and its downstream signals (45). It is also important to note that PREX1 is both activated by isoproterenol in cells and negatively regulated by PKA in vitro (10,31,32,46).…”

Inhibitor of MYC identified in a Kröhnke pyridine library

“…Rluc bioluminescence signals were integrated for 10 s following addition of the Rluc substrate benzylcoelenterazine to intact cells (5 μM; NanoLight). For immunoblot analyses, anti-Renilla luciferase antibodies to detect either F[1]-fused (Millipore; MAB4410) or F[2]-fused (Millipore; MAB4400) hybrid proteins were used (23,24).…”

“…Rluc-based PCA sentinels have been designed and used to study the dynamics of PPIs in vivo (23,24). An advantage of this assay is that it reports absolute values of protein-complex formation in real time.…”

Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling

“…To identify the specific amino acid residues of Gpr161 that are responsible for RIα binding, we used a peptide array assay. Overlapping 25-mer peptides derived from the human Gpr161-CT sequence were spotted onto a membrane and overlaid with recombinant RIα-his 6 , as previously described (27,28). In the Gpr161-CT we identified only one possible binding site for RIα.…”