Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling.

Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.

Systematic identification of signal integration by protein kinase A.

Cellular processes and homeostasis control in eukaryotic cells is achieved by the action of regulatory proteins such as protein kinase A (PKA). Although the outbound signals from PKA directed to processes such as metabolism, growth, and aging have been well charted, what regulates this conserved regulator remains to be systematically identified to understand how it coordinates biological processes. Using a yeast PKA reporter assay, we identified genes that influence PKA activity by measuring protein-protein interactions between the regulatory and the two catalytic subunits of the PKA complex in 3,726 yeast genetic-deletion backgrounds grown on two carbon sources. Overall, nearly 500 genes were found to be connected directly or indirectly to PKA regulation, including 80 core regulators, denoting a wide diversity of signals regulating PKA, within and beyond the described upstream linear pathways. PKA regulators span multiple processes, including the antagonistic autophagy and methionine biosynthesis pathways. Our results converge toward mechanisms of PKA posttranslational regulation by lysine acetylation, which is conserved between yeast and humans and that, we show, regulates protein complex formation in mammals and carbohydrate storage and aging in yeast. Taken together, these results show that the extent of PKA input matches with its output, because this kinase receives information from upstream and downstream processes, and highlight how biological processes are interconnected and coordinated by PKA.

Reciprocal regulation of PKA and Rac signaling.

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, ß-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 GTP-Rac 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 GTP-Rac 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 ß-adrenergic receptor-controlled PKA activities enhance GTP-Rac-mediated activation of nuclear Erk1/2 signaling.

Counterregulation of cAMP-directed kinase activities controls ciliogenesis.

The primary cilium emanates from the cell surface of growth-arrested cells and plays a central role in vertebrate development and tissue homeostasis. The mechanisms that control ciliogenesis have been extensively explored. However, the intersection between GPCR signaling and the ubiquitin pathway in the control of cilium stability are unknown. Here we observe that cAMP elevation promotes cilia resorption. At centriolar satellites, we identify a multimeric complex nucleated by PCM1 that includes two kinases, NEK10 and PKA, and the E3 ubiquitin ligase CHIP. We show that NEK10 is essential for ciliogenesis in mammals and for the development of medaka fish. PKA phosphorylation primes NEK10 for CHIP-mediated ubiquitination and proteolysis resulting in cilia resorption. Disarrangement of this control mechanism occurs in proliferative and genetic disorders. These findings unveil a pericentriolar kinase signalosome that efficiently links the cAMP cascade with the ubiquitin-proteasome system, thereby controlling essential aspects of ciliogenesis.

Interplay of PKA and Rac: fine-tuning of Rac localization and signaling.

Cellular membrane receptors sense environmental changes and relay the reshaped signal through spatially and temporally organized protein-protein interactions (PPI). Many of such PPI are transient and occur in a certain cell-dependent context. Molecular switches such as kinases and GTPases are engaged in versatile PPI. Recently, we have identified dynamic interaction and reciprocal regulation of cAMP-dependent protein kinase A (PKA) and Rho-GTPase Rac signaling. We demonstrated that GTP-activated Rac acts as a dual kinase-tuning scaffold for p21-activated kinase (PAK) and PKA activities. We showed that receptor-triggered PKA trans-phosphorylation of GTP-Rac-organized PAK contributes to elevations of nuclear Erk1/2 signaling and proliferation. We discuss these recent observations and we provide additional insights how the cAMP-PKA axis might also participate in the regulation of Rac localization.

Proteolysis of MOB1 by the ubiquitin ligase praja2 attenuates Hippo signalling and supports glioblastoma growth.

Human glioblastoma is the most frequent and aggressive form of brain tumour in the adult population. Proteolytic turnover of tumour suppressors by the ubiquitin-proteasome system is a mechanism that tumour cells can adopt to sustain their growth and invasiveness. However, the identity of ubiquitin-proteasome targets and regulators in glioblastoma are still unknown. Here we report that the RING ligase praja2 ubiquitylates and degrades Mob, a core component of NDR/LATS kinase and a positive regulator of the tumour-suppressor Hippo cascade. Degradation of Mob through the ubiquitin-proteasome system attenuates the Hippo cascade and sustains glioblastoma growth in vivo. Accordingly, accumulation of praja2 during the transition from low- to high-grade glioma is associated with significant downregulation of the Hippo pathway. These findings identify praja2 as a novel upstream regulator of the Hippo cascade, linking the ubiquitin proteasome system to deregulated glioblastoma growth.