Feedback inhibition of cAMP effector signaling by a chaperone-assisted ubiquitin system.

Activation of G-protein coupled receptors elevates cAMP levels promoting dissociation of protein kinase A (PKA) holoenzymes and release of catalytic subunits (PKAc). This results in PKAc-mediated phosphorylation of compartmentalized substrates that control central aspects of cell physiology. The mechanism of PKAc activation and signaling have been largely characterized. However, the modes of PKAc inactivation by regulated proteolysis were unknown. Here, we identify a regulatory mechanism that precisely tunes PKAc stability and downstream signaling. Following agonist stimulation, the recruitment of the chaperone-bound E3 ligase CHIP promotes ubiquitylation and proteolysis of PKAc, thus attenuating cAMP signaling. Genetic inactivation of CHIP or pharmacological inhibition of HSP70 enhances PKAc signaling and sustains hippocampal long-term potentiation. Interestingly, primary fibroblasts from autosomal recessive spinocerebellar ataxia 16 (SCAR16) patients carrying germline inactivating mutations of CHIP show a dramatic dysregulation of PKA signaling. This suggests the existence of a negative feedback mechanism for restricting hormonally controlled PKA activities.

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.

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.

Synthetic Glycosphingolipids for Live-Cell Labeling.

Glycosphingolipids are an important component of cell membranes that are involved in many biological processes. Fluorescently labeled glycosphingolipids are frequently used to gain insight into their localization. However, the attachment of a fluorophore to the glycan part or-more commonly-to the lipid part of glycosphingolipids is known to alter the biophysical properties and can perturb the biological function of the probe. Presented here is the synthesis of novel glycosphingolipid probes with mono- and disaccharide head groups and ceramide moieties containing fatty acids of varying chain length (C4 to C20). These glycosphingolipids bear an azide or an alkyne group as chemical reporter to which a fluorophore can be attached through a bioorthogonal ligation reaction. The fluorescent tag and any linker connected to it can be chosen in a flexible manner. We demonstrate the suitability of the probes by selective visualization of the plasma membrane of living cells by confocal microscopy techniques. Whereas the derivatives with the shorter fatty acids can be directly applied to HEK 293T cells, the hydrophobic glycosphingolipids with longer fatty acids can be delivered to cells using fusogenic liposomes.

Corrigendum: Impact of kinase activating and inactivating patient mutations on binary PKA interactions.

[This corrects the article on p. 170 in vol. 6, PMID: 26347651.].

Impact of kinase activating and inactivating patient mutations on binary PKA interactions.

The second messenger molecule cAMP links extracellular signals to intracellular responses. The main cellular cAMP effector is the compartmentalized protein kinase A (PKA). Upon receptor initiated cAMP-mobilization, PKA regulatory subunits (R) bind cAMP thereby triggering dissociation and activation of bound PKA catalytic subunits (PKAc). Mutations in PKAc or RIa subunits manipulate PKA dynamics and activities which contribute to specific disease patterns. Mutations activating cAMP/PKA signaling contribute to carcinogenesis or hormone excess, while inactivating mutations cause hormone deficiency or resistance. Here we extended the application spectrum of a Protein-fragment Complementation Assay based on the Renilla Luciferase to determine binary protein:protein interactions (PPIs) of the PKA network. We compared time- and dose-dependent influences of cAMP-elevation on mutually exclusive PPIs of PKAc with the phosphotransferase inhibiting RIIb and RIa subunits and the protein kinase inhibitor peptide (PKI). We analyzed PKA dynamics following integration of patient mutations into PKAc and RIa. We observed that oncogenic modifications of PKAc(L206R) and RIa(Δ184-236) as well as rare disease mutations in RIa(R368X) affect complex formation of PKA and its responsiveness to cAMP elevation. With the cell-based PKA PPI reporter platform we precisely quantified the mechanistic details how inhibitory PKA interactions and defined patient mutations contribute to PKA functions.

Bile Salts Modulate the Mucin-Activated Type VI Secretion System of Pandemic Vibrio cholerae.

The causative agent of cholera, Vibrio cholerae, regulates its diverse virulence factors to thrive in the human small intestine and environmental reservoirs. Among this pathogen's arsenal of virulence factors is the tightly regulated type VI secretion system (T6SS). This system acts as an inverted bacteriophage to inject toxins into competing bacteria and eukaryotic phagocytes. V. cholerae strains responsible for the current 7th pandemic activate their T6SS within the host. We established that T6SS-mediated competition occurs upon T6SS activation in the infant mouse, and that this system is functional under anaerobic conditions. When investigating the intestinal host factors mucins (a glycoprotein component of mucus) and bile for potential regulatory roles in controlling the T6SS, we discovered that once mucins activate the T6SS, bile acids can further modulate T6SS activity. Microbiota modify bile acids to inhibit T6SS-mediated killing of commensal bacteria. This interplay is a novel interaction between commensal bacteria, host factors, and the V. cholerae T6SS, showing an active host role in infection.

In-vivo detection of binary PKA network interactions upon activation of endogenous GPCRs.

Membrane receptor-sensed input signals affect and modulate intracellular protein-protein interactions (PPIs). Consequent changes occur to the compositions of protein complexes, protein localization and intermolecular binding affinities. Alterations of compartmentalized PPIs emanating from certain deregulated kinases are implicated in the manifestation of diseases such as cancer. Here we describe the application of a genetically encoded Protein-fragment Complementation Assay (PCA) based on the Renilla Luciferase (Rluc) enzyme to compare binary PPIs of the spatially and temporally controlled protein kinase A (PKA) network in diverse eukaryotic model systems. The simplicity and sensitivity of this cell-based reporter allows for real-time recordings of mutually exclusive PPIs of PKA upon activation of selected endogenous G protein-coupled receptors (GPCRs) in cancer cells, xenografts of mice, budding yeast, and zebrafish embryos. This extends the application spectrum of Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems.

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.

The Vibrio cholerae type VI secretion system employs diverse effector modules for intraspecific competition.

Vibrio cholerae is a Gram-negative bacterial pathogen that consists of over 200 serogroups with differing pathogenic potential. Only strains that express the virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) are capable of pandemic spread of cholera diarrhoea. Regardless, all V. cholerae strains sequenced to date harbour genes for the type VI secretion system (T6SS) that translocates effectors into neighbouring eukaryotic and prokaryotic cells. Here we report that the effectors encoded within these conserved gene clusters differ widely among V. cholerae strains, and that immunity proteins encoded immediately downstream from the effector genes protect their host from neighbouring bacteria producing corresponding effectors. As a consequence, strains with matching effector-immunity gene sets can coexist, while strains with different sets compete against each other. Thus, the V. cholerae T6SS contributes to the competitive behaviour of this species.

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.

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.

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.

Type VI secretion system regulation as a consequence of evolutionary pressure.

The type VI secretion system (T6SS) is a mechanism evolved by Gram-negative bacteria to negotiate interactions with eukaryotic and prokaryotic competitors. T6SSs are encoded by a diverse array of bacteria and include plant, animal, human and fish pathogens, as well as environmental isolates. As such, the regulatory mechanisms governing T6SS gene expression vary widely from species to species, and even from strain to strain within a given species. This review concentrates on the four bacterial genera that the majority of recent T6SS regulatory studies have been focused on: Vibrio, Pseudomonas, Burkholderia and Edwardsiella.

Lytic activity of the Vibrio cholerae type VI secretion toxin VgrG-3 is inhibited by the antitoxin TsaB.

The type VI secretion system (T6SS) of Gram-negative bacteria has been implicated in microbial competition; however, which components serve purely structural roles, and which serve as toxic effectors remains unresolved. Here, we present evidence that VgrG-3 of the Vibrio cholerae T6SS has both structural and toxin activity. Specifically, we demonstrate that the C-terminal extension of VgrG-3 acts to degrade peptidoglycan and hypothesize that this assists in the delivery of accessory T6SS toxins of V. cholerae. To avoid self-intoxication, V. cholerae expresses an anti-toxin encoded immediately downstream of vgrG-3 that inhibits VgrG-3-mediated lysis through direct interaction.

Constitutive type VI secretion system expression gives Vibrio cholerae intra- and interspecific competitive advantages.

The type VI secretion system (T6SS) mediates protein translocation across the cell membrane of Gram-negative bacteria, including Vibrio cholerae - the causative agent of cholera. All V. cholerae strains examined to date harbor gene clusters encoding a T6SS. Structural similarity and sequence homology between components of the T6SS and the T4 bacteriophage cell-puncturing device suggest that the T6SS functions as a contractile molecular syringe to inject effector molecules into prokaryotic and eukaryotic target cells. Regulation of the T6SS is critical. A subset of V. cholerae strains, including the clinical O37 serogroup strain V52, express T6SS constitutively. In contrast, pandemic strains impose tight control that can be genetically disrupted: mutations in the quorum sensing gene luxO and the newly described regulator gene tsrA lead to constitutive T6SS expression in the El Tor strain C6706. In this report, we examined environmental V. cholerae isolates from the Rio Grande with regard to T6SS regulation. Rough V. cholerae lacking O-antigen carried a nonsense mutation in the gene encoding the global T6SS regulator VasH and did not display virulent behavior towards Escherichia coli and other environmental bacteria. In contrast, smooth V. cholerae strains engaged constitutively in type VI-mediated secretion and displayed virulence towards prokaryotes (E. coli and other environmental bacteria) and a eukaryote (the social amoeba Dictyostelium discoideum). Furthermore, smooth V. cholerae strains were able to outcompete each other in a T6SS-dependent manner. The work presented here suggests that constitutive T6SS expression provides V. cholerae with an advantage in intraspecific and interspecific competition.

Extracellular IgC2 constant domains of CEACAMs mediate PI3K sensitivity during uptake of pathogens.

BACKGROUND: Several pathogenic bacteria utilize receptors of the CEACAM family to attach to human cells. Binding to different members of this receptor family can result in uptake of the bacteria. Uptake of Neisseria gonorrhoeae, a gram-negative human pathogen, via CEACAMs found on epithelial cells, such as CEACAM1, CEA or CEACAM6, differs mechanistically from phagocytosis mediated by CEACAM3, a CEACAM family member expressed selectively by human granulocytes. PRINCIPAL FINDINGS: We find that CEACAM1- as well as CEACAM3-mediated bacterial internalization are accompanied by a rapid increase in phosphatidylinositol-3,4,5 phosphate (PI(3,4,5)P) at the site of bacterial entry. However, pharmacological inhibition of phosphatidylinositol-3' kinase (PI3K) selectively affects CEACAM1-mediated uptake of Neisseria gonorrhoeae. Accordingly, overexpression of the PI(3,4,5)P phosphatase SHIP diminishes and expression of a constitutive active PI3K increases CEACAM1-mediated internalization of gonococci, without influencing uptake by CEACAM3. Furthermore, bacterial uptake by GPI-linked members of the CEACAM family (CEA and CEACAM6) and CEACAM1-mediated internalization of N. meningitidis by endothelial cells require PI3K activity. Sensitivity of CEACAM1-mediated uptake toward PI3K inhibition is independent of receptor localization in cholesterol-rich membrane microdomains and does not require the cytoplasmic or the transmembrane domain of CEACAM1. However, PI3K inhibitor sensitivity requires the Ig(C2)-like domains of CEACAM1, which are also present in CEA and CEACAM6, but which are absent from CEACAM3. Accordingly, overexpression of CEACAM1 Ig(C2) domains blocks CEACAM1-mediated internalization. CONCLUSIONS: Our results provide novel mechanistic insight into CEACAM1-mediated endocytosis and suggest that epithelial CEACAMs associate in cis with other membrane receptor(s) via their extracellular domains to trigger bacterial uptake in a PI3K-dependent manner.

PKA regulatory subunits mediate synergy among conserved G-protein-coupled receptor cascades.

G-protein-coupled receptors sense extracellular chemical or physical stimuli and transmit these signals to distinct trimeric G-proteins. Activated Gα-proteins route signals to interconnected effector cascades, thus regulating thresholds, amplitudes and durations of signalling. Gαs- or Gαi-coupled receptor cascades are mechanistically conserved and mediate many sensory processes, including synaptic transmission, cell proliferation and chemotaxis. Here we show that a central, conserved component of Gαs-coupled receptor cascades, the regulatory subunit type-II (RII) of protein kinase A undergoes adenosine 3'-5'-cyclic monophosphate (cAMP)-dependent binding to Gαi. Stimulation of a mammalian Gαi-coupled receptor and concomitant cAMP-RII binding to Gαi, augments the sensitivity, amplitude and duration of Gαi:ßγ activity and downstream mitogen-activated protein kinase signalling, independent of protein kinase A kinase activity. The mechanism is conserved in budding yeast, causing nutrient-dependent modulation of a pheromone response. These findings suggest a direct mechanism by which coincident activation of Gαs-coupled receptors controls the precision of adaptive responses of activated Gαi-coupled receptor cascades.

Phosphatidylinositol 3'-kinase activity is critical for initiating the oxidative burst and bacterial destruction during CEACAM3-mediated phagocytosis.

Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is an immunoglobulin-related receptor expressed on human granulocytes. CEACAM3 functions as a single chain phagocytotic receptor recognizing gram-negative bacteria such as Neisseria gonorrhoeae, which possess CEACAM-binding adhesins on their surface. The cytoplasmic domain of CEACAM3 contains an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is phosphorylated upon receptor engagement. Here we show that the SH2 domains of the regulatory subunit of phosphatidylinositol 3'-kinase (PI3K) bind to tyrosine residue 230 of CEACAM3 in a phosphorylation-dependent manner. PI3K is rapidly recruited and directly associates with CEACAM3 upon bacterial binding as shown by FRET analysis. Although PI3K activity is not required for efficient uptake of the bacteria by CEACAM3-transfected cells or primary human granulocytes, it is critical for the stimulated production of reactive oxygen species by infected phagocytes and the intracellular degradation of CEACAM-binding bacteria. Together, our results highlight the ability of CEACAM3 to coordinate signaling events that not only mediate bacterial uptake, but also trigger the killing of internalized pathogens.