Use of a cAMP BRET Sensor to Characterize a Novel Regulation of cAMP by the Sphingosine 1-Phosphate/G13 Pathway

Regulation of intracellular cyclic adenosine 3',5'-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed Gs-dependent receptors for isoproterenol and prostaglandin E2. Whereas two ligands, uridine 5'-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via Gq/calcium and Gi, the action of sphingosine 1-phosphate (S1P) is novel. In these cells, S1P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for Gs-coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S1P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S1P is mediated by the S1P2 receptor and the heterotrimeric G13 protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP

Deciphering Signaling Outcomes from a System of Complex Networks

Cellular signal transduction machinery integrates information from multiple inputs to actuate discrete cellular behaviors. Interaction complexity exists when an input modulates the output behavior that results from other inputs. To address whether this machinery is iteratively complex—that is, whether increasing numbers of inputs produce exponential increases in discrete cellular behaviors—we examined the modulated secretion of six cytokines from macrophages in response to up to five-way combinations of an agonist of Toll-like receptor 4, three cytokines, and conditions that activated the cyclic adenosine monophosphate pathway. Although all of the selected ligands showed synergy in paired combinations, few examples of nonadditive outputs were found in response to higher-order combinations. This suggests that most potential interactions are not realized and that unique cellular responses are limited to discrete subsets of ligands and pathways that enhance specific cellular functions

Raf kinase activation of adenylyl cyclases: isoform-selective regulation

ABSTRACT Adenylyl cyclases (AC), a family of enzymes that catalyze the synthesis of cyclic AMP, are critical regulators of cellular functions. The activity of adenylyl cyclase is stimulated by a range of hormone receptors, primarily via interactions with G-proteins; however, recently we identified an alternate mechanism by which growth factors sensitize adenylyl cyclase activation. We suggested that this mechanism might involve a Raf kinasemediated serine phosphorylation of adenylyl cyclase. However, the direct involvement of a specific form of Raf kinase is yet to be demonstrated. Furthermore, whether this mechanism is generalized to other isoforms of adenylyl cyclase is unknown. In human embryonic kidney 293 cells, we now demonstrate that in reconstitution studies, c-Raf kinase can mediate phosphorylation of AC VI. Furthermore, AC VI coimmunoprecipitates with c-Raf. Raf kinase-dependent regulation of adenylyl cyclase VI is dependent on the integrity of Ser750 in the fourth intracellular loop of the enzyme and Ser603/Ser608 in the C1b region of the molecule. To examine how generalized this effect is, we studied representative isoforms of the major subfamilies of adenylyl cyclase viz., AC I, AC II, and AC V. Raf kinase-dependent sensitization/ phosphorylation of adenylyl cyclases is common to AC VI, AC V, and AC II isoforms but not AC I. In aggregate, these studies indicate that Raf kinase associates with adenylyl cyclases. Furthermore, Raf kinase regulation of adenylyl cyclase is isoform-selective. These functional interactions (as well as the physical association) between adenylyl cyclases and Raf kinases suggest an important but previously unrecognized interaction between these two key regulatory enzymes

Rethinking Polanyi’s concept of tacit knowledge: From personal knowing to imagined institutions

Half a century after Michael Polanyi conceptualised ‘the tacit component’ in personal knowing, management studies has reinvented ‘tacit knowledge’—albeit in ways that squander the advantages of Polanyi’s insights and ignore his faith in ‘spiritual reality’. While tacit knowing challenged the absurdities of sheer objectivity, expressed in a ‘perfect language’, it fused rational knowing, based on personal experience, with mystical speculation about an un-experienced ‘external reality’. Faith alone saved Polanyi’s model from solipsism. But Ernst von Glasersfeld’s radical constructivism provides scope to rethink personal tacit knowing with regard to ‘other people’ and the intersubjectively viable construction of ‘experiential reality’. By separating tacit knowing from Polanyi’s metaphysical realism and drawing on Benedict Anderson’s concept of ‘imagined communities’, it is possible to conceptualise ‘imagined institutions’ as the tacit dimension of power that shapes human interaction. Whereas Douglass North claimed institutions could be reduced to rules, imagined institutions are known in ways we cannot tell

Minimum information about a protein affinity reagent (MIAPAR)

This is a proposal developed within the community as an important first step in formalizing standards in reporting the production and properties of protein binding reagents, such as antibodies, developed and sold for the identification and detection of specific proteins present in biological samples. It defines a checklist of required information, intended for use by producers of affinity reagents, qualitycontrol laboratories, users and databases. We envision that both commercial and freely available affinity reagents, as well as published studies using these reagents, could include a MIAPAR-compliant document describing the product’s properties with every available binding partner. This would enable the user or reader to make a fully informed evaluation of the validity of conclusions drawn using this reagent

Evidence that a protein–protein interaction ‘hot spot’ on heterotrimeric G protein βγ subunits is used for recognition of a subclass of effectors

To understand the requirements for binding to G protein βγ subunits, phage-displayed random peptide libraries were screened using immobilized biotinylated βγ as the target. Selected peptides were grouped into four different families based on their sequence characteristics. One group (group I) had a clear conserved motif that has significant homology to peptides derived from phospholipase C β (PLC β) and to a short motif in phosducin that binds to G protein β subunits. The other groups had weaker sequence homologies or no homology to the group I sequences. A synthetic peptide from the strongest consensus group blocked activation of PLC by G protein βγ subunits. The peptide did not block βγ-mediated inhibition of voltage-gated calcium channels and had little effect on βγ-mediated inhibition of Gs-stimulated type I adenylate cyclase. Competition experiments indicated that peptides from all four families bound to a single site on βγ. These peptides may bind to a protein–protein interaction ‘hot spot’ on the surface of βγ subunits that is used by a subclass of effectors