Signals mediated by heterotrimeric G proteins often develop over the course of tens of milliseconds, and could require either conformational rearrangement or complete physical dissociation of Gα βγ heterotrimers. Although it is known that some active heterotrimers are dissociated (into Gα and Gβγ) at steady-state, it is not clear that dissociation occurs quickly enough to participate in rapid signaling. Here we show that fusion proteins containing the c-terminus of GPCR kinase 3 (GRK3ct) and either the fluorescent protein cerulean or Renilla luciferase bind to venus-labeled Gβγ dimers (Gβγ-V), resulting in Förster or bioluminescence resonance energy transfer (FRET or BRET). GRK3ct fusion proteins are freely-diffusible, and do not form preassembled complexes with G proteins. GRK3ct fusion proteins bind to free Gβγ-V dimers but not to rearranged heterotrimers, and thus can report G protein dissociation with high temporal resolution. We find that heterotrimer dissociation can occur in living cells in less than 100 milliseconds. Under the conditions of these experiments diffusion and collision of masGRK3ct fusion proteins and Gβγ-V were not rate-limiting. These results indicate that G protein heterotrimers can dissociate quickly enough to participate in rapid signaling.

G-protein heterotrimers, composed of a guanine nucleotide-binding Gα subunit and an obligate Gβγ dimer, regulate signal transduction pathways by cycling between GDP- and GTP-bound states. Signal deactivation is achieved by Gα-mediated GTP hydrolysis (GTPase activity) which is enhanced by the GTPase-accelerating protein (GAP) activity of “regulator of G-protein signaling” (RGS) proteins. In a cellular context, RGS proteins have also been shown to speed up the onset of signaling, and to accelerate deactivation without changing amplitude or sensitivity of the signal. This latter paradoxical activity has been variably attributed to GAP/enzymatic or non-GAP/scaffolding functions of these proteins. Here, we validated and exploited a Gα switch-region point mutation, known to engender increased GTPase activity, to mimic in cis the GAP function of RGS proteins. While the transition-state, GDP·AlF 4 − -bound conformation of the G202A mutant was found to be nearly identical to wild-type, Gα i1 (G202A)·GDP assumed a divergent conformation more closely resembling the GDP·AlF 4 − -bound state. When placed within Saccharomyces cerevisiae Gα subunit Gpa1, the fast-hydrolysis mutation restored appropriate dose–response behaviors to pheromone signaling in the absence of RGS-mediated GAP activity. A bioluminescence resonance energy transfer (BRET) readout of heterotrimer activation with high temporal resolution revealed that fast intrinsic GTPase activity could recapitulate in cis the kinetic sharpening (increased onset and deactivation rates) and blunting of sensitivity also engendered by RGS protein action in trans . Thus Gα-directed GAP activity, the first biochemical function ascribed to RGS proteins, is sufficient to explain the activation kinetics and agonist sensitivity observed from G-protein–coupled receptor (GPCR) signaling in a cellular context.

Background: Innate immune signaling requires multiple mechanisms to suppress signaling in the absence of stimulation. Results: TNF receptor associated factor 6 (TRAF6) activity is regulated by reversible arginine methylation. Conclusion: Arginine methylation of TRAF6 inhibits signaling in the absence of Toll-like receptor ligands. Significance: Reversible TRAF6 methylation is a novel mechanism that controls innate immune responses.

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Hepatitis C infection produces chronic liver injury that is significantly exacerbated by alcohol consumption. While multiple mechanisms contribute to this synergy, a viral-induced loss of antioxidant responses has been shown to play an important role. This study examined the effects of HCV infection and alcohol on the regulation of the transcription factor FOXO3, an important regulator of SOD2 expression, a tumor suppressor, and a component of the hepatic antioxidant response system. FOXO3 was activated by either HCV or alcohol alone but suppressed by the combination. To understand this paradoxical result, we applied a capillary isoelectric focusing (IEF) method to determine the pattern of FOXO3 post-translational modifications (PTMs) induced by HCV and alcohol. We observed the presence of multiple different nuclear and cytosolic species of FOXO3 and used anti phosphoserine, acetyl-lysine, methylarginine and ubiquitin antibodies to identify the PTM patterns present in each species. HCV caused multiple changes including phosphorylation of FOXO3 at S-574, a novel JNK site, which promoted nuclear translocation and transcription. Ethanol suppressed arginine-methylation of FOXO3 promoting nuclear export and degradation of the JNK phosphorylated form. Human liver biopsy samples showed the presence of the HCV-specific form of FOXO3 in HCV-infected livers but not in normal liver or nonalcoholic steatohepatitis. Conclusion The development of this novel IEF method for the simultaneous quantification of differently modified FOXO3 species allowed us to demonstrate how HCV and alcohol combine to modify a complex pattern of FOXO3 PTMs that contribute to pathogenesis. This approach will allow further dissection of the role of protein PTMs in viral liver disease.

G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that β2-adrenergic receptors (β2ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of β2ARs between subcellular compartments. BRET between β2ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between β2ARs and endosome markers increases. Energy transfer between β2ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled β2ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that β2ARs associate transiently with each other in the plasma membrane, or that β2AR dimers or oligomers are actively disrupted during internalization.

Forkhead box O3 (FOXO3) is a multispecific transcription factor that is responsible for multiple and conflicting transcriptional programs such as cell survival and apoptosis. The protein is heavily post-translationally modified and there is considerable evidence that post-transcriptional modifications (PTMs) regulate protein stability and nuclear-cytosolic translocation. Much less is known about how FOXO3 PTMs determine the specificity of its transcriptional program. In this study we demonstrate that exposure of hepatocytes to ethanol or exposure of macrophages to lipopolysaccharide (LPS) induces the c-Jun N-terminal kinase (JNK)-dependent phosphorylation of FOXO3 at serine-574. Chromatin immunoprecipitation (ChIP), mRNA and protein measurements demonstrate that p-574-FOXO3 selectively binds to promoters of pro-apoptotic genes but not to other well-described FOXO3 targets. Both unphosphorylated and p-574-FOXO3 bound to the B-cell lymphoma 2 (Bcl-2) promoter, but the unphosphorylated form was a transcriptional activator, whereas p-574-FOXO3 was a transcriptional repressor. The combination of increased TRAIL (TNF-related apoptosis-inducing ligand) and decreased Bcl-2 was both necessary and sufficient to induce apoptosis. LPS treatment of a human monocyte cell line (THP-1) induced FOXO3 S-574 phosphorylation and apoptosis. LPS-induced apoptosis was prevented by knockdown of FOXO3. It was restored by overexpressing wild-type FOXO3 but not by overexpressing a nonphosphorylatable S-574A FOXO3. Expression of an S-574D phosphomimetic form of FOXO3 induced apoptosis even in the absence of LPS. A similar result was obtained with mouse peritoneal macrophages where LPS treatment increased TRAIL, decreased Bcl-2 and induced apoptosis in wild-type but not FOXO3 −/− cells. This work thus demonstrates that S-574 phosphorylation generates a specifically apoptotic form of FOXO3 with decreased transcriptional activity for other well-described FOXO3 functions. Forkhead box O3 (FOXO3) is a multispecific transcription factor that serves as a longevity factor 1,2 and tumor suppressor and is critically involved in multiple seemingly independent biological process including cell-cycle arrest, 3 DNA repair, 4 antioxidant and stress responses, 5 apoptosis, 6 autophagy, glucose metabolism and aging. 7 Its many transcriptional programs are frequently in opposition to each other as it induces apoptosis, yet it is also critical for cell survival and longevity. Although there is considerable information regarding the mechanisms that regulate the nuclear/cytosolic distribution and protein stability of FOXO3, the control mechanisms that regulate transcriptional specificity are poorly understood.FOXO3 function is tightly regulated by multiple posttranslational modifications (PTMs) including phosphorylation, acetylation, ubiquitination and arginine and lysine methylation. Specific PTMs regulate the partitioning of FOXO3 between the cytosol and the nucleus 8-15 and its stability and degradation, 16 but the links between specific PTMs and FOXO3 tran...

Many of the molecules that mediate G-protein signaling are thought to constitutively associate with each other in variably stable signaling complexes. Much of the evidence for signaling complexes has come from Förster resonance energy transfer and bioluminescence resonance energy transfer (BRET) studies. However, detection of constitutive protein association with these methods is hampered by nonspecific energy transfer that occurs when donor and acceptor molecules are in close proximity by chance. We show that chemically-induced recruitment of local third-party BRET donors or acceptors reliably separates nonspecific and specific BRET. We use this method to reexamine the constitutive association of class A G-protein-coupled receptors (GPCRs) with other GPCRs and with heterotrimeric G-proteins. We find that beta2 adrenoreceptors constitutively associate with each other and with several other class A GPCRs. In contrast, GPCRs and G-proteins are unlikely to exist in stable constitutive preassembled complexes.