Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gα_i/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gα_i1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gα_i1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gα_o-AlF₄⁻. Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gα_i1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gα_o-AlF₄⁻ and an AlF₄⁻-insensitive mutant (G42R) of Gα_i1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gα_i1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gα_o-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity.     

Cleavage of Signal Regulatory Protein α (SIRPα) Enhances Inflammatory Signaling

Signal regulatory protein α (SIRPα) is a membrane glycoprotein immunoreceptor abundant in cells of monocyte lineage. SIRPα ligation by a broadly expressed transmembrane protein, CD47, results in phosphorylation of the cytoplasmic immunoreceptor tyrosine-based inhibitory motifs, resulting in the inhibition of NF-κB signaling in macrophages. Here we observed that proteolysis of SIRPα during inflammation is regulated by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), resulting in the generation of a membrane-associated cleavage fragment in both THP-1 monocytes and human lung epithelia. We mapped a charge-dependent putative cleavage site near the membrane-proximal domain necessary for ADAM10-mediated cleavage. In addition, a secondary proteolytic cleavage within the membrane-associated SIRPα fragment by γ-secretase was identified. Ectopic expression of a SIRPα mutant plasmid encoding a proteolytically resistant form in HeLa cells inhibited activation of the NF-κB pathway and suppressed STAT1 phosphorylation in response to TNFα to a greater extent than expression of wild-type SIRPα. Conversely, overexpression of plasmids encoding the proteolytically cleaved SIRPα fragments in cells resulted in enhanced STAT-1 and NF-κB pathway activation. Thus, the data suggest that combinatorial actions of ADAM10 and γ-secretase on SIRPα cleavage promote inflammatory signaling.     

Huntingtin-associated Protein 1 (HAP1) Is a cGMP-dependent Kinase Anchoring Protein (GKAP) Specific for the cGMP-dependent Protein Kinase Iβ Isoform

Protein-protein interactions are important in providing compartmentalization and specificity in cellular signal transduction. Many studies have hallmarked the well designed compartmentalization of the cAMP-dependent protein kinase (PKA) through its anchoring proteins. Much less data are available on the compartmentalization of its closest homolog, cGMP-dependent protein kinase (PKG), via its own PKG anchoring proteins (GKAPs). For the enrichment, screening, and discovery of (novel) PKA anchoring proteins, a plethora of methodologies is available, including our previously described chemical proteomics approach based on immobilized cAMP or cGMP. Although this method was demonstrated to be effective, each immobilized cyclic nucleotide did not discriminate in the enrichment for either PKA or PKG and their secondary interactors. Hence, with PKG signaling components being less abundant in most tissues, it turned out to be challenging to enrich and identify GKAPs. Here we extend this cAMP-based chemical proteomics approach using competitive concentrations of free cyclic nucleotides to isolate each kinase and its secondary interactors. Using this approach, we identified Huntingtin-associated protein 1 (HAP1) as a putative novel GKAP. Through sequence alignment with known GKAPs and secondary structure prediction analysis, we defined a small sequence domain mediating the interaction with PKG Iβ but not PKG Iα. In vitro binding studies and site-directed mutagenesis further confirmed the specificity and affinity of HAP1 binding to the PKG Iβ N terminus. These data fully support that HAP1 is a GKAP, anchoring specifically to the cGMP-dependent protein kinase isoform Iβ, and provide further evidence that also PKG spatiotemporal signaling is largely controlled by anchoring proteins.     

IQGAP1 Protein Regulates Nuclear Localization of β-Catenin via Importin-β5 Protein in Wnt Signaling

In the canonical Wnt signaling pathway, the translocation of β-catenin is important for the activation of target genes in the nucleus. However, the molecular mechanisms underlying its nuclear localization remain unclear. In the present study, we found IQGAP1 to be a regulator of β-catenin function via importin-β5. In Xenopus embryos, depletion of IQGAP1 reduced Wnt-induced nuclear accumulation of β-catenin and expression of Wnt target genes during early embryogenesis. Depletion of endogenous importin-β5 associated with IQGAP1 also reduced expression of Wnt target genes and the nuclear localization of IQGAP1 and β-catenin. Moreover, a small GTPase, Ran1, contributes to the nuclear translocation of β-catenin and the activation of Wnt target genes. These results suggest that IQGAP1 functions as a regulator of translocation of β-catenin in the canonical Wnt signaling pathway.     

NLRP3 Protein Deficiency Exacerbates Hyperoxia-induced Lethality through Stat3 Protein Signaling Independent of Interleukin-1β

Supplemental oxygen inhalation is frequently used to treat severe respiratory failure; however, prolonged exposure to hyperoxia causes hyperoxic acute lung injury (HALI), which induces acute respiratory distress syndrome and leads to high mortality rates. Recent investigations suggest the possible role of NLRP3 inflammasomes, which regulate IL-1β production and lead to inflammatory responses, in the pathophysiology of HALI; however, their role is not fully understood. In this study, we investigated the role of NLRP3 inflammasomes in mice with HALI. Under hyperoxic conditions, NLRP3^−/− mice died at a higher rate compared with wild-type and IL-1β^−/− mice, and there was no difference in IL-1β production in their lungs. Under hyperoxic conditions, the lungs of NLRP3^−/− mice exhibited reduced inflammatory responses, such as inflammatory cell infiltration and cytokine expression, as well as increased and decreased expression of MMP-9 and Bcl-2, respectively. NLRP3^−/− mice exhibited diminished expression and activation of Stat3, which regulates MMP-9 and Bcl-2, in addition to increased numbers of apoptotic alveolar epithelial cells. In vitro experiments revealed that alveolar macrophages and neutrophils promoted Stat3 activation in alveolar epithelial cells. Furthermore, NLRP3 deficiency impaired the migration of neutrophils and chemokine expression by macrophages. These findings demonstrate that NLRP3 regulates Stat3 signaling in alveolar epithelial cells by affecting macrophage and neutrophil function independent of IL-1β production and contributes to the pathophysiology of HALI.     

HOIL-1L Interacting Protein (HOIP) as an NF-κB Regulating Component of the CD40 Signaling Complex

The tumor necrosis factor receptor (TNFR) superfamily mediates signals critical for regulation of the immune system. One family member, CD40, is important for the efficient activation of antibody-producing B cells and other antigen-presenting cells. The molecules and mechanisms that mediate CD40 signaling are only partially characterized. Proteins known to interact with the cytoplasmic domain of CD40 include members of the TNF receptor-associated factor (TRAF) family, which regulate signaling and serve as links to other signaling molecules. To identify additional proteins important for CD40 signaling, we used a combined stimulation/immunoprecipitation procedure to isolate CD40 signaling complexes from B cells and characterized the associated proteins by mass spectrometry. In addition to known CD40-interacting proteins, we detected SMAC/DIABLO, HTRA2/Omi, and HOIP/RNF31/PAUL/ZIBRA. We found that these previously unknown CD40-interacting partners were recruited in a TRAF2-dependent manner. HOIP is a ubiquitin ligase capable of mediating NF-κB activation through the ubiquitin-dependent activation of IKKγ. We found that a mutant HOIP molecule engineered to lack ubiquitin ligase activity inhibited the CD40-mediated activation of NF-κB. Together, our results demonstrate a powerful approach for the identification of signaling molecules associated with cell surface receptors and indicate an important role for the ubiquitin ligase activity of HOIP in proximal CD40 signaling.     

Ubiquitin-associated Domain-containing Ubiquitin Regulatory X (UBX) Protein UBXN1 Is a Negative Regulator of Nuclear Factor κB (NF-κB) Signaling

Excessive nuclear factor κB (NF-κB) activation should be precisely controlled as it contributes to multiple immune and inflammatory diseases. However, the negative regulatory mechanisms of NF-κB activation still need to be elucidated. Various types of polyubiquitin chains have proved to be involved in the process of NF-κB activation. Many negative regulators linked to ubiquitination, such as A20 and CYLD, inhibit IκB kinase activation in the NF-κB signaling pathway. To find new NF-κB signaling regulators linked to ubiquitination, we used a small scale siRNA library against 51 ubiquitin-associated domain-containing proteins and screened out UBXN1, which contained both ubiquitin-associated and ubiquitin regulatory X (UBX) domains as a negative regulator of TNFα-triggered NF-κB activation. Overexpression of UBXN1 inhibited TNFα-triggered NF-κB activation, although knockdown of UBXN1 had the opposite effect. UBX domain-containing proteins usually act as valosin-containing protein (VCP)/p97 cofactors. However, knockdown of VCP/p97 barely affected UBXN1-mediated NF-κB inhibition. At the same time, we found that UBXN1 interacted with cellular inhibitors of apoptosis proteins (cIAPs), E3 ubiquitin ligases of RIP1 in the TNFα receptor complex. UBXN1 competitively bound to cIAP1, blocked cIAP1 recruitment to TNFR1, and sequentially inhibited RIP1 polyubiquitination in response to TNFα. Therefore, our findings demonstrate that UBXN1 is an important negative regulator of the TNFα-triggered NF-κB signaling pathway by mediating cIAP recruitment independent of VCP/p97.     

Role of α7 Nicotinic Acetylcholine Receptor in Calcium Signaling Induced by Prion Protein Interaction with Stress-inducible Protein 1

The prion protein (PrP^C) is a conserved glycosylphosphatidylinositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrP^C extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrP^C-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrP^C engagement induces an increase in intracellular Ca²⁺ levels. This effect was not detected in PrP^C-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrP^C. Using a best candidate approach to test for potential channels involved in Ca²⁺ influx evoked by STI1-PrP^C, we found that α-bungarotoxin, a specific inhibitor for α7 nicotinic acetylcholine receptor (α7nAChR), was able to block PrP^C-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when α7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrP^C and allowed reconstitution of signaling by PrP^C-STI1 interaction. These results indicate that STI1 can interact with the PrP^C·α7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.     

Amyloid-β Protein Modulates Insulin Signaling in Presynaptic Terminals

Synaptic loss is a major neuropathological correlate of memory decline as a result of Alzheimer's disease (AD). This phenomenon appears to be aggravated by soluble amyloid-β (Aβ) oligomers causing presynaptic terminals to be particularly vulnerable to damage. Furthermore, insulin is known to participate in synaptic plasticity through the activation of the insulin receptor (IR) and the PI3K signaling pathway, while low concentrations of soluble Aβ and Aβ oligomers aberrantly modulate IR function in cultured neurons. To further examine how Aβ and insulin interact in the pathology of AD, the present work analyzes the effect of insulin and Aβ in the activation of the IR/PI3K pathway in synaptosomes. We found that insulin increased mitochondrial activity and IR/Akt phosphorylation in synaptosomes taken from both hippocampus and cortex. Also, pretreatment with Aβ antagonized insulin's effect on hippocampal synaptosomes, but not vice versa. These results show that Aβ can reduce responsiveness to insulin. Combined with evidence that insulin desensitization can increase the risk of developing AD, our results suggest that the initial mechanism that impairs synaptic maintenance in AD might start with Aβ changes in insulin sensitivity.     

Hypoxia-inducible Factor-1α (HIF-1α) Protein Diminishes Sodium Glucose Transport 1 (SGLT1) and SGLT2 Protein Expression in Renal Epithelial Tubular Cells (LLC-PK1) under Hypoxia

In this work, we demonstrated the regulation of glucose transporters by hypoxia inducible factor-1α (HIF-1α) activation in renal epithelial cells. LLC-PK₁ monolayers were incubated for 1, 3, 6, or 12 h with 0% or 5% O₂ or 300 μm cobalt (CoCl₂). We evaluated the effects of hypoxia on the mRNA and protein expression of HIF-1α and of the glucose transporters SGLT1, SGLT2, and GLUT1. The data showed an increase in HIF-1α mRNA and protein expression under the three evaluated conditions (p < 0.05 versus t = 0). An increase in GLUT1 mRNA (12 h) and protein expression (at 3, 6, and 12 h) was observed (p < 0.05 versus t = 0). SGLT1 and SGLT2 mRNA and protein expression decreased under the three evaluated conditions (p < 0.05 versus t = 0). In conclusion, our results suggest a clear decrease in the expression of the glucose transporters SGLT1 and SGLT2 under hypoxic conditions which implies a possible correlation with increased expression of HIF-1α.     

Leptin Regulates KATP Channel Trafficking in Pancreatic β-Cells by a Signaling Mechanism Involving AMP-activated Protein Kinase (AMPK) and cAMP-dependent Protein Kinase (PKA)

Pancreatic β-cells secrete insulin in response to metabolic and hormonal signals to maintain glucose homeostasis. Insulin secretion is under the control of ATP-sensitive potassium (K_ATP) channels that play key roles in setting β-cell membrane potential. Leptin, a hormone secreted by adipocytes, inhibits insulin secretion by increasing K_ATP channel conductance in β-cells. We investigated the mechanism by which leptin increases K_ATP channel conductance. We show that leptin causes a transient increase in surface expression of K_ATP channels without affecting channel gating properties. This increase results primarily from increased channel trafficking to the plasma membrane rather than reduced endocytosis of surface channels. The effect of leptin on K_ATP channels is dependent on the protein kinases AMP-activated protein kinase (AMPK) and PKA. Activation of AMPK or PKA mimics and inhibition of AMPK or PKA abrogates the effect of leptin. Leptin activates AMPK directly by increasing AMPK phosphorylation at threonine 172. Activation of PKA leads to increased channel surface expression even in the presence of AMPK inhibitors, suggesting AMPK lies upstream of PKA in the leptin signaling pathway. Leptin signaling also leads to F-actin depolymerization. Stabilization of F-actin pharmacologically occludes, whereas destabilization of F-actin simulates, the effect of leptin on K_ATP channel trafficking, indicating that leptin-induced actin reorganization underlies enhanced channel trafficking to the plasma membrane. Our study uncovers the signaling and cellular mechanism by which leptin regulates K_ATP channel trafficking to modulate β-cell function and insulin secretion.     

Tumor Suppressor PTEN Inhibits Integrin- and Growth Factor–mediated Mitogen-activated Protein (MAP) Kinase Signaling Pathways

The tumor suppressor PTEN dephosphorylates focal adhesion kinase (FAK) and inhibits integrin-mediated cell spreading and cell migration. We demonstrate here that expression of PTEN selectively inhibits activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. PTEN expression in glioblastoma cells lacking the protein resulted in inhibition of integrin-mediated MAP kinase activation. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)- induced MAPK activation were also blocked. To determine the specific point of inhibition in the Ras/Raf/ MEK/ERK pathway, we examined these components after stimulation by fibronectin or growth factors. Shc phosphorylation and Ras activity were inhibited by expression of PTEN, whereas EGF receptor autophosphorylation was unaffected. The ability of cells to spread at normal rates was partially rescued by coexpression of constitutively activated MEK1, a downstream component of the pathway. In addition, focal contact formation was enhanced as indicated by paxillin staining. The phosphatase domain of PTEN was essential for all of these functions, because PTEN with an inactive phosphatase domain did not suppress MAP kinase or Ras activity. In contrast to its effects on ERK, PTEN expression did not affect c-Jun NH₂-terminal kinase (JNK) or PDGF-stimulated Akt. Our data suggest that a general function of PTEN is to down-regulate FAK and Shc phosphorylation, Ras activity, downstream MAP kinase activation, and associated focal contact formation and cell spreading.     

Commensal Bacteria-induced Interleukin 1β (IL-1β) Secreted by Macrophages Up-regulates Hepcidin Expression in Hepatocytes by Activating the Bone Morphogenetic Protein Signaling Pathway

The liver hormone hepcidin is the central regulator of systemic iron metabolism. Its increased expression in inflammatory states leads to hypoferremia and anemia. Elucidation of the mechanisms that up-regulate hepcidin during inflammation is essential for developing rational therapies for this anemia. Using mouse models of inflammatory bowel disease, we have shown previously that colitis-associated hepcidin induction is influenced by intestinal microbiota composition. Here we investigate how two commensal bacteria, Bifidobacterium longum and Bacteroides fragilis, representative members of the gut microbiota, affect hepcidin expression. We found that supernatants of a human macrophage cell line infected with either of the bacteria up-regulated hepcidin when added to a human hepatocyte cell line. This activity was abrogated by neutralization of IL-1β. Moreover, purified IL-1β increased hepcidin expression when added to the hepatocyte line or primary human hepatocytes and when injected into mice. IL-1β activated the bone morphogenetic protein (BMP) signaling pathway in hepatocytes and in mouse liver, as indicated by increased phosphorylation of small mothers against decapentaplegic proteins. Activation of BMP signaling correlated with IL-1β-induced expression of BMP2 in human hepatocytes and activin B in mouse liver. Treatment of hepatocytes with two different chemical inhibitors of BMP signaling or with a neutralizing antibody to BMP2 prevented IL-1β-induced up-regulation of hepcidin. Our results clarify how commensal bacteria affect hepcidin expression and reveal a novel connection between IL-1β and activation of BMP signaling. They also suggest that there may be differences between mice and humans with respect to the mechanism by which IL-1β up-regulates hepcidin.     

Cellular FLICE-like Inhibitory Protein (c-FLIP) and PS1-associated Protein (PSAP) Mediate Presenilin 1-induced γ-Secretase-dependent and -independent Apoptosis,Respectively

Presenilin 1 (PS1) has been implicated in apoptosis; however, its mechanism remains elusive. We report that PS1-induced apoptosis was associated with cellular FLICE-like inhibitory protein (c-FLIP) turnover and that γ-secretase inhibitor blocked c-FLIP turnover and also partially blocked PS1-induced apoptosis. A complete inhibition of PS1-induced apoptosis was achieved by knockdown of PS1-associated protein (PSAP), a mitochondrial proapoptotic protein that forms a complex with Bax upon induction of apoptosis, in the presence of γ-secretase inhibitor. PS1-induced apoptosis was partially inhibited by knockdown of caspase-8, Fas-associated protein with death domain (FADD), or Bid. However, knockdown of Bax or overexpression of Bcl-2 resulted in complete inhibition of PS1-induced apoptosis. These data suggest that PS1 induces apoptosis through two pathways: the γ-secretase-dependent pathway mediated by turnover of c-FLIP and the γ-secretase-independent pathway mediated by PSAP-Bax complex formation. These two pathways converge on Bax to activate mitochondria-dependent apoptosis. These findings provide new insight into the mechanisms by which PS1 is involved in apoptosis and the mechanism by which PS1 exerts its pathogenic effects. In addition, our results suggest that PS2 induces apoptosis through a pathway that is different from that of PS1.     

Insulin-Like Growth Factor (IGF) Binding Protein 2 Functions Coordinately with Receptor Protein Tyrosine Phosphatase β and the IGF-I Receptor To Regulate IGF-I-Stimulated Signaling

Insulin-like growth factor I (IGF-I) is a mitogen for vascular smooth muscle cells (VSMC) and has been implicated in the development and progression of atherosclerosis. IGF binding proteins (IGFBPs) modify IGF-I actions independently of IGF binding, but a receptor-based mechanism by which they function has not been elucidated. We investigated the role of IGFBP-2 and receptor protein tyrosine phosphatase β (RPTPβ) in regulating IGF-I signaling and cellular proliferation. IGFBP-2 bound RPTPβ, which led to its dimerization and inactivation. This enhanced PTEN tyrosine phosphorylation and inhibited PTEN activity. Utilization of substrate trapping and phosphatase-dead mutants showed that RPTPβ bound specifically to PTEN and dephosphorylated it. IGFBP-2 knockdown led to decreased PTEN tyrosine phosphorylation and decreased AKT Ser473 activation. IGFBP-2 enhanced IGF-I-stimulated VSMC migration and proliferation. Analysis of aortas obtained from IGFBP-2^−/− mice showed that RPTPβ was activated, and this was associated with inhibition of IGF-I stimulated AKT Ser473 phosphorylation and VSMC proliferation. These changes were rescued following administration of IGFBP-2. These findings present a novel mechanism for coordinate regulation of IGFBP-2 and IGF-I signaling functions that lead to stimulation of VSMC proliferation. The results have important implications for understanding how IGFBPs modulate the cellular response to IGF-I.