Homo-oligomerization and Activation of AMP-activated Protein Kinase Are Mediated by the Kinase Domain ��G-Helix

AMP-activated protein kinase (AMPK) is a heterotrimeric complex playing a crucial role in maintaining cellular energy homeostasis. Recently, homodimerization of mammalian AMPK and yeast ortholog SNF1 was shown by us and others. In SNF1, it involved specific hydrophobic residues in the kinase domain αG-helix. Mutation of the corresponding AMPK α-subunit residues (Val-219 and Phe-223) to glutamate reduced the tendency of the kinase to form higher order homo-oligomers, as was determined by the following three independent techniques in vitro: (i) small angle x-ray scattering, (ii) surface plasmon resonance spectroscopy, and (iii) two-dimensional blue native/SDS-PAGE. Recombinant protein as well as AMPK in cell lysates of primary cells revealed distinct complexes of various sizes. In particular, the assembly of very high molecular mass complexes was dependent on both the αG-helix-mediated hydrophobic interactions and kinase activation. In vitro and when overexpressed in double knock-out (α1^−/−, α2^−/−) mouse embryonic fibroblast cells, activation of mutant AMPK was impaired, indicating a critical role of the αG-helix residues for AMPK activation via its upstream kinases. Also inactivation by protein phosphatase 2Cα was affected in mutant AMPK. Importantly, activation of mutant AMPK by LKB1 was restored by exchanging the corresponding and conserved hydrophobic αG-helix residues of LKB1 (Ile-260 and Phe-264) to positively charged amino acids. These mutations functionally rescued LKB1-dependent activation of mutant AMPK in vitro and in cell culture. Our data suggest a physiological role for the hydrophobic αG-helix residues in homo-oligomerization of heterotrimers and cellular interactions, in particular with upstream kinases, indicating an additional level of AMPK regulation.The maintenance of energy homeostasis is a basic requirement of all living organisms. The AMP-activated protein kinase (AMPK)² is crucially involved in this essential process by playing a central role in sensing and regulating energy metabolism on the cellular and whole body level (1–6). AMPK is also participating in several signaling pathways associated with cancer and metabolic diseases, like type 2 diabetes mellitus, obesity, and other metabolic disorders (7–9).Mammalian AMPK belongs to a highly conserved family of serine/threonine protein kinases with homologs found in all eukaryotic organisms examined (1, 3, 10). Its heterotrimeric structure includes a catalytic α-subunit and regulatory β- and γ-subunits. These subunits exist in different isoforms (α1, α2, β1, β2, γ1, γ2, and γ3) and splice variants (for γ2 and γ3) and can thus assemble to a broad variety of heterotrimeric isoform combinations. The α- and β-subunits possess multiple autophosphorylation sites, which have been implicated in regulation of subcellular localization and kinase activation (11–15). The most critical step of AMPK activation, however, is phosphorylation of Thr-172 within the activation segment of the α-subunit kinase domain. At least two AMPK upstream kinases (AMPKKs) have been identified so far, namely the tumor suppressor kinase LKB1 in complex with MO25 and STRAD (16) and Ca²⁺/calmodulin-dependent protein kinase kinase-2 (CamKK2) (17). Furthermore, the transforming growth factor-β-activated kinase 1 was also shown to activate AMPK using a variety of in vitro approaches (18), but the physiological relevance of these findings remains unclear. Besides direct phosphorylation of Thr-172, AMPK activity is stimulated by the allosteric activator AMP, which can bind to two Bateman domains formed by two pairs of CBS domains within the γ-subunit (19–22). Hereby bound AMP not only allosterically stimulates AMPK but also protects Thr-172 from dephosphorylation by protein phosphatase 2Cα (PP2Cα) and thus hinders inactivation of the kinase (19, 22, 23). Consequently, on the cellular level, AMPK is activated upon metabolic stress increasing the AMP/ATP ratio. Furthermore, AMPK activation can also be induced by several chemical compounds, like nucleoside 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (24) and the anti-diabetic drug Metformin (25–28). In addition, the small molecule compound A-769662 was recently developed as a direct allosteric activator of AMPK (29, 30).Previous work in our groups proposed a model of AMPK regulation by AMP, which incorporates the major functional features and the latest structural information (31). The latter mainly included truncated core complexes of AMPK from different species (32–35). Further valuable structural information is provided by the x-ray structures of the isolated catalytic domains, in particular of the human AMPK α2-subunit (Protein Data Bank code 2H6D) and its yeast ortholog SNF1 (36, 37). The kinase domain of SNF1 is capable of forming homodimers in the protein crystal, as well as in vitro in solution, in a unique way, which has not been observed previously in any other kinase (36). The dimer interface is predominantly formed by hydrophobic interactions of the loop-αG region, also known as subdomain X situated on the large kinase lobe (36, 38, 39), and it mainly involves Ile-257 and Phe-261. Because the T-loop activation segment was buried within the dimer interface, it was suggested that the dimeric state of the SNF1 catalytic domain represents the inactive form of the kinase. Intriguingly, it was shown in our groups by small angle x-ray scattering that AMPK self-organizes in a concentration-dependent manner to form homo-oligomers in solution (31). However, the interface responsible for oligomerization of the AMPK heterotrimer has remained elusive.Here we further investigate the distinct oligomeric states of the AMPK heterotrimer and suggest a possible regulatory function for this process. Most importantly, we provide conclusive evidence for participation of αG-helix residues in the recognition of AMPK by its upstream kinases LKB1 and CamKK2.     

Structure of the Nuclear Factor κB-inducing Kinase (NIK) Kinase Domain Reveals a Constitutively Active Conformation

NF-κB-inducing kinase (NIK) is a central component in the non-canonical NF-κB signaling pathway. Excessive NIK activity is implicated in various disorders, such as autoimmune conditions and cancers. Here, we report the first crystal structure of truncated human NIK in complex with adenosine 5′-O-(thiotriphosphate) at a resolution of 2.5 Å. This truncated protein is a catalytically active construct, including an N-terminal extension of 60 residues prior to the kinase domain, the kinase domain, and 20 residues afterward. The structure reveals that the NIK kinase domain assumes an active conformation in the absence of any phosphorylation. Analysis of the structure uncovers a unique role for the N-terminal extension sequence, which stabilizes helix αC in the active orientation and keeps the kinase domain in the catalytically competent conformation. Our findings shed light on the long-standing debate over whether NIK is a constitutively active kinase. They also provide a molecular basis for the recent observation of gain-of-function activity for an N-terminal deletion mutant (ΔN324) of NIK, leading to constitutive non-canonical NF-κB signaling with enhanced B-cell adhesion and apoptosis resistance.     

Signaling Cascade of Diacylglycerol Kinase �� in the Pituitary Intermediate Lobe: Dopamine D2 Receptor/Phospholipase C��4/Diacylglycerol Kinase ��/Protein Kinase C��

The pituitary gland dynamically changes its hormone output under various pathophysiological conditions. One of the pathways implicated in the regulatory mechanism of this gland is a dopaminergic system that operates the phosphoinositide (PI) cycle to transmit downstream signal through second messengers. We have previously shown that diacylglycerol kinase β (DGKβ) is coexpressed with dopamine D1 and D2 receptors in medium spiny neurons of the striatum, suggesting a plausible implication of DGKβ in dopaminergic transmission. However, it remains elusive whether DGKβ is involved in the dopaminergic system in the pituitary gland. The aim of this study is to investigate the expression and localization of DGK in the pituitary gland, together with the molecular components involved in the PI signaling cascade, including dopamine receptors, phospholipase C (PLC), and a major downstream molecule, protein kinase C (PKC). Here we show that DGKβ and the dopamine D2 receptor are coexpressed in the intermediate lobe and localize to the plasma membrane side by side. In addition, we reveal that PLCβ4 and PKCα are the subtypes expressed in the intermediate lobe among those families. These findings will substantiate and further extend our understanding of the molecular-anatomical pathway of PI signaling and the functional roles of DGK in the pituitary intermediate lobe. (J Histochem Cytochem 58:119–129, 2010)     

Evolution of Phosphagen Kinase VII. Isolation of Glycocyamine Kinase from the Polychaete Neanthes diversicolor and the cDNA-Derived Amino Acid Sequences of α and β Chains

Glycocyamine kinase (GK) was isolated from the marine polychaete Neanthes diversicolor by gel filtration, DEAE-cellulose chromatography, butyl-Toyopearl hydrophobic chromatography, and chromatofocusing. The GK was eluted as a single peak on the latter three chromatographies, and the molecular mass for the native GK was estimated to be about 80 kDa. The SDS–PAGE showed that the isolated GK consists of two distinct subunits in equal proportion, α and β chains, with molecular masses of 42.2 and 43.8 kDa, respectively. The present results suggest that the Neanthes GK has a heterodimeric structure. The cDNAs for α and β chains of Neanthes GK were amplified by PCR and their cDNA-derived amino acid sequences were determined. The α and β chains are composed of 374 and 390 amino acids, and the molecular masses were calculated to be 42,392 and 43,966 Da, respectively, in good agreement with the apparent masses on SDS–PAGE. The β chain has a characteristic N-terminal extension of 15 amino acids, and all of the sequence differences between α and β chains were restricted in the N-terminal region of 50 residues. The overall sequence identity was 92%. The occurrence of heterodimeric nature in Neanthes GK is of great interest from the evolutionary point of view, because the heterodimeric structure is only known for creatine kinase MB-isozyme specific for mammalian heart muscle among phosphagen kinases.     

Structural Basis of Protein Kinase Cα Regulation by the C-Terminal Tail

Protein kinase C (PKC) isoenzymes are multi-modular proteins activated at the membrane surface to regulate signal transduction processes. When activated by second messengers, PKC undergoes a drastic conformational and spatial transition from the inactive cytosolic state to the activated membrane-bound state. The complete structure of either state of PKC remains elusive. We demonstrate, using NMR spectroscopy, that the isolated Ca²⁺-sensing membrane-binding C2 domain of the conventional PKCα interacts with a conserved hydrophobic motif of the kinase C-terminal region, and we report a structural model of the complex. Our data suggest that the C-terminal region plays a dual role in regulating the PKC activity: activating, through sensitization of PKC to intracellular Ca²⁺ oscillations; and auto-inhibitory, through its interaction with a conserved positively charged region of the C2 domain.     

Evidence That Atypical Protein Kinase C-λ and Atypical Protein Kinase C-ζ Participate in Ras-mediated Reorganization of the F-actin Cytoskeleton

Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound morphological alterations which include a disassembly of actin stress fibers. The Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations which inhibit the activity of the atypical aPKC isotypes λ and ζ, whereas lower concentrations of the inhibitor which block conventional and novel PKC isotypes are ineffective. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant-negative (DN) mutants of aPKC-λ and aPKC-ζ, as well as antisense constructs encoding RNA-directed against isotype-specific 5′ sequences of the corresponding mRNA, abrogates the Ha-Ras–induced reorganization of the actin cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-α was unable to counteract Ras with regard to the dissolution of actin stress fibers. Transfection of cells with constructs encoding constitutively active (CA) mutants of atypical aPKC-λ and aPKC-ζ lead to a disassembly of stress fibers independent of oncogenic Ha-Ras. Coexpression of (DN) Rac-1 N17 and addition of the phosphatidylinositol 3′-kinase (PI3K) inhibitors wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 are in agreement with a tentative model suggesting that, in the signaling pathway from Ha-Ras to the cytoskeleton aPKC-λ acts upstream of PI3K and Rac-1, whereas aPKC-ζ functions downstream of PI3K and Rac-1.This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-λ or aPKC-ζ results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-ζ was abrogated by coexpression of DN Rac-1 N17.     

Direct Interactions with the Integrin β1 Cytoplasmic Tail Activate the Abl2/Arg Kinase

Integrins are heterodimeric α/β extracellular matrix adhesion receptors that couple physically to the actin cytoskeleton and regulate kinase signaling pathways to control cytoskeletal remodeling and adhesion complex formation and disassembly. β1 integrins signal through the Abl2/Arg (Abl-related gene) nonreceptor tyrosine kinase to control fibroblast cell motility, neuronal dendrite morphogenesis and stability, and cancer cell invasiveness, but the molecular mechanisms by which integrin β1 activates Arg are unknown. We report here that the Arg kinase domain interacts directly with a lysine-rich membrane-proximal segment in the integrin β1 cytoplasmic tail, that Arg phosphorylates the membrane-proximal Tyr-783 in the β1 tail, and that the Arg Src homology domain then engages this phosphorylated region in the tail. We show that these interactions mediate direct binding between integrin β1 and Arg in vitro and in cells and activate Arg kinase activity. These findings provide a model for understanding how β1-containing integrins interact with and activate Abl family kinases.     

Tryptophan Depletion and the Kinase GCN2 Mediate IFN-γ-Induced Autophagy

IFN-γ is a master regulator of the immune responses that occur in the transplanted kidney, acting both on the immune system and on the graft itself. The cellular responses to IFN-γ are complex, and emerging evidence suggests that IFN-γ may regulate autophagic functions. Conversely, autophagy modulates innate and adaptive immune functions in various contexts. In this study, we identify a novel mechanism by which IFN-γ activates autophagy in human kidney epithelial cells and provide new insights into how autophagy regulates immune functions in response to IFN-γ. Our results indicate that IFN-γ promotes tryptophan depletion, activates the eIF2α kinase general control nonderepressible-2 (GCN2), and leads to an increase in the autophagic flux. Further, tryptophan supplementation and RNA interference directed against GCN2 inhibited IFN-γ-induced autophagy. This process is of functional relevance because autophagy regulates the secretion of inflammatory cytokines and growth factors by human kidney epithelial cells in response to IFN-γ. These findings assign to IFN-γ a novel function in the regulation of autophagy, which, in turn, modulates IFN-γ-induced secretion of inflammatory cytokines.     

Structure of the CaMKIIδ/Calmodulin Complex Reveals the Molecular Mechanism of CaMKII Kinase Activation

Long-term potentiation (LTP), a long-lasting enhancement in communication between neurons, is considered to be the major cellular mechanism underlying learning and memory. LTP triggers high-frequency calcium pulses that result in the activation of Calcium/Calmodulin (CaM)-dependent kinase II (CaMKII). CaMKII acts as a molecular switch because it remains active for a long time after the return to basal calcium levels, which is a unique property required for CaMKII function. Here we describe the crystal structure of the human CaMKIIδ/Ca²⁺/CaM complex, structures of all four human CaMKII catalytic domains in their autoinhibited states, as well as structures of human CaMKII oligomerization domains in their tetradecameric and physiological dodecameric states. All four autoinhibited human CaMKIIs were monomeric in the determined crystal structures but associated weakly in solution. In the CaMKIIδ/Ca²⁺/CaM complex, the inhibitory region adopted an extended conformation and interacted with an adjacent catalytic domain positioning T287 into the active site of the interacting protomer. Comparisons with autoinhibited CaMKII structures showed that binding of calmodulin leads to the rearrangement of residues in the active site to a conformation suitable for ATP binding and to the closure of the binding groove for the autoinhibitory helix by helix αD. The structural data, together with biophysical interaction studies, reveals the mechanism of CaMKII activation by calmodulin and explains many of the unique regulatory properties of these two essential signaling molecules.

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Differential Roles of the Glycogen-Binding Domains of β Subunits in Regulation of the Snf1 Kinase Complex

Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic α subunit and regulatory β and γ subunits. In this study, the role of the β subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (α), Snf4 (γ), and one of three alternative β subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three β subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the β subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.The Snf1 protein kinase of Saccharomyces cerevisiae is the yeast ortholog of the AMP-activated protein kinase (AMPK) found in mammals and other eukaryotes. AMPK acts as a nutrient and energy sensor, becoming activated under conditions of nutrient and energy depletion (6). In mammals, AMPK plays a key role in glucose homeostasis and is a target for drugs used to treat metabolic syndrome and type 2 diabetes (34). In yeast, the Snf1 kinase plays an essential role during aerobic growth and fermentative growth on alternative carbon sources. Cells lacking Snf1 kinase activity are viable but display numerous phenotypes including poor or no growth on alternative carbon sources, defects in meiosis and sporulation, defects in response to ion stress, and defects in pseudohyphal growth (7).The Snf1 kinase and all members of the AMPK family function as heterotrimers composed of a catalytic α subunit complexed with regulatory β and γ subunits (2). The γ subunit in mammalian enzymes directly binds three molecules of AMP (26, 33), which stimulates enzyme activity by inhibiting the dephosphorylation of the conserved threonine residue in the kinase activation loop (23). In yeast, there is no evidence that the γ subunit binds AMP; however, similar to mammals, the key glucose-regulated step is the dephosphorylation of the kinase activation loop (22).In this study, we examine the role of the β subunits in the regulation of the Snf1 kinase activity. Yeasts express three isoforms of the Snf1 kinase that differ depending on which of the three distinct β subunits, Sip1, Sip2, and Gal83, is incorporated into the enzyme. Previous studies have shown that the Snf1 isoforms have distinct substrate preferences (24), subcellular localizations (32), and stress response capacities (9). Only the Snf1 isoform containing Gal83 as the β subunit is able to localize to the cell nucleus in a process that requires Sak1, one of the three Snf1-activating protein kinases. Since all three of the Snf1-activating kinases (SAKs) are capable of phosphorylating Snf1 on its activation loop (3), it has remained a mystery as to why the Sak1 kinase is specifically required for Snf1 nuclear localization.The β subunits of Snf1 as well as mammalian AMPK contain a domain that is referred to as either a carbohydrate-binding module (CBM) (11) or a glycogen-binding domain (GBD) (19). The structure of this domain has been solved (20), and it was previously shown that this domain binds most tightly to branched oligosaccharides like glycogen that contain α1→6 branches (12). The binding of glycogen to the β subunit causes an allosteric inhibition of AMPK activity and inhibits phosphorylation by the upstream activating kinase. The β subunits of yeast contain the GBDs, but the importance of binding glycogen is questionable since cells that lack the ability to make glycogen show a normal regulation of Snf1 kinase in response to glucose limitation (15). Nonetheless, the deletion of the GBD from the Gal83 protein caused an increased activity of the Snf1 enzyme and release from glucose repression. Therefore, the GBD acts as a negative regulator of kinase activity in both mammalian and fungal cells.In this study we examine the role of the GBD present in the Sip2 and Sip1 proteins. We also extend the characterization of the Gal83 GBD by determining what connection this domain has with the regulated dephosphorylation of the Snf1 kinase. Finally, we have characterized other N-terminal domains in the β subunits that control accumulation and phosphorylation.     

Acetylcorynoline Impairs the Maturation of Mouse Bone Marrow-Derived Dendritic Cells via Suppression of IκB Kinase and Mitogen-Activated Protein Kinase Activities

Background

Dendritic cells (DCs) are major modulators in the immune system. One active field of research is the manipulation of DCs as pharmacological targets to screen novel biological modifiers for the treatment of inflammatory and autoimmune disorders. Acetylcorynoline is the major alkaloid component derived from Corydalis bungeana herbs. We assessed the capability of acetylcorynoline to regulate lipopolysaccharide (LPS)-stimulated activation of mouse bone marrow-derived DCs.

Methodology/Principal Findings

Our experimental data showed that treatment with up to 20 µM acetylcorynoline does not cause cytotoxicity in cells. Acetylcorynoline significantly inhibited the secretion of tumor necrosis factor-α, interleukin-6, and interleukin-12p70 by LPS-stimulated DCs. The expression of LPS-induced major histocompatibility complex class II, CD40, and CD86 on DCs was also decreased by acetylcorynoline, and the endocytic capacity of LPS-stimulated DCs was restored by acetylcorynoline. In addition, LPS-stimulated DC-elicited allogeneic T-cell proliferation was blocked by acetylcorynoline, and the migratory ability of LPS-stimulated DCs was reduced by acetylcorynoline. Moreover, acetylcorynoline significantly inhibits LPS-induced activation of IκB kinase and mitogen-activated protein kinase. Importantly, administration of acetylcorynoline significantly attenuates 2,4-dinitro-1-fluorobenzene-induced delayed-type hypersensitivity.

Conclusions/Significance

Acetylcorynoline may be one of the potent immunosuppressive agents through the blockage of DC maturation and function.     

Protein Kinase Cα Isoform Regulates the Activation of the MAP Kinase ERK1/2 in Human Glioma Cells: Involvement in Cell Survival and Gene Expression

Protein kinase C is a family of serine/threonine protein kinases involved in many cellular responses, including cell survival and apoptosis. We have recently found that specific inhibition of the PKCα isoform by nucleic acid enzymes induced apoptosis in sensitive cells. Here we show that in PKCα DNA enzyme-treated glioma cells the activation of MAP kinases ERK1/2 is inhibited, whereas their total level was not significantly affected by the treatment. Similar results were obtained when the overall activity of the PKC was inhibited by calphostin, a specific inhibitor for PKC. These results would indicate that the ERK1/2 signaling pathway plays an important role in glioma cell survival and that the PKCα isoform is the main modulator of this pathway. Furthermore, we show that the ERK1/2 signaling pathway is required for the constitutive expression of the basic fibroblast growth factor, a potent mitogen for glioma cell growth.     

AMP-activated Protein Kinase Mediates the Interferon-��-induced Decrease in Intestinal Epithelial Barrier Function

Impaired epithelial barrier function plays a crucial role in the pathogenesis of inflammatory bowel disease. Elevated levels of the pro-inflammatory cytokine, interferon-γ (IFNγ), are believed to be prominently involved in the pathogenesis of Crohn disease. Treatment of T₈₄ intestinal epithelial cells with IFNγ severely impairs their barrier properties measured as transepithelial electrical resistance (TER) or permeability and reduces the expression of tight junction proteins such as occludin and zonula occludens-1 (ZO-1). However, little is known about the signaling events that are involved. The cellular energy sensor, AMP-activated protein kinase (AMPK), is activated in response to cellular stress, as occurs during inflammation. The aim of this study was to investigate a possible role for AMPK in mediating IFNγ-induced effects on the intestinal epithelial barrier. We found that IFNγ activates AMPK by phosphorylation, independent of intracellular energy levels. Inhibition of AMPK prevents, at least in part, the IFNγ-induced decrease in TER. Furthermore, AMPK knockdown prevented the increased epithelial permeability, the decreased TER, and the decrease in occludin and ZO-1 caused by IFNγ treatment of T₈₄ cells. However, AMPK activity alone was not sufficient to cause alterations in epithelial barrier function. These data show a novel role for AMPK, in concert with other signals induced by IFNγ, in mediating reduced epithelial barrier function in a cell model of chronic intestinal inflammation. These findings may implicate AMPK in the pathogenesis of chronic intestinal inflammatory conditions, such as inflammatory bowel disease.Inflammatory bowel disease (IBD)² consists of two major subgroups, ulcerative colitis and Crohn disease (CD). A complex cascade of genetic, immunological, and bacterial factors contributes to IBD pathogenesis (1). In the healthy intestine, the epithelial barrier separates the luminal bacterial microbiota and other aspects of the external environment from cells of the mucosal immune system. In CD in particular, an impaired epithelial barrier (2, 3) leads to increased exposure of the immune system to commensal bacteria. Along with possible genetic defects in bacterial sensing, this might contribute to a dysregulated immune response leading to further epithelial damage and active episodes of IBD (4). Epithelial barrier dysfunction in CD is characterized by alterations in intercellular tight junctions (5), as well as by an excessive loss of water and salt into the lumen. An important immunological marker in CD is the existence of excessively high levels of the pro-inflammatory cytokine, interferon gamma (IFNγ) (6).IFNγ treatment of intestinal epithelial cell monolayers severely compromises their barrier integrity. Most importantly from a functional perspective, IFNγ causes a decrease in transepithelial electrical resistance (TER) and increases epithelial permeability (7, 8). These defects closely resemble observations in CD, where there is a disruption of intercellular tight junctional complexes. This effect is due to disruption of the apical actin cytoskeleton in conjunction with decreased expression, as well as increased internalization, of important tight junction proteins such as occludin and zonula occludens-1 (ZO-1) (8–11). Conversely, induction of epithelial apoptosis by IFNγ is believed to contribute little to barrier dysfunction (12). IFNγ also induces further alterations in epithelial function that include reduced expression of various ion transporters and associated decreases in epithelial ion transport (13, 14). Despite the influence of IFNγ on a number of epithelial functions, relatively little is known about intracellular signaling mechanisms mediating its effects following receptor activation. Recent studies demonstrated the involvement of phosphatidylinositol 3′-kinase (PI3K) in mediating IFNγ-induced effects on epithelial barrier function (11, 15). However, this is unlikely to be the only regulatory pathway involved. Indeed, increased expression of receptors for tumor necrosis factor core family members, such as the tumor necrosis factor receptor and LIGHT (homologous to lymphotoxin, shows inducible expression and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator (HVEM), a receptor expressed by T lymphocytes), can also occur in response to IFNγ and lead to changes in intestinal barrier function (16–18).The effects of IFNγ in intestinal epithelial cells resemble, at least in part, those of the cellular energy sensor, AMP-activated protein kinase (AMPK). Upon activation, AMPK restores intracellular ATP levels by stimulating energy-producing pathways, such as glucose uptake (19) and glycolysis, while inhibiting energy-consuming pathways, such as the synthesis of fatty acids or triglycerides (20, 21). In the intestine, energy-consuming processes include epithelial ion transport, and, indeed, AMPK has been shown to decrease intestinal ATP-consuming ion transport as well as the synthesis of various proteins (22, 23). Moreover, it has previously been demonstrated that ion transport processes are suppressed in intestinal biopsies from IBD patients (24–26).AMPK is usually activated in response to cellular stress that depletes intracellular ATP and elevates the AMP:ATP ratio (27, 28). AMPK-activating conditions include oxidative stress (29), hypoxia (30), and hypoglycemia (31). Binding of AMP to AMPK causes an increase in activity of 5-fold or less (32). Further, binding of AMP to AMPK makes AMPK a better substrate for upstream kinase activation, resulting in phosphorylation of the catalytic α-subunit of AMPK on the Thr¹⁷² residue and subsequently in a 50- to 100-fold activation of the enzyme (32). A number of upstream kinases for AMPK have been identified, with LKB1 (33, 34) or calmodulin kinase II (35–37) being the most important and well studied. However, recent studies also indicate that PI3K can activate AMPK (38, 39).The goal of this study was to determine whether AMPK mediates IFNγ-induced alterations in intestinal epithelial barrier function. We found that IFNγ activates AMPK in intestinal epithelial cells and AMPK inhibition prevents, at least in part, IFNγ-induced barrier dysfunction. Our data indicate a novel role for the cellular energy sensor, AMPK, in the regulation of intestinal epithelial barrier properties in a cell model of chronic inflammation. These findings may have implications for barrier function in the setting of chronic inflammatory processes, such as IBD.     

Bacillus licheniformis Zhengchangsheng® Inhibits Obesity by Regulating the AMP-Activated Protein Kinase Signaling Pathway

Probiotics and Antimicrobial Proteins - As a metabolic syndrome, obesity has become a global public health problem. Bacillus licheniformis has been shown to inhibit obesity by regulating the gut...     

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.