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.     

Skeletal Muscle AMP-activated Protein Kinase Is Essential for the Metabolic Response to Exercise in Vivo

AMP-activated protein kinase (AMPK) has been postulated as a super-metabolic regulator, thought to exert numerous effects on skeletal muscle function, metabolism, and enzymatic signaling. Despite these assertions, little is known regarding the direct role(s) of AMPK in vivo, and results obtained in vitro or in situ are conflicting. Using a chronically catheterized mouse model (carotid artery and jugular vein), we show that AMPK regulates skeletal muscle metabolism in vivo at several levels, with the result that a deficit in AMPK activity markedly impairs exercise tolerance. Compared with wild-type littermates at the same relative exercise capacity, vascular glucose delivery and skeletal muscle glucose uptake were impaired; skeletal muscle ATP degradation was accelerated, and arterial lactate concentrations were increased in mice expressing a kinase-dead AMPKα2 subunit (α2-KD) in skeletal muscle. Nitric-oxide synthase (NOS) activity was significantly impaired at rest and in response to exercise in α2-KD mice; expression of neuronal NOS (NOSμ) was also reduced. Moreover, complex I and IV activities of the electron transport chain were impaired 32 ± 8 and 50 ± 7%, respectively, in skeletal muscle of α2-KD mice (p < 0.05 versus wild type), indicative of impaired mitochondrial function. Thus, AMPK regulates neuronal NOSμ expression, NOS activity, and mitochondrial function in skeletal muscle. In addition, these results clarify the role of AMPK in the control of muscle glucose uptake during exercise. Collectively, these findings demonstrate that AMPK is central to substrate metabolism in vivo, which has important implications for exercise tolerance in health and certain disease states characterized by impaired AMPK activation in skeletal muscle.The ubiquitously expressed serine/threonine AMP-activated protein kinase (AMPK)² is an αβγ heterotrimer postulated to play a key role in the response to energetic stress (1, 2), because of its sensitivity to increased cellular AMP levels (3). Pharmacological activation of AMPK (primarily via the AMP analogue ZMP) increases catabolic processes such as GLUT4 translocation (4, 5), glucose uptake (6, 7), long chain fatty acid (LCFA) uptake (8), and substrate oxidation (6). Concomitantly, pharmacological activation of AMPK inhibits anabolic processes, and in skeletal muscle genetic reduction of the catalytic AMPKα2 subunit eliminates these pharmacological effects (9–12). Thus, AMPK has been proposed to act as a metabolic master switch (2, 13, 14). Physiologically, exercise at intensities sufficient to increase free cytosolic AMP (AMP_free) levels is a potent stimulus of AMPK, preferentially activating AMPKα2 in skeletal muscle (15–17). The metabolic profile of skeletal muscle during moderate to high intensity exercise is remarkably similar to skeletal muscle in which AMPK has been pharmacologically activated (i.e. increases in catabolic processes). This is consistent with the hypothesis that AMPK activation is required for the metabolic response to increased cellular stress. Given this, it is surprising that the direct role(s) of skeletal muscle AMPK during exercise under physiological in vivo conditions is unknown.A number of studies have tried to attribute causality to the AMPK and metabolic responses to exercise using transgenic models. In mouse models in which AMPKα2 protein expression and/or activity has been impaired, contractions performed in isolated skeletal muscle in vitro, ex vivo, or in situ have demonstrated that skeletal muscle glucose uptake (MGU) is normal (9, 10), partially impaired (11, 18), or ablated (19). Furthermore, ex vivo skeletal muscle LCFA uptake and oxidation in response to contraction appears to be AMPK-independent (20, 21). A key limitation of these studies is that the experimental models were not physiological. Under in vivo conditions, mice expressing a kinase-dead (18) or inactive (22) AMPKα2 subunit in cardiac and skeletal muscle have impaired voluntary and maximal physical activity, respectively, indicative of a physiological role for AMPK during exercise. In this context, obese non-diabetic and diabetic individuals have impaired skeletal muscle AMPK activation during moderate intensity exercise (23) as well as during the post-exercise period (24), yet the contribution of this impairment to the disease state is unclear. Thus, in vivo studies are essential to define the role of AMPK in skeletal muscle during exercise.Physical exercise of a moderate intensity is an effective adjunct treatment for chronic metabolic diseases such as obesity and type 2 diabetes (25). Given the importance of elucidating the molecular mechanism(s) regulating skeletal muscle substrate metabolism during exercise and the putative role of AMPK as a critical mediator in this process, we tested the hypothesis that AMPKα2 is functionally linked to substrate metabolism in vivo.     

Regulation of the Interaction between Protein Kinase C-related Protein Kinase 2 (PRK2) and Its Upstream Kinase, 3-Phosphoinositide-dependent Protein Kinase 1 (PDK1)

The members of the AGC kinase family frequently exhibit three conserved phosphorylation sites: the activation loop, the hydrophobic motif (HM), and the zipper (Z)/turn-motif (TM) phosphorylation site. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates the activation loop of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by ³²P_i revealed phosphorylation at two sites, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do not apply for other AGC kinases.The regulation of protein function by phosphorylation and dephosphorylation is a key mechanism of intracellular signaling pathways in eukaryotic organisms. Protein phosphorylation is catalyzed by protein kinases, which are themselves often regulated by phosphorylation (1). The specificity of protein kinases is essential for their cellular functions. In some groups of protein kinases, the specificity is achieved by means of “docking interactions.” Protein kinase docking interactions involve a recognition site on the kinase or a flanking domain that is different from the active site. The most notable example, MAP kinases, uses a docking interaction to specifically recognize substrates, upstream kinases, and phosphatases. Despite the large amount of data on protein kinase docking interactions, e.g. in the MAP kinase field, there is very little information on how these essential interactions are regulated (2–4).3-Phosphoinositide-dependent protein kinase 1 (PDK1)³ belongs to the AGC family of protein kinases and is the activation loop kinase for several other AGC kinases (5). A key feature of the AGC kinase family members except PDK1 is the presence of a C-terminal extension (CT) to the catalytic core that contains a conserved hydrophobic motif (HM) harboring a phosphorylation site. In many AGC kinases, the HM mediates a docking interaction with PDK1. For example, p90 ribosomal S6 kinase (RSK), p70 S6 kinase (S6K) and serum- and glucocorticoid-induced protein kinase (SGK) interact with PDK1 upon phosphorylation of the HM site (6–9). The phosphorylated HM binds to a HM-binding pocket in the catalytic core of PDK1 that was originally termed the PIF-binding pocket (6, 10).Besides its role in the docking of substrates to PDK1, the HM/PIF-binding pocket was also identified as a ubiquitous and key regulatory site in likely all AGC kinases (7, 11). Thus, in AGC kinases studied up to now, the HM/PIF-binding pocket serves as an intramolecular docking site for the phosphorylated HM. In summary, the HM has a dual function in AGC kinase activation, (i) mediating the intermolecular interaction with PDK1 and (ii) acting as an intramolecular allosteric activator that stabilizes the active conformation of the kinase domain via binding to the HM/PIF-binding pocket.The CT of AGC kinases additionally contains a second regulatory phosphorylation site traditionally termed the “turn motif” (TM), and more recently the zipper (Z) site. The Z/TM phosphate interacts with a binding site within the kinase domain, acting like a zipper which serves to support the intramolecular binding of the phosphorylated HM to the HM/PIF-binding pocket (12). Hence, AGC kinases are synergistically activated by phosphorylation at the activation loop, the HM, and the Z/TM sites.Protein kinase C-related protein kinases (PRKs) (13) (also named PAK for protease-activated kinase (14–16) and PKN for protein kinase N (17)) represent a subfamily of AGC kinases. So far, three PRK isoforms were identified, PRK1, PRK2, and PKN3, which are effectors of the small GTP-binding protein Rho. PRKs, as well as the Rho-associated kinases (ROCKs), are considered to be the protein kinases that mediate the phosphorylation events downstream of Rho activation and both can be inhibited by the highly specific protein kinase inhibitor Y27632 (18). The most notable role described for PRK2 is the control of entry into mitosis and exit from cytokinesis (19). In addition, PRK2 phosphorylates the hepatitis C virus (HCV) RNA polymerase (20). In support of a function in HCV RNA replication, PRK2 inhibitors like Y27632 suppress HCV replication (21).The N-terminal region of PRK2 possesses three Rho effector (HR1) domains (13), a pseudosubstrate region that is thought to have an autoinhibitory function (22) and a C2-like domain, which is a potential binding site for lipid activators. The C-terminal region of PRK2 harbors the HM that mediates the docking interaction with the HM/PIF-binding pocket in its upstream kinase PDK1 (10, 23). Interestingly, PRKs and also atypical protein kinase Cs (PKCs, PKCζ, and PKCι/λ), contain an acidic residue instead of a phosphorylatable amino acid at the site equivalent to the HM phosphorylation site in other AGC kinases. Therefore, the molecular events that regulate the interaction of PRK2 and PKCζ with PDK1 must be different from the mechanism characterized for S6K, SGK, and RSK.In the present work we extended and refined the model of docking interaction between PRK2 and PDK1 and characterized C-terminal regions of PRK2 that participate in the regulation of this interaction. The work sheds light on the common as well as specific mechanisms that operate in the regulation of PDK1 docking interaction with its different substrates.     

Sgt1 Dimerization Is Negatively Regulated by Protein Kinase CK2-mediated Phosphorylation at Ser361

The kinetochore, which consists of centromere DNA and structural proteins, is essential for proper chromosome segregation in eukaryotes. In budding yeast, Sgt1 and Hsp90 are required for the binding of Skp1 to Ctf13 (a component of the core kinetochore complex CBF3) and therefore for the assembly of CBF3. We have previously shown that Sgt1 dimerization is important for this kinetochore assembly mechanism. In this study, we report that protein kinase CK2 phosphorylates Ser³⁶¹ on Sgt1, and this phosphorylation inhibits Sgt1 dimerization.The kinetochore is a structural protein complex located in the centromeric region of the chromosome coupled to spindle microtubules (1, 2). The kinetochore generates a signal to arrest cells during mitosis when it is not properly attached to microtubules, thereby preventing chromosome missegregation, which can lead to aneuploidy (3, 4). The molecular structure of the kinetochore complex of the budding yeast Saccharomyces cerevisiae has been well characterized; it is composed of more than 70 proteins, many of which are conserved in mammals (2).The centromere DNA in the budding yeast is a 125-bp region that contains three conserved regions, CDEI, CDEII, and CDEIII (5, 6). CDEIII (25 bp) is essential for centromere function (7) and is bound to a key component of the centromere, the CBF3 complex. The CBF3 complex contains four proteins, Ndc10, Cep3, Ctf13 (8–15), and Skp1 (14, 15), all essential for viability. Mutations in any of the CBF3 proteins abolish the ability of CDEIII to bind to CBF3 (16, 17). All of the kinetochore proteins, except the CDEI-binding Cbf1 (18–20), localize to the kinetochores in a CBF3-dependent manner (2). Thus, CBF3 is a fundamental kinetochore complex, and its mechanism of assembly is of great interest.We have previously found that Sgt1 and Skp1 activate Ctf13; thus, they are required for assembly of the CBF3 complex (21). The molecular chaperone Hsp90 is also required to form the active Ctf13-Skp1 complex (22). Sgt1 has two highly conserved motifs that are required for protein-protein interaction: the tetratricopeptide repeat (21) and the CHORD protein and Sgt1-specific motif. We and others have found that both domains are important for the interaction of Sgt1 with Hsp90 (23–26), which is required for assembly of the core kinetochore complex. This interaction is an initial step in kinetochore activation (24, 26, 27), which is conserved between yeast and humans (28, 29).We have recently shown that Sgt1 dimerization is important for Sgt1-Skp1 binding and therefore for kinetochore assembly (30). In this study, we have found that protein kinase CK2 phosphorylates Sgt1 at Ser³⁶¹, and this phosphorylation inhibits Sgt1 dimerization. Therefore, CK2 appears to regulate kinetochore assembly negatively in budding yeast.     

A Ca2+ Release-activated Ca2+ (CRAC) Modulatory Domain (CMD) within STIM1 Mediates Fast Ca2+-dependent Inactivation of ORAI1 Channels

STIM1 and ORAI1, the two limiting components in the Ca²⁺ release-activated Ca²⁺ (CRAC) signaling cascade, have been reported to interact upon store depletion, culminating in CRAC current activation. We have recently identified a modulatory domain between amino acids 474 and 485 in the cytosolic part of STIM1 that comprises 7 negatively charged residues. A STIM1 C-terminal fragment lacking this domain exhibits enhanced interaction with ORAI1 and 2–3-fold higher ORAI1/CRAC current densities. Here we focused on the role of this CRAC modulatory domain (CMD) in the fast inactivation of ORAI1/CRAC channels, utilizing the whole-cell patch clamp technique. STIM1 mutants either with C-terminal deletions including CMD or with 7 alanines replacing the negative amino acids within CMD gave rise to ORAI1 currents that displayed significantly reduced or even abolished inactivation when compared with STIM1 mutants with preserved CMD. Consistent results were obtained with cytosolic C-terminal fragments of STIM1, both in ORAI1-expressing HEK 293 cells and in RBL-2H3 mast cells containing endogenous CRAC channels. Inactivation of the latter, however, was much more pronounced than that of ORAI1. The extent of inactivation of ORAI3 channels, which is also considerably more prominent than that of ORAI1, was also substantially reduced by co-expression of STIM1 constructs missing CMD. Regarding the dependence of inactivation on Ca²⁺, a decrease in intracellular Ca²⁺ chelator concentrations promoted ORAI1 current fast inactivation, whereas Ba²⁺ substitution for extracellular Ca²⁺ completely abrogated it. In summary, CMD within the STIM1 cytosolic part provides a negative feedback signal to Ca²⁺ entry by triggering fast Ca²⁺-dependent inactivation of ORAI/CRAC channels.The Ca²⁺ release-activated Ca²⁺ (CRAC)⁵ channel is one of the best characterized store-operated entry pathways (1–7). Substantial efforts have led to identification of two key components of the CRAC channel machinery: the stromal interaction molecule 1 (STIM1), which is located in the endoplasmic reticulum and acts as a Ca²⁺ sensor (8–10), and ORAI1/CRACM1, the pore-forming subunit of the CRAC channel (11–13). Besides ORAI1, two further homologues named ORAI2 and ORAI3 belong to the ORAI channel family (12, 14).STIM1 senses endoplasmic reticulum store depletion primarily by its luminal EF-hand in its N terminus (8, 15), redistributes close to the plasma membrane, where it forms puncta-like structures, and co-clusters with ORAI1, leading to inward Ca²⁺ currents (12, 16–19). The STIM1 C terminus, located in the cytosol, contains two coiled-coil regions overlapping with an ezrin-radixin-moesin (ERM)-like domain followed by a serine/proline- and a lysine-rich region (2, 8, 20–22). Three recent studies have described the essential ORAI-activating region within the ERM domain, termed SOAR (Stim ORAI-activating region) (23), OASF (ORAI-activating small fragment) (24), and CAD (CRAC-activating domain) (25), including the second coiled coil domain and the following ∼55 amino acids. We and others have provided evidence that store depletion leads to a dynamic coupling of STIM1 to ORAI1 (26–28) that is mediated by a direct interaction of the STIM1 C terminus with ORAI1 C terminus probably involving the putative coiled-coil domain in the latter (27).Furthermore, different groups have proven that the C terminus of STIM1 is sufficient to activate CRAC as well as ORAI1 channels independent of store depletion (22–25, 27, 29). We have identified that OASF-(233–474) or shorter fragments exhibit further enhanced coupling to ORAI1 resulting in 3-fold increased constitutive Ca²⁺ currents. A STIM1 fragment containing an additional cluster of anionic amino acids C-terminal to position 474 displays weaker interaction with ORAI1 as well as reduced Ca²⁺ current comparable with that mediated by wild-type STIM1 C terminus. Hence, we have suggested that these 11 amino acids (474–485) act in a modulatory manner onto ORAI1; however, their detailed mechanistic impact within the STIM1/ORAI1 signaling machinery has remained so far unclear.In this study, we focused on the impact of this negative cluster on fast inactivation of STIM1-mediated ORAI Ca²⁺ currents. Lis et al. (30) have shown that all three ORAI homologues display distinct inactivation profiles, where ORAI2 and ORAI3 show a much more pronounced fast inactivation than ORAI1. Moreover, it has been reported (31) that different expression levels of STIM1 to ORAI1 affect the properties of CRAC current inactivation. Yamashita et al. (32) have demonstrated a linkage between the selectivity filter of ORAI1 and its Ca²⁺-dependent fast inactivation. Here we provide evidence that a cluster of acidic residues within the C terminus of STIM1 is involved in the fast inactivation of ORAI1 and further promotes that of ORAI3 and native CRAC currents.     

Heterodimerization of the Sialidase NEU1 with the Chaperone Protective Protein/Cathepsin A Prevents Its Premature Oligomerization

Lysosomal neuraminidase-1 (NEU1) forms a multienzyme complex with β-galactosidase and protective protein/cathepsin A (PPCA). Because of its association with PPCA, which acts as a molecular chaperone, NEU1 is transported to the lysosomal compartment, catalytically activated, and stabilized. However, the mode(s) of association between these two proteins both en route to the lysosome and in the multienzyme complex has remained elusive. Here, we have analyzed the hydrodynamic properties of PPCA, NEU1, and a complex of the two proteins and identified multiple binding sites on both proteins. One of these sites on NEU1 that is involved in binding to PPCA can also bind to other NEU1 molecules, albeit with lower affinity. Therefore, in the absence of PPCA, as in the lysosomal storage disease galactosialidosis, NEU1 self-associates into chain-like oligomers. Binding of PPCA can reverse self-association of NEU1 by causing the disassembly of NEU1-oligomers and the formation of a PPCA-NEU1 heterodimeric complex. The identification of binding sites between the two proteins allowed us to create innovative structural models of the NEU1 oligomer and the PPCA-NEU1 heterodimeric complex. The proposed mechanism of interaction between NEU1 and its accessory protein PPCA provides a rationale for the secondary deficiency of NEU1 in galactosialidosis.Mammalian neuraminidases have been classified as lysosomal (NEU1),⁴ cytosolic (NEU2), plasma membrane (NEU3), and mitochondria/lysosomal (NEU4) based on their subcellular distributions, pH optimum, kinetic properties, responses to ions and detergents, and substrate specificities (1–3). Of the four sialidases, only NEU1 is ubiquitously expressed at different levels in various tissues and cell types (4–7). The importance of these proteins in normal cellular physiology is illustrated by the numerous metabolic processes that they control, including cell proliferation and differentiation, cell adhesion, membrane fusion and fluidity, immunocyte function, and receptor modification (8–21).NEU1 initiates the intralysosomal hydrolysis of sialo-oligosaccharides, -glycolipids, and -glycoproteins by removing their terminal sialic acid residues. In human and murine tissues, NEU1 forms a complex with at least two other proteins, β-galactosidase and the protective protein/cathepsin A (PPCA) (22). By virtue of their association with PPCA, NEU1 and β-galactosidase acquire their active and stable conformation in lysosomes. However, PPCA appears to function as a crucial chaperone/transport protein for NEU1. Because NEU1 is poorly mannose 6-phosphorylated, it depends on PPCA for correct compartmentalization and catalytic activation in lysosomes (23–25). Only a small amount of PPCA and β-galactosidase activities is found in the NEU1-PPCA-β-galactosidase complex, which instead contains all of the NEU1 catalytic activity (24–27). By understanding how and when NEU1 and PPCA interact, how they regulate each other in different cell types, and what determinants control their association, we may gain important insight into their significance in physiologic and pathologic conditions.The absence of NEU1 is associated with two neurodegenerative diseases that involve glycoprotein metabolism; sialidosis, which is caused by structural lesions in the lysosomal NEU1 locus (28), and galactosialidosis (GS), a combined deficiency of NEU1 and β-galactosidase which is caused by the absence of PPCA (22). Patients with sialidosis and those with GS have similar clinical and biochemical features, and both diseases are characterized by multiple phenotypes that are classified according to the age of onset and severity of the symptoms.Previously, we generated two animal models of primary or secondary NEU1 deficiency, Neu1^−/− mice and Ppca^−/− mice. Both mouse models have a profound loss of Neu1 activity in multiple tissues and develop clinical, biochemical, and pathologic manifestations resembling those seen in patients with severe sialidosis and GS (29–31). Neu1^−/− mice are phenotypically similar but not identical to Ppca^−/− mice and, like children with the disease, exhibit a time-dependent splenomegaly associated with extramedullary hematopoiesis (30, 31). We found that the cause of these phenotypic abnormalities is the gradual loss of retention of hematopoietic progenitors within the bone niche due to exacerbated lysosomal exocytosis of bone marrow cells. The latter process is negatively regulated by NEU1 activity (31).The mode of interaction between PPCA and NEU1 and the mechanism of catalytic activation are not well understood. Here we present biochemical, analytical, and structural analyses of NEU1, PPCA, and the PPCA-NEU1 complex by using purified baculovirus (BV)-expressed wild-type and mutagenized recombinant enzymes and synthetic peptides.     

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.     

Thromboxane A2 Receptor Activates a Rho-associated Kinase/LKB1/PTEN Pathway to Attenuate Endothelium Insulin Signaling

This study was conducted to elucidate the molecular mechanisms of thromboxane A2 receptor (TP)-induced insulin resistance in endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) or mouse aortic endothelial cells to either IBOP or U46619, two structurally related thromboxane A₂ mimetics, significantly reduced insulin-stimulated phosphorylation of endothelial nitric-oxide synthase (eNOS) at Ser¹¹⁷⁷ and Akt at Ser⁴⁷³. These effects were abolished by pharmacological or genetic inhibitors of TP. TP-induced suppression of both eNOS and Akt phosphorylation was accompanied by up-regulation of PTEN (phosphatase and tension homolog deleted on chromosome 10), Ser³⁸⁰/Thr^382/383 PTEN phosphorylation, and PTEN lipid phosphatase activity. PTEN-specific small interference RNA restored insulin signaling in the face of TP activation. The small GTPase, Rho, was also activated by TP stimulation, and pretreatment of HUVECs with Y27632, a Rho-associated kinase inhibitor, rescued TP-impaired insulin signaling. Consistent with this result, pertussis toxin abrogated IBOP-induced dephosphorylation of both Akt and eNOS, implicating the G_i family of G proteins in the suppressive effects of TP. In mice, high fat diet-induced diabetes was associated with aortic PTEN up-regulation, PTEN-Ser³⁸⁰/Thr^382/383 phosphorylation, and dephosphorylation of both Akt (at Ser⁴⁷³) and eNOS (at Ser¹¹⁷⁷). Importantly, administration of TP antagonist blocked these changes. We conclude that TP stimulation impairs insulin signaling in vascular endothelial cells by selectively activating the Rho/Rho-associated kinase/LKB1/PTEN pathway.Insulin exerts multiple biological actions relating to not only metabolism but also to endothelial functions (1, 2). Insulin has beneficial effects on the vasculature, primarily because of its ability to enhance endothelial nitric-oxide synthase (eNOS)² activation and expression. These effects, in turn, enhance the bioavailability of nitric oxide (3), which engenders a wide array of antiatherogenic effects. Global insulin resistance is a key feature of the metabolic syndrome leading to cardiovascular disease. In an insulin-resistant state, a systemic deregulation of the insulin signal leads to a combined deregulation of insulin-regulated metabolism and endothelial functions (4), resulting in glucose intolerance and cardiovascular disease. Insulin resistance is associated with endothelial dysfunction (5), a hallmark of atherosclerosis, and predicts adverse cardiovascular events (6). Therefore, endothelium-specific insulin resistance (impaired insulin-stimulated phosphorylation of Akt and eNOS) may play an important role in the development of cardiovascular diseases.Prostanoids have critical roles in the development of endothelial dysfunction (7). Thromboxane A₂ (TXA₂) is believed to be a prime mediator of a variety of cardiovascular and pulmonary diseases such as atherosclerosis, myocardial infarction, and primary pulmonary hypertension. TXA₂ perturbs the normal quiescent phenotype of endothelial cells (ECs). This results in leukocyte adhesion to the vessel wall as well as increased vascular permeability and expression of adhesion molecules on ECs, all important components of the inflammatory response. In smooth muscle cells, TXA₂ promotes proliferation (8) and migration, contributing to neointima formation (9). TXA₂ binds to the thromboxane A₂ receptor (TP), which has two isoforms TPα and TPβ in human (10–12), activation of which is implicated in atherosclerosis and inflammation (13–16). Atherosclerosis is accelerated by diabetes and is associated with increased levels of TXA₂ and other eicosanoids that stimulate TP (14). TP expression and plasma levels of TP ligands are elevated, both locally and systemically, in several vascular and thrombotic diseases (17). Importantly, TP activation induces EC apoptosis (15, 18) and prevents tube formation (19) by inhibiting Akt phosphorylation (18). TP activation also inhibits vascular endothelial growth factor-induced EC migration and angiogenesis by decreasing Akt and eNOS phosphorylation (20). However, the regulatory mechanisms underlying Akt inhibition by TP stimulation remain largely undefined. Moreover, whether TP activation impairs endothelial insulin signaling is also unclear.Here, we investigated whether TP ligands interfere with insulin signaling. Our results reveal that activation of TP using a potent and stable ligand (IBOP) abrogates insulin signaling in ECs. We also show that Rho/ROCK/LKB1-mediated PTEN (phosphatase and tensin homolog deleted on chromosome ten) up-regulation is required for TP-induced inhibition of insulin signaling in ECs.     

Semaphorin3A Signaling Mediated by Fyn-dependent Tyrosine Phosphorylation of Collapsin Response Mediator Protein 2 at Tyrosine 32

Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr³² residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr³² of CRMP showed that Tyr³²-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr³² to Phe³²) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr³² is involved in Sema3A signaling.Collapsin response mediator proteins (CRMPs)⁴ have been identified as intracellular proteins that mediate Semaphorin3A (Sema3A) signaling in the nervous system (1). CRMP2 is one of the five members of the CRMP family. CRMPs also mediate signal transduction of NT3, Ephrin, and Reelin (2–4). CRMPs interact with several intracellular molecules, including tubulin, Numb, kinesin1, and Sra1 (5–8). CRMPs are involved in axon guidance, axonal elongation, cell migration, synapse maturation, and the generation of neuronal polarity (1, 2, 4, 5).CRMP family proteins are known to be the major phosphoproteins in the developing brain (1, 9). CRMP2 is phosphorylated by several Ser/Thr kinases, such as Rho kinase, cyclin-dependent kinase 5 (Cdk5), and glycogen synthase kinase 3β (GSK3β) (2, 10–13). The phosphorylation sites of CRMP2 by these kinases are clustered in the C terminus and have already been identified. Rho kinase phosphorylates CRMP2 at Thr⁵⁵⁵ (10). Cdk5 phosphorylates CRMP2 at Ser⁵²², and this phosphorylation is essential for sequential phosphorylations by GSK3β at Ser⁵¹⁸, Thr⁵¹⁴, and Thr⁵⁰⁹ (2, 11–13). These phosphorylations disrupt the interaction of CRMP2 with tubulin or Numb (2, 3, 13). The sequential phosphorylation of CRMP2 by Cdk5 and GSK3β is an essential step in Sema3A signaling (11, 13). Furthermore, the neurofibrillary tangles in the brains of people with Alzheimer disease contain hyperphosphorylated CRMP2 at Thr⁵⁰⁹, Ser⁵¹⁸, and Ser⁵²² (14, 15).CRMPs are also substrates of several tyrosine kinases. The phosphorylation of CRMP2 by Fes/Fps and Fer has been shown to be involved in Sema3A signaling (16, 17). Phosphorylation of CRMP2 at Tyr⁴⁷⁹ by a Src family tyrosine kinase Yes regulates CXCL12-induced T lymphocyte migration (18). We reported previously that Fyn is involved in Sema3A signaling (19). Fyn associates with PlexinA2, one of the components of the Sema3A receptor complex. Fyn also activates Cdk5 through the phosphorylation at Tyr¹⁵ of Cdk5 (19). In dorsal root ganglion (DRG) neurons from fyn-deficient mice, Sema3A-induced growth cone collapse response is attenuated compared with control mice (19). Furthermore, we recently found that Fyn phosphorylates CRMP1 and that this phosphorylation is involved in Reelin signaling (4). Although it has been shown that CRMP2 is involved in Sema3A signaling (1, 11, 13), the relationship between Fyn and CRMP2 in Sema3A signaling and the tyrosine phosphorylation site(s) of CRMPs remain unknown.Here, we show that Fyn phosphorylates CRMP2 at Tyr³². Using a phospho-specific antibody against Tyr³², we determined that the residue is phosphorylated in vivo. A nonphosphorylated mutant CRMP2Y32F inhibits Sema3A-induced growth cone collapse. These results indicate that tyrosine phosphorylation by Fyn at Tyr³² is involved in Sema3A signaling.