-AMP-activated protein kinase (AMPK) is a meta-bolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific ␥3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK ␥3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse ␥3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK ␥3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK ␥3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK ␥3 isoform as a therapeutic target for prevention and treatment of insulin resistance.AMPK 1 is a heterotrimeric serine/threonine protein kinase composed of a catalytic ␣ subunit and non-catalytic ␤ and ␥ subunits (1, 2). The mammalian genome contains seven AMPK genes encoding two ␣, two ␤, and three ␥ isoforms. AMPK signaling is elicited by cellular stresses that deplete ATP (and consequently elevate AMP) by either inhibiting ATP production (e.g. hypoxia) or accelerating ATP consumption (e.g. muscle contraction). AMPK is activated allosterically by AMP and through phosphorylation of Thr 172 in the ␣ subunit by an upstream AMPK kinase, the tumor-suppressor protein kinase LKB1 (3, 4). AMPK is likely to be important for diverse functions in many cell types, but particular interest has been focused on elucidating the role of AMPK in the regulation of lipid and carbohydrate metabolism in skeletal muscle (5-10). AMPK activity has been correlated with an increase in glucose uptake and fatty acid oxidation and an inhibition of glycogen synthase activity and fatty acid synthesis. Exercise, as well as skeletal muscle contractions in vitro, leads to AMPK activation. Pharmacological activation of AMPK also can be achieved using 5-aminoimidazole-4-carboxamide-1-␤-D-ribonucleoside (AICAR). Once taken up by the cell, AICAR is phosphorylated to 5-aminoimidazole-4-carboxamide riboside monophosphate (ZMP) and mimics effects of AMP on AMPK (1, 2). AMPK function is closely related to glycogen storage. AMPK phosphorylates glycogen synthase in vitro (11) and co-immunoprecipitates with glycogen synthase and glycogen phosphorylase from skeletal muscle (12). Mutations of the ␥3 or ␥2 subunit, respectively, affect glycogen storage in pigs (13, 14) or glycogen storage associated with cardiac abnormalities in humans (15). The recent identification of a glycogen-binding domain in the AMPK ␤1 subunit provides a molecular relationship between AMPK and glycogen (16,17). The formation of heterotrimers appears to be...

The AMP-activated protein kinase (AMPK) is a metabolic-stress-sensing protein kinase that regulates metabolism in response to energy demand and supply by directly phosphorylating rate-limiting enzymes in metabolic pathways as well as controlling gene expression.

Background-Mutations in the ␥ 2 subunit (PRKAG2) of AMP-activated protein kinase produce an unusual human cardiomyopathy characterized by ventricular hypertrophy and electrophysiological abnormalities: Wolff-ParkinsonWhite syndrome (WPW) and progressive degenerative conduction system disease. Pathological examinations of affected human hearts reveal vacuoles containing amylopectin, a glycogen-related substance. Methods and Results-To elucidate the mechanism by which PRKAG2 mutations produce hypertrophy with electrophysiological abnormalities, we constructed transgenic mice overexpressing the PRKAG2 cDNA with or without a missense N488I human mutation. Transgenic mutant mice showed elevated AMP-activated protein kinase activity, accumulated large amounts of cardiac glycogen (30-fold above normal), developed dramatic left ventricular hypertrophy, and exhibited ventricular preexcitation and sinus node dysfunction. Electrophysiological testing demonstrated alternative atrioventricular conduction pathways consistent with WPW. Cardiac histopathology revealed that the annulus fibrosis, which normally insulates the ventricles from inappropriate excitation by the atria, was disrupted by glycogen-filled myocytes. These anomalous microscopic atrioventricular connections, rather than morphologically distinct bypass tracts, appeared to provide the anatomic substrate for ventricular preexcitation. Conclusions-Our data establish PRKAG2 mutations as a glycogen storage cardiomyopathy, provide an anatomic explanation for electrophysiological findings, and implicate disruption of the annulus fibrosis by glycogen-engorged myocytes as the cause of preexcitation in Pompe, Danon, and other glycogen storage diseases.

JAK2, a member of the Janus kinase (JAK) family of protein tyrosine kinases (PTKs), is an important intracellular mediator of cytokine signaling. Mutations of the JAK2 gene are associated with hematologic cancers, and aberrant JAK activity is also associated with a number of immune diseases, including rheumatoid arthritis. Accordingly, the development of JAK2-specific inhibitors has tremendous clinical relevance. Critical to the function of JAK2 is its PTK domain. We report the 2.0 Å crystal structure of the active conformation of the JAK2 PTK domain in complex with a high-affinity, pan-JAK inhibitor that appears to bind via an induced fit mechanism. This inhibitor, the tetracyclic pyridone 2-tert-butyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-1, was buried deep within a constricted ATP-binding site, in which extensive interactions, including residues that are unique to JAK2 and the JAK family, are made with the inhibitor. We present a structural basis of high-affinity JAK-specific inhibition that will undoubtedly provide an invaluable tool for the further design of novel, potent, and specific therapeutics against the JAK family. IntroductionThe Janus kinases (JAKs) are an important family of intracellular protein tyrosine kinases (PTKs), with 4 mammalian members, JAK1, JAK2, JAK3, and TYK2, 1-5 as well as homologs in chicken, 6 fish, 7 and Drosophila. 8 The JAKs play critical roles in several important intracellular signaling pathways, including the eponymous JAK/STAT pathway, 9 central to the mediation of cytokine signaling. 10,11 It is this pivotal role in cytokine signaling that underpins the notion that specific JAK inhibitors may be therapeutically deployed in situations where cytokine activity results in disease. Important examples of this include autoimmune diseases such as rheumatoid arthritis and psoriasis, 12,13 myeloproliferative syndromes such as polycythemia vera, 14-17 leukemias, 18-20 lymphomas, 21 and cardiovascular disease 22,23 inter alia.Members of the JAK family each share a characteristic domain structure, 2 with a C-terminal PTK domain (known as the JAK homology-1 [JH1] domain), immediately adjacent to a kinase-like domain (JH2), and 5 additional JAK homology domains (JH3-JH7). While the JH2 domain appears to possess an important regulatory role on the PTK activity of the JH1 domain, 24-29 the precise mechanism by which this control is exerted is currently poorly understood. The role of a putative SH2-like domain (JH3/JH4) 2,30 is also unknown at present, whereas the function of a well-defined band F ezrin-radixin-moesin homology (FERM) domain (JH7) 31,32 appears to be critical for interaction of the JAKs with their cognate receptors and regulatory proteins.The JAKs coordinate specifically to different receptors, for example, JAK3 appears to be associated with cytokine receptors that include the ␥c chain of the interleukin-2 (IL-2) receptor (eg, IL-4, IL-7, etc), whereas JAK2 is associated with a wide range of cytokine receptors, including those activated by growth horm...

AMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable ␣␤␥ heterotrimer comprising a catalytic ␣ and two non-catalytic subunits, ␤ and ␥. The ␤ subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here we find that the conserved C-terminal 85-residue sequence of the ␤ subunit, ␤1-(186 -270), is sufficient to form an active AMP-dependent heterotrimer ␣1␤1- AMPK 1 is a multi-substrate enzyme that is activated in response to both hormones and intracellular metabolic stress generated by exercise, hypoxia, and nutrient deprivation. There are multiple isoforms of each AMPK subunit, with ␣1, ␣2, ␤1, ␤2, ␥1, ␥2, and ␥3 forming heterotrimers that differ in tissue and subcellular localization (reviewed in Ref. 1). The ␣ subunit contains an N-terminal catalytic core (1-312) and a C-terminal sequence (313-548) responsible for autoregulation and binding the ␤␥ subunits (2). Maximum activity requires all three subunits (3). The catalytic AMPK ␣1-(1-312) fragment is constitutively active whereas the ␣1-(1-392) fragment is autoinhibited, and neither bind ␤␥ subunits (2). The three ␥ subunits each contain four CBS sequence repeats that were named after the corresponding sequences in cystathionine ␤-synthase (CBS) along with variable N-terminal extensions (4). AMP binds to the ␥ subunit and is responsible for the allosteric regulation of AMPK (5). There are two binding sites for AMP formed by the CBS1/2 and CBS3/4 sequence pairs (5), and because pairs of the CBS sequences form a discrete functional structure, they have now been termed Bateman modules (6). The ␤ subunit N-terminal myristoyl group is responsible for targeting AMPK to the membrane (7), and an internal glycogen-binding domain (68 -163) targets AMPK to glycogen (8, 9).The AMPK ␣ subunit is a homolog of yeast Snf1p kinase (10), which also binds ␤␥ subunit homologs (11, 12). There are three yeast ␤ subunit homologs, Gal83p, Sip1p, and Sip2p, and a single ␥ subunit homolog, Snf4p (13). In contrast to mammalian AMPK, which requires all three subunits for optimal activity (3), Snf1p/Snf4p forms a stable active complex that can be readily isolated from bakers' yeast without a ␤ homolog (Gal83p, Sip1p, or Sip2p) (11,14). Deletion of either Snf1p or Snf4p blocks growth on sucrose, as does deletion of all three ␤ subunit homologs (15). Studies of the subunit interactions in yeast using the two-hybrid approach have identified two regions within the yeast ␤ homologs termed KIS (kinase interacting sequence) and ASC (association with Snf1p complex), respectively. The C-terminal 85-residue ASC sequence of Gal83p was shown to bind Snf4p by two-hybrid analysis (13). The internal KIS sequence of Gal83p, Sip1p, or Sip2p interacted with the non-catalytic C terminus of Snf1p (13). In contrast, studies on mammalian AMPK have shown that the corresponding int...

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Single-chain variable fragment of the murine monoclonal antibody NC10 specific to influenza virus N9 neuraminidase, joined directly in the V(L) to V(H) orientation (scFv-0), forms an equilibrium mixture of tetramer and trimer with the tetramer as the preferred multimeric species. In contrast, the V(H)-V(L) isomer was previously shown to exist exclusively as a trimer. Computer-generated trimeric and tetrameric scFv models, based on the refined crystal structure for NC10 Fv domain, were constructed and used to evaluate factors influencing the transition between V(L)-V(H) trimer and tetramer. These model structures indicated that steric restrictions between loops spanning amino acid residues L55-L59 and L13-L17 from the two adjacent V(L) domains within the V(L)-V(H) trimer were responsible for four scFv-0 molecules assembling to form a tetramer. In particular, leucine at position L15 and glutamate at position L57 appeared to interfere significantly with each other. To minimize this steric interference, the site-directed mutagenesis technique was used to construct several NC10 scFv-0 clones with mutations at these positions. Size-exclusion chromatographic analyses revealed that several of these mutations resulted in the production of NC10 scFv-0 proteins with significantly altered tetramer-trimer equilibrium ratios. In particular, introduction of a polar residue, such as asparagine or threonine, at position L15 generated a highly stable NC10 scFv-0 trimer.

AMP-activated protein kinase (AMPK) plays multiple roles in the body's overall metabolic balance and response to exercise, nutritional stress, hormonal stimulation, and the glucose-lowering drugs metformin and rosiglitazone. AMPK consists of a catalytic ␣ subunit and two non-catalytic subunits, ␤ and ␥, each with multiple isoforms that form active 1:1:1 heterotrimers. Here we show that recombinant human AMPK ␣1␤1␥1 expressed in insect cells is monomeric and displays specific activity and AMP responsiveness similar to rat liver AMPK. The previously determined crystal structure of the core of mammalian ␣␤␥ complex shows that ␤ binds ␣ and ␥. Here we show that a ␤1(186 -270)␥1 complex can form in the absence of detectable ␣ subunit. Moreover, using alanine mutagenesis we show that ␤1 Thr-263 and Tyr-267 are required for ␤␥ association but not ␣␤ association.Mammalian AMP-activated protein kinase (AMPK) 6 is a metabolite-sensing serine/threonine protein kinase that plays a central role in whole body energy homeostasis. The enzyme is activated in response to intracellular stresses that lead to increased AMP:ATP ratios (e.g. hypoxia, exercise, nutrient deprivation) and hormones that regulate whole-body energy metabolism, including adiponectin and leptin (1, 2). AMPK is also activated by metformin and rosiglitazone, glucose lowering drugs used to treat type II diabetes (3, 4). Activation of AMPK leads to phosphorylation of multiple downstream targets that restore energy imbalances broadly by switching off anabolic processes and stimulating energy producing pathways (2).Fully functional AMPK is a heterotrimer composed of ␣, ␤, and ␥ subunits (5), with distinct genes encoding each of the subunits (␣1, ␣2, ␤1, ␤2, ␥1, ␥2, and ␥3). The ␣ subunits each contain a highly conserved N-terminal catalytic core (1-312), an autoinhibitory sequence (313-335) (6, 7), and a less conserved C-terminal ␤ binding sequence (313-473) with the remainder of the ␣ C terminus required for ␥ binding (7, 8). The three ␥ subunits each contain four conserved CBS sequence repeats, named after the corresponding regions in cystathionine-␤-synthase. Pairs of CBS sequences (CBS1/CBS2 and CBS3/CBS4) form discrete functional structures termed Bateman domains (9) that bind nucleotides (10). Four nucleotide binding sites have been identified with each CBS sequence contributing one site (11). The mammalian ␥1 subunit crystal structure has 3 of these 4 sites occupied, with 2 sites capable of binding either AMP or ATP (12). It is presumed ligand binding to the Bateman domains induces conformational changes within the ␣ subunit and provides a mechanism for allosteric activation of the kinase. The ␥ subunits possess non-conserved N-terminal extensions of up to 94 residues, the functions of which are poorly understood but may be involved in subcellular targeting. The ␤ subunits contain an N-terminal myristoylation site responsible for localization of AMPK to the membrane (13,14) and an internal glycogen binding domain (residues 68 -163) (15,16).Studies using AM...