Laura A. Castelli

β-Subunit myristoylation is the gatekeeper for initiating metabolic stress sensing by AMP-activated protein kinase (AMPK)

The AMP-activated protein kinase (AMPK) is an αβγ heterotrimer that acts as a master metabolic regulator to maintain cellular energy balance following increased energy demand and increases in the AMP/ATP ratio. This regulation provides dynamic control of energy metabolism, matching energy supply with demand that is essential for the function and survival of organisms. AMPK is inactive unless phosphorylated on Thr172 in the α-catalytic subunit activation loop by upstream kinases (LKB1 or calcium-calmodulin-dependent protein kinase kinase β). How a rise in AMP levels triggers AMPK α-Thr172 phosphorylation and activation is incompletely understood. Here we demonstrate unequivocally that AMP directly stimulates α-Thr172 phosphorylation provided the AMPK β-subunit is myristoylated. Loss of the myristoyl group abolishes AMP activation and reduces the extent of α-Thr172 phosphorylation. Once AMPK is phosphorylated, AMP further activates allosterically but this activation does not require β-subunit myristoylation. AMP and glucose deprivation also promote membrane association of myristoylated AMPK, indicative of a myristoyl-switch mechanism. Our results show that AMP regulates AMPK activation at the initial phosphorylation step, and that β-subunit myristoylation is important for transducing the metabolic stress signal.

Expression and analysis of the NS2 protein of influenza A virus

SummaryInfluenza NS2 protein was expressed inSaccharomyces cerevisiae using a copper-inducible promoter. The protein produced had a molecular weight of 13 kDa, was reactive with anti-NS2 antiserum and was localised to the yeast cell nucleus. Two-hybrid analysis identified a direct protein-protein interaction between NS2 and the M2 protein of the virus, involving the C-terminal 163 residues of M1. A filter-binding assay localised the M1 binding region to the C-terminal 70 amino acids of NS2.

HIV-1 protein Vpr causes gross mitochondrial dysfunction in the yeast Saccharomyces cerevisiae

The biological effects of the HIV‐1 accessory protein, Vpr, have been studied in yeast expression systems. In our previous study [1] , employing the pCUP1‐vpr copper‐inducible expression cassette, Vpr was shown to cause growth arrest and structural defects. In this study yeast constitutively expressing vpr, through elevated copy number and/or elevated transcription levels, displayed no growth arrest in fermentative growth conditions while Vpr was produced at much lower levels than in the inducible expression system. However, such cells were respiratory deficient and unable to utilise ethanol or glycerol as the sole carbon source. They exhibited gross mitochondrial dysfunction displayed in the loss of respiratory chain complex I, II, III, IV and citrate synthase activities. The effects on mitochondria required a C‐terminal domain of Vpr that contains a conserved amino acid sequence motif HFRIGCRHSRIG. These results suggest that the widely observed phenomenon of ‘Vpr‐induced growth arrest’ in human cells could be due to mitochondrial dysfunction.

Adipose Triglyceride Lipase Regulation of Skeletal Muscle Lipid Metabolism and Insulin Responsiveness

Adipose triglyceride lipase (ATGL) is important for triglyceride (TG) metabolism in adipose tissue, and ATGL-null mice show increased adiposity. Given the apparent importance of ATGL in TG metabolism and the association of lipid deposition with insulin resistance, we examined the role of ATGL in regulating skeletal muscle lipid metabolism and insulin-stimulated glucose disposal. ATGL expression in myotubes was reduced by small interfering RNA and increased with a retrovirus encoding GFP-HA-ATGL. ATGL was also overexpressed in rats by in vivo electrotransfer. ATGL was down-regulated in skeletal muscle of obese, insulin-resistant mice and negatively correlated with intramyocellular TG levels. ATGL small interfering RNA in myotubes reduced TG hydrolase activity and increased TG content, whereas ATGL overexpression induced the reciprocal response, indicating that ATGL is an essential TG lipase in skeletal muscle. ATGL overexpression in myotubes increased the oxidation of fatty acid liberated from TG and diglyceride and ceramide contents. These responses in cells were largely recapitulated in rats overexpressing ATGL. When ATGL protein expression and TG hydrolase activity in obese, insulin-resistant rats were restored to levels observed in lean rats, TG content was reduced; however, the insulin resistance induced by the high-fat diet persisted. In conclusion, ATGL TG hydrolysis in skeletal muscle is a critical determinant of lipid metabolism and storage. Although ATGL content and TG hydrolase activity are decreased in obese, insulin-resistant phenotypes, overexpression does not rescue the condition, indicating reduced ATGL is unlikely to be a primary cause of obesity-associated insulin resistance.

Insulin stimulates movement of sorting nexin 9 between cellular compartments: a putative role mediating cell surface receptor expression and insulin action

SNX9 (sorting nexin 9) is one member of a family of proteins implicated in protein trafficking. This family is characterized by a unique PX (Phox homology) domain that includes a proline-rich sequence and an upstream phospholipid binding domain. Many sorting nexins, including SNX9, also have a C-terminal coiled region. SNX9 additionally has an N-terminal SH3 (Src homology 3) domain. Here we have investigated the cellular localization of SNX9 and the potential role it plays in insulin action. SNX9 had a cytosolic and punctate distribution, consistent with endosomal and cytosolic localization, in 3T3L1 adipocytes. It was excluded from the nucleus. The SH3 domain was responsible, at least in part, for the membrane localization of SNX9, since expression of an SH3-domain-deleted GFP (green fluorescent protein)-SNX9 fusion protein in HEK293T cells rendered the protein cytosolic. Membrane localization may also be attributed in part to the PX domain, since in vitro phospholipid binding studies demonstrated SNX9 binding to polyphosphoinositides. Insulin induced movement of SNX9 to membrane fractions from the cytosol. A GST (glutathione S-transferase)-SNX9 fusion protein was associated with IGF1 (insulin-like growth factor 1) and insulin receptors in vitro. A GFP-SNX9 fusion protein, overexpressed in 3T3L1 adipocytes, co-immunoprecipitated with insulin receptors. Furthermore, overexpression of this GFP-SNX9 fusion protein in CHOT cells decreased insulin binding, consistent with a role for SNX9 in the trafficking of insulin receptors. Microinjection of 3T3L1 cells with an antibody against SNX9 inhibited stimulation by insulin of GLUT4 translocation. These results support the involvement of SNX9 in insulin action, via an influence on the processing/trafficking of insulin receptors. A secondary role in regulation of the cellular processing, transport and/or subcellular localization of GLUT4 is also suggested.

AMP-activated Protein Kinase Subunit Interactions β1:γ1 ASSOCIATION REQUIRES β1 Thr-263 AND Tyr-267

AMP-activated protein kinase (AMPK) plays multiple roles in the bodys overall metabolic balance and response to exercise, nutritional stress, hormonal stimulation, and the glucose-lowering drugs metformin and rosiglitazone. AMPK consists of a catalytic α subunit and two non-catalytic subunits, β and γ, each with multiple isoforms that form active 1:1:1 heterotrimers. Here we show that recombinant human AMPK α1β1γ1 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 αβγ complex shows that β binds α and γ. Here we show that a β1(186–270)γ1 complex can form in the absence of detectable α subunit. Moreover, using alanine mutagenesis we show that β1 Thr-263 and Tyr-267 are required for βγ association but not αβ association.

AMPK subunit interactions; β1:γ1 association requires β1 THR-263 and TYR-267

AMP-activated protein kinase (AMPK) plays multiple roles in the bodys overall metabolic balance and response to exercise, nutritional stress, hormonal stimulation, and the glucose-lowering drugs metformin and rosiglitazone. AMPK consists of a catalytic α subunit and two non-catalytic subunits, β and γ, each with multiple isoforms that form active 1:1:1 heterotrimers. Here we show that recombinant human AMPK α1β1γ1 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 αβγ complex shows that β binds α and γ. Here we show that a β1(186–270)γ1 complex can form in the absence of detectable α subunit. Moreover, using alanine mutagenesis we show that β1 Thr-263 and Tyr-267 are required for βγ association but not αβ association.

Cellular munc18c levels can modulate glucose transport rate and GLUT4 translocation in 3T3L1 cells

Munc18c has been shown to bind syntaxin 4 and to play a role in GLUT4 translocation and glucose transport, although this role is as yet poorly defined. In the present study, the effects of modulating the available level of munc18c on glucose transport and GLUT4 translocation were examined. Over‐expression of munc18c in 3T3L1 adipocytes inhibited insulin‐stimulated glucose transport by approximately 50%. Basal glucose transport rates were also decreased by approximately 25%. In contrast, microinjection of a munc18c polyclonal antibody stimulated GLUT4 translocation by approximately 60% over basal levels without affecting insulin‐stimulated GLUT4 levels. Microinjection of a control antibody had no effect. These data are consistent with the likelihood that antibody microinjection sequesters munc18c enabling translocation/fusion of GLUT4 vesicles. Mutagenesis of a potential proline‐directed kinase phosphorylation site in munc18c, T569, that in previous studies of its neuronal counterpart munc18a caused its dissociation from its complex with syntaxin 1a, had no effect on munc18cs association with syntaxin 4 or its inhibition of glucose transport, indicative that phosphorylation of this residue is not important for insulin regulation of glucose transport. The over‐expression and microinjection sequestration data support an inhibitory role for munc18c on translocation/fusion of GLUT4 vesicles. They further show that altering the level of available munc18c in 3T3L1 cells can modulate glucose transport rates, indicating its potential as a target for therapeutics in diabetes.

Identification of elongation factor 1α as a potential associated binding partner for Akt2

Akt protein kinase has been shown to play a pivotal role in diverse cell functions, including motility, apoptosis, growth and metabolism. How it differentially regulates these diverse functions is of significant interest. Three isoforms have been well characterized, Akt1, 2, and 3, encoded by separate genes, but showing high homology over the entire coding sequence (> 80%). An area of variability between the three isoforms is the C-terminal tail. To find potentially regulating binding partners of Akt2, the isoform implicated in metabolic control, we used a glutathione-S-transferase (GST) fusion protein expressing the C-terminal 75 residues of Akt2 (GST-Akt2 tail) to screen for proteins that specifically bound the Akt2 tail. Elongation factor 1α (EF1α) and β-tubulin were identified as binding partners for the Akt2 tail by peptide mass fingerprinting. These two proteins have themselves been previously identified as interacting partners (Nakazawa et al.FEBS Lett. 453,29-34, 1999). Using CHOT cells that overexpress insulin receptors and HA-tagged Akt2, we showed that EF1α co-immunoprecipitated with HA-tagged Akt2. It is thus possible that these proteins colocalise as part of a regulatory signaling complex with the cytoskeleton directing them to sites of cell activity.

Expression of HIV-1 nef in Yeast Causes Membrane Perturbation and Release of the Myristylated Nef Protein

The human immunodeficiency virus type 1 (HIV-1) Nef protein is essential for AIDS pathogenesis, but its function remains highly controversial. During stresses such as growth in the presence of copper or at elevated temperature, myristylated Nef is released from yeast cells and, after extended culture in stationary phase, it accumulates in the supernatant as a dense membranous material that can be centrifuged into a discrete layer above the cell pellet. This material is unique to Nef-producing cells and represents a convenient source of Nef that may have application in further biological studies. Within the yeast cell, electron microscopic examination shows that Nef localises in novel, membrane-bound bodies. These data support the evidence for a role of Nef in membrane perturbation and suggest that there may be a similar localisation for myristylated Nef in HIV-1 infected cells.