AMP-activated protein kinase (AMPK) is a heterotrimericT he 5ЈAMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is ubiquitously expressed and functions as an intracellular fuel sensor activated by depletion of highenergy phospho-compounds (1). Activation of AMPK initiates a complex series of signaling events that trigger increases in the uptake and oxidation of substrates important for ATP synthesis and decreases in ATP-consuming biosynthetic processes such as protein, lipid, and glycogen synthesis (2). Activation of AMPK has been linked to the regulation of glucose transport (3), but the substrates linking these events are elusive.AMPK regulates GLUT4-dependent glucose transport across the sarcolemma in skeletal muscle in response to diverse forms of cellular stress including exercise, hypoxia, and agents that disrupt the intracellular ATP-to-AMP ratio (4 -6). Evidence linking AMPK and glucose uptake has been provided by the use of different genetic approaches, including knockout (KO) and transgenic dominant-negative kinase mouse models (4 -6). Expression of a dominant-negative ␣2 AMPK construct in skeletal muscle suppresses ␣2 and ␣1 isoform-specific AMPK activity and completely prevents 5-aminoimidazole-4-carboxamide 1 ␤-D-ribonucleoside (AICAR)-induced glucose transport (6). This observation was reinforced by recent reports showing that knockout of either the catalytic ␣2 (but not ␣1 AMPK isoform) or the regulatory ␥3 AMPK subunit completely abolishes AICAR-induced glucose transport (4,5). Collectively, these data provide evidence to suggest that ␣2 and ␥3 containing AMPK heterotrimeric complexes are involved in AICAR-induced glucose transport. Although AICAR-induced glucose transport in resting skeletal muscle is mediated by GLUT4 translocation to the plasma membrane (7,8), the link between AMPK signaling and GLUT4 translocation is presently unknown.AS160 is a substrate for the protein kinase Akt that links insulin signaling and GLUT4 trafficking (9 -11). AS160 contains a GTPase-activating protein homology domain that has been shown to regulate the GTPase activity of certain Rab proteins in vitro (12). Phosphorylation of AS160 by Akt is likely to inhibit its GTPase-activating protein activity, such that as a consequence, the GTP form of a Rab protein is elevated and this elevation in turn increases GLUT4 vesicle movement to, and/or fusion with, From the

DNA damage-induced cell-cycle checkpoints have a critical role in maintaining genomic stability. A key target of the checkpoints is the CDC25A (cell division cycle 25 homologue A) phosphatase, which is essential for the activation of cyclin-dependent kinases and cell-cycle progression. To identify new genes involved in the G2/M checkpoint we performed a large-scale short hairpin RNA (shRNA) library screen. We show that NIMA (never in mitosis gene A)-related kinase 11 (NEK11) is required for DNA damage-induced G2/M arrest. Depletion of NEK11 prevents proteasome-dependent degradation of CDC25A, both in unperturbed and DNA-damaged cells. We show that NEK11 directly phosphorylates CDC25A on residues whose phosphorylation is required for beta-TrCP (beta-transducin repeat-containing protein)-mediated polyubiquitylation and degradation of CDC25A. Furthermore, we demonstrate that CHK1 (checkpoint kinase 1) directly activates NEK11 by phosphorylating it on Ser 273, indicating that CHK1 and NEK11 operate in a single pathway that controls proteolysis of CDC25A. Taken together, these results demonstrate that NEK11 is an important component of the pathway enforcing the G2/M checkpoint, suggesting that genetic mutations in NEK11 may contribute to the development of human cancer.

Dysregulation of GS phosphorylation plays a major role in impaired insulin regulation of GS in obesity and T2DM. In obesity, independent of T2DM, this is associated with impaired regulation of site 2+2a and likely site 3, whereas the exaggerated insulin resistance to activate GS in T2DM is linked to hyperphosphorylation of at least site 1b. Thus, T2DM per se seems unrelated to defects in the glycogen synthase kinase-3 regulation of GS.

To identify key connections between DNA-damage repair and checkpoint pathways, we performed RNA interference screens for regulators of the ionizing radiation-induced G2 checkpoint, and we identified the breast cancer gene BRCA2. The checkpoint was also abrogated following depletion of PALB2, an interaction partner of BRCA2. BRCA2 and PALB2 depletion led to premature checkpoint abrogation and earlier activation of the AURORA A-PLK1 checkpoint-recovery pathway. These results indicate that the breast cancer tumour suppressors and homologous recombination repair proteins BRCA2 and PALB2 are main regulators of G2 checkpoint maintenance following DNA-damage.

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Cells respond to DNA damage by activating cell cycle checkpoints to delay proliferation and facilitate DNA repair. Here, to uncover new checkpoint regulators, we perform RNA interference screening targeting genes involved in ubiquitylation processes. We show that the F-box protein cyclin F plays an important role in checkpoint control following ionizing radiation. Cyclin F-depleted cells initiate checkpoint signalling after ionizing radiation, but fail to maintain G2 phase arrest and progress into mitosis prematurely. Importantly, cyclin F suppresses the B-Myb-driven transcriptional programme that promotes accumulation of crucial mitosis-promoting proteins. Cyclin F interacts with B-Myb via the cyclin box domain. This interaction is important to suppress cyclin A-mediated phosphorylation of B-Myb, a key step in B-Myb activation. In summary, we uncover a regulatory mechanism linking the F-box protein cyclin F with suppression of the B-Myb/cyclin A pathway to ensure a DNA damage-induced checkpoint response in G2.

activated protein kinase (AMPK) was recently suggested to regulate pyruvate dehydrogenase (PDH) activity and thus pyruvate entry into the mitochondrion. We aimed to provide evidence for a direct link between AMPK and PDH in resting and metabolically challenged (exercised) skeletal muscle. Compared with rest, treadmill running increased AMPK␣1 activity in ␣2KO mice (90%, P Ͻ 0.01) and increased AMPK␣2 activity in wild-type (WT) mice (110%, P Ͻ 0.05), leading to increased AMPK␣ Thr 172 (WT: 40%, ␣2KO: 100%, P Ͻ 0.01) and ACC␤ Ser 227 phosphorylation (WT: 70%, ␣2KO: 210%, P Ͻ 0.01). Compared with rest, exercise significantly induced PDH-E1␣ site 1 (WT: 20%, ␣2KO: 62%, P Ͻ 0.01) and site 2 (only ␣2KO: 83%, P Ͻ 0.01) dephosphorylation and PDHa [ϳ200% in both genotypes (P Ͻ 0.01)]. Compared with WT, PDH dephosphorylation and activation was markedly enhanced in the ␣2KO mice both at rest and during exercise. The increased PDHa activity during exercise was associated with elevated glycolytic flux, and muscles from the ␣2KO mice displayed marked lactate accumulation and deranged energy homeostasis. Whereas mitochondrial DNA content was normal, the expression of several mitochondrial proteins was significantly decreased in muscle of ␣2KO mice. In isolated resting EDL muscles, activation of AMPK signaling by AICAR did not change PDH-E1␣ phosphorylation in either genotype. PDH is activated in mouse skeletal muscle in response to exercise and is independent of AMPK␣2 expression. During exercise, ␣2KO muscles display deranged energy homeostasis despite enhanced glycolytic flux and PDHa activity. This may be linked to decreased mitochondrial oxidative capacity.adenosine 5Ј-monophosphate-activated protein kinase; 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside; treadmill running; glucose metabolism THE PYRUVATE DEHYDROGENASE (PDH) COMPLEX (PDC) is a mitochondrial enzyme that catalyzes the irreversible conversion of pyruvate to acetyl-CoA and entry of carbohydrate-derived substrate into the citric acid cycle for oxidation. Therefore, it is a key regulator of carbohydrate metabolism in cells. PDC consists of several copies of three catalytic proteins denoted E 1 , E 2 , and E 3 , where the PDH activity in the active form (PDH a activity) is associated with the E 1 subunit, also denoted PDH. PDH is composed of two ␣-and two ␤-subunits (14), and the PDH activity is regulated by phosphorylation and dephosphorylation on three specific serine residues on the E 1 ␣ subunit (25, 32). In skeletal muscle, PDH is inactivated by phosphorylation of site 1 and site 2 (15), the level of which is regulated by PDH kinases (PDK1-4) and PDH phosphatases (PDP1/2). A range of allosteric factors modulates the action of the upstream kinases and phosphatases (reviewed in Refs. 14 and 31). PKC␦ was recently shown (5) to translocate to the mitochondrion, and phosphorylation/activation of PDP led to dephosphorylation and activation of PDH. These observations introduce an unexplored field of PDH regulation by covalent modification of upstream regulator...

Exercising muscle releases interleukin-6 (IL-6), but the mechanisms controlling this process are poorly understood. This study was performed to test the hypothesis that the IL-6 release differs in arm and leg muscle during whole-body exercise, owing to differences in muscle metabolism. Sixteen subjects (10 men and six women, with body mass index 24 ± 1 kg m −2 and peak oxygen uptake 3.4 ± 0.6 l min −1 ) performed a 90 min combined arm and leg cycle exercise at 60% of maximal oxygen uptake. The subjects arrived at the laboratory having fasted overnight, and catheters were placed in the femoral artery and vein and in the subclavian vein. During exercise, arterial and venous limb blood was sampled and arm and leg blood flow were measured by thermodilution. Lean limb mass was measured by dual-energy X-ray absorbtiometry scanning. Before and after exercise, biopsies were obtained from vastus lateralis and deltoideus. During exercise, IL-6 release was similar between men and women and higher (P < 0.05) from arms than legs (1.01 ± 0.42 and 0.33 ± 0.12 ng min −1 (kg lean limb mass) −1 , respectively). Blood flow (425 ± 36 and 554 ± 35 ml min −1 (kg lean limb mass) −1 ) and fatty acid uptake (26 ± 7 and 47 ± 7 μmol min −1 (kg lean limb mass) −1 ) were lower, glucose uptake similar (51 ± 12 and 41 ± 8 mmol min −1 (kg lean limb mass) −1 ) and lactate release higher (82 ± 32 and −2 ± 12 μmol min −1 (kg lean limb mass) −1 ) in arms than legs, respectively, during exercise (P < 0.05). No correlations were present between IL-6 release and exogenous substrate uptakes. Muscle glycogen was similar in arms and legs before exercise (388 ± 22 and 428 ± 25 mmol (kg dry weight) −1 ), but after exercise it was only significantly lower in the leg (219 ± 29 mmol (kg dry weight) −1 ). The novel finding of a markedly higher IL-6 release from the exercising arm compared with the leg during whole-body exercise was not directly correlated to release or uptake of exogenous substrate, nor to muscle glycogen utilization.