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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