Elongation factor 2 kinase (eEF2k) phosphorylates and inactivates eEF2. Insulin induces dephosphorylation of eEF2 and inactivation of eEF2 kinase, and these effects are blocked by rapamycin, which inhibits the mammalian target of rapamycin, mTOR. However, the signalling mechanisms underlying these effects are unknown. Regulation of eEF2 phosphorylation and eEF2k activity is lost in cells in which phosphoinositide-dependent kinase 1 (PDK1) has been genetically knocked out. This is not due to loss of mTOR function since phosphorylation of another target of mTOR, initiation factor 4E-binding protein 1, is not defective. PDK1 is required for activation of members of the AGC kinase family; we show that two such kinases, p70 S6 kinase (regulated via mTOR) and p90 RSK1 (activated by Erk), phosphorylate eEF2k at a conserved serine and inhibit its activity. In response to insulin-like growth factor 1, which activates p70 S6 kinase but not Erk, regulation of eEF2 is blocked by rapamycin. In contrast, regulation of eEF2 by stimuli that activate Erk is insensitive to rapamycin, but blocked by inhibitors of MEK/Erk signalling, consistent with the involvement of p90 RSK1 .

PDK1 mediates activation of PKB, p70 S6 kinase and p90 Rsk in vivo, but is not rate-limiting for activation of PKA, MSK1 and AMPK. Another kinase phosphorylates PKB at its hydrophobic motif in PDK1(-/-) cells. PDK1 phosphorylates the hydrophobic motif of p70 S6 kinase either directly or by activation of another kinase.

PDK1 functions as a master kinase, phosphorylating and activating PKB/Akt, S6K and RSK. To learn more about the roles of PDK1, we generated mice that either lack PDK1 or possess PDK1 hypomorphic alleles, expressing only approximately 10% of the normal level of PDK1. PDK1(-/-) embryos die at embryonic day 9.5, displaying multiple abnormalities including lack of somites, forebrain and neural crest derived tissues; however, development of hind- and midbrain proceed relatively normally. In contrast, hypomorphic PDK1 mice are viable and fertile, and insulin injection induces the normal activation of PKB, S6K and RSK. Nevertheless, these mice are 40-50% smaller than control animals. The organ volumes from the PDK1 hypomorphic mice are reduced proportionately. We also establish that the volume of a number of PDK1-deficient cells is reduced by 35-60%, and show that PDK1 deficiency does not affect cell number, nuclear size or proliferation. We provide genetic evidence that PDK1 is essential for mouse embryonic development, and regulates cell size independently of cell number or proliferation, as well as insulin's ability to activate PKB, S6K and RSK.

did not alter the activity of LKB1 to phosphorylate itself or the tumor suppressor protein p53 or alter the amount of LKB1 associated with cell membranes. The reintroduction of wild-type LKB1 into a cancer cell line that lacks LKB1 suppressed growth, but mutants of LKB1 in which Ser 431 was mutated to Ala to prevent phosphorylation of LKB1 were ineffective in inhibiting growth. In contrast, a mutant of LKB1 that cannot be prenylated was still able to suppress the growth of cells.Peutz-Jeghers syndrome is an autosomal dominantly inherited disorder that predisposes to a wide spectrum of benign and malignant tumors (1, 2). It is caused by mutation of a widely expressed protein kinase of unknown function termed LKB1 (also known as STK11) (3, 4). To date, over 60 different mutations have been mapped to LKB1, many of which would be expected to impair LKB1 activity. These discoveries have aroused great interest because they indicate that LKB1 is likely to function in cells as a tumor suppressor, and consistent with this, overexpression of LKB1 in a number of tumor cell lines has been shown to suppress cell growth by inducing a G 1 cell cycle block (5). However, little is known regarding the mechanism by which LKB1 activity is regulated in cells, and no substrates for LKB1 have thus far been identified.LKB1 is a 436-amino acid protein possessing a kinase domain (residues 50 -337) that is only distantly related to other mammalian kinases. The N-terminal non-catalytic domain comprises both a nuclear localization signal (6) and a putative cytoplasmic retention signal (7). There are no yeast homologs of LKB1, but there are putative homologs in Xenopus (termed XEEK1, with 84% overall identity to LKB1) (8) and Caenorhabditis elegans (termed PAR-4, with 26% overall identity to LKB1 and 41% identity in the kinase domain) (9). In Drosophila, an uncharacterized protein kinase listed in the NCBI Protein Database (NCBI accession number AAF54972) possesses 44% overall identity to LKB1.Recently, a C-terminal fragment of LKB1 was shown to be phosphorylated at Ser 431 by the cAMP-dependent protein kinase (10). Ser 431 of LKB1 lies in the sequence Lys-Xaa-ArgArg-Xaa-Ser (where Xaa is any amino acid), which is conserved in all known mammalian LKB1 sequences and in Xenopus XEEK1. This study did not establish whether full-length or endogenously expressed LKB1 was phosphorylated at Ser 431 in response to stimuli that activated cAMP-dependent protein kinase (PKA) 1 or the role that this phosphorylation played in enabling LKB1 to suppress cell growth. Ser 431 lies in the consensus sequence for phosphorylation by a group of kinases related to PKA, viz. p90 ribosomal S6 kinase (p90 RSK ), mitogen-and stress-stimulated protein kinase (MSK1), and p70 ribosomal S6 kinase (S6K1) (11)(12)(13), that collectively belong to the AGC kinase subfamily. p90 RSK is activated in cells by growth factors and phorbol esters and by ERK1/2 MAPK family members (14), whereas MSK1 is activated in vivo by two dif-* The costs of publication of this article were defr...

The multi-site phosphorylation of the protein kinase C (PKC) superfamily plays an important role in the regulation of these enzymes. One of the key phosphorylation sites required for the activation of all PKC isoforms lies in the T-loop of the kinase domain. Recent in vitro and transfection experiments indicate that phosphorylation of this residue can be mediated by the 3-phosphoinositide-dependent protein kinase-1 (

In most epithelial tissues, accumulating mutations (i.e., genetic progression)and loss of cellular control functions are observed as the phenotype changes from normal histology, to early intraepithelial neoplasia (1EN)then to increasingly severe IEN, superficial cancers, and finally invasive disease. IEN, such as colorectal adenomas, PIN, and CIN are primary examples of histologic biomarkers suitable for following carcinogenesis. Useful genomic biomarkers include LOH and gene amplification at panels of microsatellite loci where mutations indicate increasing genomic instability. Both phenotypic and genomic changes during carcinogenesis can also be demonstrated by molecular biomarkers. For example, excess proliferation may be measured by increased levels of cellular antigens such as PCNA and MIB-1 or overexpression of growth factors such as EGF, TGFa, and IGF-1; reduced apoptosis may be detected by increased expression of bcl-2. Aberrant differentiation may result in changes in Gactin, cytokeratins, and blood group antigens. Other molecular biomarkers may reflect general changes in cell growth control. These include TGFP, cyclins, p53 and other tumor suppressors, as well as overexpression and mutation of oncogenes such as rusand the transcription factors myc, fos, and jun. Tissue-and drug-specific biomarkers may also be useful. Examples of tissue-specific biomarkers are hormone receptors in breast and prostate. Currently, there are more than 50 clinical chemoprevention studies, primarily Phase I1 trials, in progress in ten major cancer sites (prostate, breast, colon, lung, head and neck, bladder, cervix, esophagus, skin, and 1iver)involving evaluation of tissue-based histologic, genomic, and molecular biomarkers as surrogate endpoints for cancer. PDKI, the missing link in insulin signal transduction MRC Protern PhosphoryIation Unit, MSI/WTB Complex, Universrty of Dwndee, 374Dwndee DDl IEH Protein kinase B (PKB, also called Akt) is activated within a minute or two when cells are stimulated with insulin or insulin-like growth factor-I (IGF-I) and mediates many of the intracellular actions of these signals by phosphorylating key regulatory proteins at serine and thrconine residues that lie in Arg-Xaa-Arg-Xaa-Xaa-SerfThr sequences.Physiological substrates for PKB include glycogen synthase kinase-3 (GSK3), the PDE3B isoform of cyclic AMP phosphodiesterase, and the cardiac isoform of 6-phosphofructo-2-kinase (PFKZ). The inhibition of GSK3 by PKB leads to the dephosphorylation and activation of glycogen synthase and protein synthesis initiation factor eIF2B and contributes to the insulin-induced stimulation of glycogen synthesis and protein synthesis. The activation of PDE3B underlies the insulin-induced decrease in the level of cyclic AMP, which inhibits lipolysis in adipose tissue. The activation of PFKZ underlies the insulin-induced stimulation of glycolysis in the heart. The insulin or IGF1-induced activation of PKB results from the phosphorylation of PKB at two residues, namely Thr308 in the activation loop of the ca...

Efficient signal transduction is important in maintaining the function of the nervous system across tissues. An intact neurotransmission process can regulate energy balance through proper communication between neurons and peripheral organs. This ensures that the right neural circuits are activated in the brain to modulate cellular energy homeostasis and systemic metabolic function. Alterations in neurotransmitters secretion can lead to imbalances in appetite, glucose metabolism, sleep, and thermogenesis. Dysregulation in dietary intake is also associated with disruption in neurotransmission and can trigger the onset of type 2 diabetes (T2D) and obesity. In this review, we highlight the various roles of neurotransmitters in regulating energy balance at the systemic level and in the central nervous system. We also address the link between neurotransmission imbalance and the development of T2D as well as perspectives across the fields of neuroscience and metabolism research.

Please refer to abstract number 82. This abstract was originally submitted as a poster, and on the basis of its scientific interest and merit, was chosen by the colloquium organizers to be presented as an oral communication, as well as a poster.