Angela Woods

We have developed a sensitive assay for the AMP-activated protein kinase kinase, the upstream component in the AMP-activated protein kinase cascade. Phosphorylation and activation of the downstream kinase by the upstream kinase absolutely requires AMP and is antagonized by high (millimolar) concentrations of ATP. We have purified the upstream kinase >1000-fold from rat liver; a variety of evidence indicates that the catalytic subunit may be a polypeptide of 58 kDa. The physical properties of the downstream and upstream kinases, e.g. catalytic subunit masses (63 versus 58 kDa) and native molecular masses (190 versus 195 kDa), are very similar. However, unlike the downstream kinase, the upstream kinase is not inactivated by protein phosphatases. The upstream kinase phosphorylates the downstream kinase at a single major site on the α subunit, i.e. threonine 172, which lies in the “activation segment” between the DFG and APE motifs. This site aligns with activating phosphorylation sites on many other protein kinases, including Thr177 on calmodulin-dependent protein kinase I. As well as suggesting a mechanism of activation of AMP-activated protein kinase, this finding is consistent with our recent report that the AMP-activated protein kinase kinase can slowly phosphorylate and activate calmodulin-dependent protein kinase I, at least in vitro (Hawley, S. A., Selbert, M. A., Goldstein, E. G., Edelman, A. M., Carling, D., and Hardie, D. G. (1995) J. Biol. Chem. 270, 27186-27191). We have developed a sensitive assay for the AMP-activated protein kinase kinase, the upstream component in the AMP-activated protein kinase cascade. Phosphorylation and activation of the downstream kinase by the upstream kinase absolutely requires AMP and is antagonized by high (millimolar) concentrations of ATP. We have purified the upstream kinase >1000-fold from rat liver; a variety of evidence indicates that the catalytic subunit may be a polypeptide of 58 kDa. The physical properties of the downstream and upstream kinases, e.g. catalytic subunit masses (63 versus 58 kDa) and native molecular masses (190 versus 195 kDa), are very similar. However, unlike the downstream kinase, the upstream kinase is not inactivated by protein phosphatases. The upstream kinase phosphorylates the downstream kinase at a single major site on the α subunit, i.e. threonine 172, which lies in the “activation segment” between the DFG and APE motifs. This site aligns with activating phosphorylation sites on many other protein kinases, including Thr177 on calmodulin-dependent protein kinase I. As well as suggesting a mechanism of activation of AMP-activated protein kinase, this finding is consistent with our recent report that the AMP-activated protein kinase kinase can slowly phosphorylate and activate calmodulin-dependent protein kinase I, at least in vitro (Hawley, S. A., Selbert, M. A., Goldstein, E. G., Edelman, A. M., Carling, D., and Hardie, D. G. (1995) J. Biol. Chem. 270, 27186-27191).

Caloric restriction (CR) protects against aging and disease, but the mechanisms by which this affects mammalian life span are unclear. We show in mice that deletion of ribosomal S6 protein kinase 1 (S6K1), a component of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway, led to increased life span and resistance to age-related pathologies, such as bone, immune, and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian life-span and suggest that therapeutic manipulation of S6K1 and AMPK might mimic CR and could provide broad protection against diseases of aging.