Ability Of AMP-activated Protein Kinase Research Articles

Abstract 193: Mechanism of the AMPK-dependent activation of eukaryotic elongation factor 2 kinase (eEF-2K)

Abstract Eukaryotic elongation factor 2 kinase (eEF-2K) negatively regulates the rate of protein translation by phosphorylating and inactivating elongation factor 2 (eEF-2). Activation of eEF-2K by AMP-activated protein kinase (AMPK) contributes to the adaptive response of cancer cells to nutrient deprivation, promoting cancer cell survival and tumor progression. However, the mechanism by which AMPK activates eEF-2K is poorly described. Using LC-MS/MS, we confirmed previous findings that AMPK phosphorylates eEF-2K on three sites in vitro: Ser78, Ser366, and Ser398. Previous reports indicate that only Ser398 is phosphorylated by AMPK in cells, suggesting phosphorylation of Ser78 and Ser366 (which are known to inhibit eEF-2K via other kinases) are in vitro artifacts. To test the notion that phosphorylation of Ser398 activates eEF-2K, we purified recombinant eEF-2K S78/S366A, which cannot be phosphorylated at Ser78 or Ser366. Incubation of eEF-2K S78/S366A with AMPK led to significant phosphorylation of eEF-2K after four hours but did not alter eEF-2K activity against a peptide substrate. We then purified a form of eEF-2K S78/S366A in which Ser398 is ∼90% phosphorylated (eEF-2K SSA-pS398), as shown by LC-MS/MS and Western blot. The activity of eEF-2K SSA-pS398 against a peptide substrate was unchanged compared to a non-phosphorylated control, as was its activity against full-length eEF-2. Similarly, the EC50 and affinity of eEF-2K S78/S366A for Ca2+/calmodulin (CaM), which eEF-2K requires for activity, were unchanged by Ser398 phosphorylation. Phosphorylation of Ser398 did not alter the rate of autophosphorylation of eEF-2K S78/S366A at Thr348 or Ser500, which modulate its activity and dependence on Ca2+/CaM, respectively. Overall, our in vitro data suggests that AMPK may not directly regulate eEF-2K through phosphorylation of Ser398. In cells, mTOR activates several kinases, including p70S6K, that phosphorylate and inactivate eEF-2K. AMPK inhibits mTOR through phosphorylation of TSC2 and Raptor, suggesting AMPK could relieve eEF-2K from mTOR-mediated inhibition by suppressing mTOR activity. In line with this notion, we found that treatment of MDA-MB-231 breast cancer cells with Torin1 (a selective inhibitor of mTOR) or AICAR (an AMP-mimetic that activates AMPK) led to similar levels of eEF-2K activation, and activation of AMPK with AICAR reduced levels of mTOR activity. Combined application of Torin1 and AICAR resulted in levels of eEF-2K activity similar to those obtained with either compound separately. Taken together, our data suggests that the AMPK-dependent activation of eEF-2K likely involves the ability of AMPK to inhibit mTOR and relieve eEF-2K from mTOR-mediated inhibition, and questions the role of Ser398 phosphorylation in activating eEF-2K directly. Citation Format: David H. Giles, Chris M. Crittenden, Jennifer S. Brodbelt, Kevin N. Dalby. Mechanism of the AMPK-dependent activation of eukaryotic elongation factor 2 kinase (eEF-2K). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 193.

Insulin Resistance and Muscle Metabolism in Chronic Kidney Disease

Insulin resistance is a common finding in chronic kidney disease (CKD) and is manifested by mild fasting hyperglycemia and abnormal glucose tolerance testing. Circulating levels of glucocorticoids are high. In muscle, changes in the insulin signaling pathway occur. An increase in the regulatory p85 subunit of Class I phosphatidylinositol 3-Kinase enzyme leads to decreased activation of the downstream effector protein kinase B (Akt). Mechanisms promoting muscle proteolysis and atrophy are unleashed. The link of Akt to the ubiquitin proteasome pathway, a major degradation pathway in muscle, is discussed. Another factor associated with insulin resistance in CKD is angiotensin II (Ang II) which appears to induce its intracellular effects through inflammatory cytokines or reactive oxygen species. Skeletal muscle ATP is depleted and the ability of AMP-activated protein kinase (AMPK) to replenish energy stores is blocked. How this can be reversed is discussed. Interleukin-6 (IL-6) levels are elevated in CKD and impair insulin signaling at the level of IRS-1. With exercise, IL-6 levels are reduced; glucose uptake and utilization are increased. For patients with CKD, exercise may improve insulin signaling and build up muscle. Treatment strategies for preventing muscle atrophy are discussed.

Impairment of PGC-1alpha expression, neuropathology and hepatic steatosis in a transgenic mouse model of Huntington's disease following chronic energy deprivation

We investigated the ability of AMP-activated protein kinase (AMPK) to activate PPARgamma coactivator-1alpha (PGC-1alpha) in the brain, liver and brown adipose tissue (BAT) of the NLS-N171-82Q transgenic mouse model of Huntington's disease (HD). In the striatum of the HD mice, the baseline levels of PGC-1alpha, NRF1, NRF2, Tfam, COX-II, PPARdelta, CREB and ERRalpha mRNA and mitochondrial DNA (mtDNA), were significantly reduced. Administration of the creatine analog beta guanidinopropionic acid (GPA) reduced ATP and PCr levels and increased AMPK mRNA in both the cerebral cortex and striatum. Treatment with GPA significantly increased expression of PGC-1alpha, NRF1, Tfam and downstream genes in the striatum and cerebral cortex of wild-type (WT) mice, but there was no effect on these genes in the HD mice. The striatum of the untreated HD mice showed microvacuolation in the neuropil, as well as gliosis and huntingtin aggregates, which were exacerbated by treatment with GPA. GPA treatment produced a significant increase in mtDNA in the cerebral cortex and striatum of WT mice, but not in HD mice. The HD mice treated with GPA had impaired activation of liver PGC-1alpha and developed hepatic steatosis with accumulation of lipids, degeneration of hepatocytes and impaired activation of gluconeogenesis. The BAT in the HD mice showed vacuolation due to accumulation of neutral lipids, and age-dependent impairment of UCP-1 activation and temperature regulation. Impaired activation of PGC-1alpha, therefore, plays an important role in the behavioral phenotype, metabolic disturbances and pathology of HD, which suggests the possibility that agents that enhance PGC-1alpha function will exert therapeutic benefits in HD patients.