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We identified a novel human AMP-activated protein kinase (AMPK) family member, designated ARK5, encoding 661 amino acids with an estimated molecular mass of 74 kDa. The putative amino acid sequence reveals 47, 45.8, 42.4, and 55% homology to AMPK-␣1, AMPK-␣2, MELK, and SNARK, respectively, suggesting that it is a new member of the AMPK family. It has a putative Akt phosphorylation motif at amino acids 595-600, and Ser 600 was found to be phosphorylated by active Akt resulting in the activation of kinase activity toward the SAMS peptide, a consensus AMPK substrate. During nutrient starvation, ARK5 supported the survival of cells in an Akt-dependent manner. In addition, we also demonstrated that ARK5, when activated by Akt, phosphorylated the ATM protein that is mutated in the human genetic disorder ataxia-telangiectasia and also induced the phosphorylation of p53. On the basis of our current findings, we propose that a novel AMPK family member, ARK5, is the tumor cell survival factor activated by Akt and acts as an ATM kinase under the conditions of nutrient starvation. AMPK1 is a mammalian homologue of sucrose non-fermenting protein kinase (SNF-1), which belongs to a serine/threonine protein kinase family, and its activation is well documented in cells under metabolic stress, hypoxia, heat shock, and ischemia (1, 2). The SNF-1/AMPK family is highly conserved in several species including mammals (3-8), and the ␣-subunit of AMPK has been shown to be the catalytic subunit (2). Four proteins, AMPK-␣1 and AMPK-␣2 (9 -12), MELK (5), and SNARK (13), have been identified as catalytic subunits of the AMPK family to date. Although it has been reported that AMPK activation is initiated by phosphorylation at Thr 172 (14), how the phosphorylation is initiated remains unclear.AMPK is activated under various stress conditions where the cellular ATP concentration decreases and plays a key role in cellular adaptive responses to maintain energy balance. The well known targets of AMPK belong to those involved in energy metabolism including 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA), acyl-CoA decarboxylase, and glucose and amino acid transporters (15). However, the cellular mechanism for survival under stress conditions is complex and includes cell cycle regulation, repair of cellular components, remodeling tissue components, and regulation of the cell death pathway. Targets of AMPK are not fully described to date. Recently, we demonstrated that some tumor cells, such as the pancreatic cancer cell line PANC-1, showed high tolerance against glucose starvation, which is compatible with hypovascular findings on clinical angiography (16), although most cell lines, including a human hepatoma cell line and the human normal fibroblast cell line, underwent cell death under glucose starvation (16,17). Similar tolerance to glucose starvation was induced by hypoxic conditions, and tolerance was found to be dependent on AMPK activity as well as . During glucose starvation, a G 1 phase cell cycle delay occurs with a concomitant i...

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AMP-activated protein kinases (AMPKs) are a class of serine/threonine protein kinases that are activated by an increase in intracellular AMP concentration. They are a sensitive indicator of cellular energy status and have been found to promote tumor cell survival during nutrient starvation. We recently identified a novel AMPK catalytic subunit family member, ARK5, whose activation is directly regulated by Akt, which, in turn, has been reported to be a key player in tumor malignancy. In this study, we attempted to determine whether ARK5 is involved in tumor malignancy under regulation by Akt. Matrigel invasion assays demonstrated that both overexpressed and endogenous ARK5 showed strong activity dependent on Akt. In addition, ARK5 expression induced activation of matrix metalloproteinase 2 (MMP-2) and MMP-9 following new expression of membrane type 1 MMP (MT1-MMP), and the MT1-MMP expression induced by ARK5 was initiated by rapamycin-sensitive signaling. In nude mice, ARK5 expression was associated with a significant increase in tumor growth and significant suppression of necrosis in tumor tissue. Interestingly, only the ARK5-overexpressing PANC-1 cell line (P/ARK) tumor showed invasion and metastasis in nude mice, although Akt was activated in tumors derived from both P/ARK and its parental cell line. We report that a novel AMPK catalytic subunit family member, ARK5, plays a key role in tumor malignancy downstream of Akt.

AMPK is a serine/threonine protein kinase family and we recently identified a novel member, ARK5. The activation of ARK5 is triggered by Akt, and ARK5 induces tumor cell survival during nutrient starvation. In the current study, we investigated the mechanisms of induction of cell survival by ARK5. Human hepatoma HepG2 cells undergo necrotic cell death within 24 h after the start of glucose starvation, and the cell death signaling has been found to be mediated by death-receptor-independent activation of caspase 8. When HepG2 cells were transfected with ARK5 expression vector and subjected to several cell death stimuli, ARK5 was found to suppress cell death by glucose starvation, TRAIL, and TNF-a, but not by ultraviolet irradiation, camptothecin, or doxorubicin. Western blotting analysis revealed that both TRAIL and glucose starvation induced Bid cleavage and FLIP degradation following caspase 8 activation in a timedependent manner, and ARK5 overexpression clearly delayed Bid cleavage, FLIP degradation, and caspase 8 activation. On the basis of the results of this study, we report that cell survival induced by ARK5 is, at least in part, due to inhibition of caspase 8 activation.