Fyn-phosphorylated PIKE-A binds and inhibits AMPK signaling,blocking its tumor suppressive activity

The AMP-activated protein kinase, a key regulator of energy homeostasis, has a critical role in metabolic disorders and cancers. AMPK is mainly regulated by cellular AMP and phosphorylation by upstream kinases. Here, we show that PIKE-A binds to AMPK and blocks its tumor suppressive actions, which are mediated by tyrosine kinase Fyn. PIKE-A directly interacts with AMPK catalytic alpha subunit and impairs T172 phosphorylation, leading to repression of its kinase activity on the downstream targets. Mutation of Fyn phosphorylation sites on PIKE-A, depletion of Fyn, or pharmacological inhibition of Fyn blunts the association between PIKE-A and AMPK, resulting in loss of its inhibitory effect on AMPK. Cell proliferation and oncogenic assays demonstrate that PIKE-A antagonizes tumor suppressive actions of AMPK. In human glioblastoma samples, PIKE-A expression inversely correlates with the p-AMPK levels, supporting that PIKE-A negatively regulates AMPK activity in cancers. Thus, our findings provide additional layer of molecular regulation of the AMPK signaling pathway in cancer progression.AMP-activated protein kinase is activated under a variety of physiological and pathological stresses that increase the intracellular AMP/ATP ratio, either by increasing ATP consumption (exercise/muscle contraction) or by decreasing ATP production (e.g., glucose deprivation, hypoxia or ischemia). It is a heterotrimeric complex consisting of a catalytic α subunit and two regulatory (β and γ) subunits. An increase in intracellular AMP/ATP ratio results in allosteric activation of the kinase by protecting T172 from dephosphorylation.¹ T172 phosphorylation in the activation loop of the α subunit is an absolute requirement for full activation of AMPK activity,^{2, 3} and is mediated by at least two distinct upstream kinases, liver kinase B1 (LKB1)^{4, 5, 6} and Ca²⁺/calmodulin-dependent kinase kinase β (CaMKKβ).^{7, 8, 9}AMPK is an evolutionarily conserved metabolic sensor that has a pivotal role in maintaining energy homeostasis by coordinating metabolic pathways to balance nutrient supply and demand.¹⁰ Regulation of AMPK in multiple tissues is controlled by a growing number of hormones and cytokines, including leptin, adiponectin, IL-6, CNTF, TNF-α, and ghrelin. Moreover, AMPK can be activated by numerous small molecules such as metformin, aminoimidazole-4-carboxymide-1-β-D-ribofuranoside (AICAR), resveratrol, thiozolidinedione (TZD), and A-769662. Activated AMPK regulates glucose uptake and fatty acid oxidation in muscle and blocks gluconeogenesis in liver, enhancing insulin sensitivity. It also regulate appetite (for review, see Dzamko and Steinberg).¹¹ In addition to these well-characterized functions in metabolic syndromes, AMPK serves as a metabolic tumor suppressor that reprograms the cellular metabolism and elicits a metabolic checkpoint on the cell cycle through its actions on mTORC1, p53, and other modulators for cell proliferation, cell growth, cell survival, and autophagy.¹² Further, LKB1 activates AMPK and represses RNA synthesis.¹³ In LKB1-deficient lung cancer cells, AMPK activity is suppressed, leading to increased cell growth, whereas the ability of AMPK to inhibit cell growth is restored when wild-type LKB1 is expressed.^{14, 15} Additionally, the express levels of AMPK inversely correlate with clinical prognosis in gastric,¹⁶ breast, and ovarian tumors, and are diminished in cancer cells by activated PI3K pathways.¹⁷ Accumulating evidence supports that the susceptibility of cancer might be attributable to the dysregulated AMPK.^{18, 19} Hence, activation of AMPK may represent a novel target for cancer treatment.PIKE-A is a GTPase that directly interacts with PI 3 kinase or Akt and enhances their kinase activities.^{20, 21, 22, 23} It is a proto-oncogene that frequently amplified in numerous human cancers.^{24, 25} It binds Akt and escalates its kinase activity and promotes cancer cell survival, invasion, and migration.^{26, 27} Interestingly, PIKE knockout (PIKE^−/−) mice are resistant to diet-induced obesity and diabetes,²⁸ strongly implicating PIKE in obesity control. Accordingly, we observed higher AMPK phosphorylation and lipid oxidation in PIKE^−/− muscle and fat tissues, which provide a mechanistic explanation to the slim phenotype of the knockout mice.²⁸ Further, PIKE-A interacts with insulin receptor and mediates its suppressive effect on AMPK activation.²⁹ Previously, we have reported that Fyn phosphorylates PIKE-A on both Y682 and Y774³⁰ and regulates its interaction with different partners, promoting neuronal survival³¹ and adiposeness.³² In this report, we provide new evidence supporting that Fyn phosphorylation of PIKE-A is critical for its association with AMPK and inhibition of its kinase activity, leading to the blockade of cell proliferation. Hence, PIKE-A promotes tumorigenesis, at least, partially through blocking the tumor suppressive activity of AMPK. This discovery highlights a previously unappreciated relationship between cell metabolism and cell proliferation mediated by PIKE-A/AMPK complex.