FLCN interacts with AMPK via FNIP1 and/or FNIP2, and regulates mTOR signalling (Baba et al., 2006; Hasumi et al., 2008, Takagi et al., 2008). Phosphorylation at S62 of FLCN increases the FLCN-FNIP complex’s affinity for AMPK, while phosphorylation at S302 decreases FLCN’s affinity for AMPK (Piao et al., 2009, Wang et al., 2010). AMPK is an important energy sensing protein, which inhibits anabolic growth via mTOR signalling and stimulates autophagy to promote cell survival when energy supply is low (Alers et al., 2012).
Loss of FLCN in mouse embryonic fibroblasts (MEFs), leads to the constitutive activation of AMPK (Yan et al., 2014). This ultimately activates HIF signalling and leads to metabolic changes consistent with the Warburg Effect within FLCN-null cells. A nonphosphorylatable FLCN S62A mutant was unable to bind and inhibit AMPK, meaning that FLCN-FNIP binding to AMPK is required for its inhibition.
Possik et al., (2014) also found that deletion of flcn-1 in nematode worms leads to constitutive activation of AMPK, which causes a subsequent increase in autophagic flux and intracellular ATP levels. This leads to increased stress resistance and protection from apoptosis in C. elegans nematodes, mouse embryonic fibroblasts, and the FLCN-null FTC-133 thyroid carcinoma cell line (Possik et al., 2014). The flcn-1 null nematodes are also more resistant to hyperosmotic stress due to increase glycogen storage, an additional result of the increased AMPK signalling (Possik et al., 2015).
Taken together, these studies suggest that FLCN inhibits AMPK signalling. However, two recent studies have shown that FLCN may also activate AMPK signalling in certain conditions.
Using a mouse model where FLCN had been deleted in alveolar epithelial type II cells (AECs) using a Cre-lox system, Goncharova et al., (2014) found that loss of FLCN in AECs led to increased apoptosis and cell permeability due to loss of E-cadherin expression, subsequent LKB1 dysregulation and consequent AMPK downregulation. The AMPK activator, AICAR, and constitutively active AMPK were able to increase cell survival in FLCN-null cells, and AICAR improved alveolar surface tension in vivo. These mice showed pulmonary developmental defects and impaired lung function consistent with a role for FLCN in branching morphogenesis.
Mice lacking FLCN in their hearts developed cardiac hypertrophy and died at 3 months. AMPKα phosphorylation at T172, was shown to be reduced in the heart cells of these mice. AMPK inactivation was caused by increased ATP levels following PGC1A overexpression and increased mitochondrial biogenesis (Hasumi et al., 2014).
FLCN loss has since been shown to have highly cell-specific outcomes and knockdown of FLCN led to reduced AMPK signalling in HBE cells, but had no effect on in SAEC cells (Khabibullin et al., 2014).
FNIP1 has been found to be required for AMPK to inhibit mTOR signalling in murine B cells by (Park et al. 2012). FNIP1 deletion in iNKT cells lead to increased mTOR signalling despite mitochondrial mass and intracellular ATP levels being reduced, suggesting that AMPK’s ability to inhibit mTOR was faulty in these cells (Park et al., 2014).
FLCN and FNIP2 activate AMPK to trigger apoptosis in response to N-Nitroso-N-methylurea treatment and FLCN and AMPK act in opposition to regulate FNIP2 protein stability (Lim et al., 2012, Sano et al., 2013).
In addition to FLCN affecting AMPK signalling, AMPK has also been shown to phosphorylate FLCN, FNIP1 and FNIP2 (Baba et al., 2006; Hasumi et al., 2008, Takagi et al., 2008, Wang et al., 2010), indicating that FLCN and AMPK signalling might form a feedback loop.