UNDERSTANDING THE ROLE OF SUMOYLATION IN REGULATING LKB1 FUNCTION

Energy homeostasis in a cell is critical for its survival during metabolic stress. Liver kinase B1 (LKB1), one of the key regulators of cellular energy balance, was initially discovered as a tumor suppressor mutated in patients with Peutz-Jeghers syndrome. Germline mutations in LKB1 predispose patients to develop several benign and malignant tumors including gastrointestinal and lung cancers. In 2003, several groups demonstrated that LKB1is a major upstream kinase of the energy sensor AMP-activated protein kinase (AMPK), directly associating it with the regulation of energy balance in cells. During energy stress, LKB1 phosphorylates AMPK at threonine 172 (T172) resulting in AMPK activation. This leads to the inhibition of anabolic pathways such as fatty acid synthesis and activation of energy-producing pathways such as glycolysis. Some of the proteins targeted include: ACC1 (fatty acid synthesis), ACC2 (fatty acid oxidation), mTORC1 (protein synthesis), ULK1 (mitophagy) and GLUT1 (glucose uptake). Therefore, LKB1 is strategically positioned as an essential kinase in maintaining cellular energy balance. A number of studies have described the influence of covalent post-translational modifications in governing LKB1 activity. However, the role of SUMOylation in regulating LKB1 function is unknown. SUMOylation is the reversible covalent modification of a SUMO (small ubiquitin-related modifier) protein to a specific lysine on the target protein. This process has been implicated in important processes such as transcription, protein stability, and protein subcellular localization. At the molecular level, SUMOylation can (1) inhibit the interaction between the target and its interacting partner, (2) enhance this interaction through creation of a binding surface where the target would recognize the partner via a SUMO-interacting motif (SIM), or (3) change the conformation of the target, thereby altering its function. Given the diverse roles SUMOylation plays in the eukaryotic cell, we hypothesized that, during energy stress, SUMOylation regulates the LKB1-AMPK interaction and that this accordingly affects the kinase activity of AMPK. Our findings here demonstrate that energy deficit triggers an increase in the modification of LKB1 by SUMO1 despite a global reduction in both SUMO1 and SUMO2/3 conjugates. During metabolic stress, LKB1 is specifically modified by SUMO1 at lysine 178 (K178) but not by SUMO2/3, acetylation, or ubiquitination. This modification is essential in promoting LKB1-AMPK interaction. On the basis of the crystal structure depicting the non-covalent recognition of SUMO1 by RanBP2, we identified a SIM in the N-terminal region of AMPK. Mutation of the hydrophobic residues necessary for SUMO1 interaction prevented LKB1 from recognizing and activating AMPK. Finally, we observed that cells with the LKB1 K178R SUMO mutant had defective AMPK signaling and mitochondrial function, inducing apoptosis in energy-deprived cells. Thus, we propose a model in which energy stress upregulates the modification of LKB1 by SUMO1, thereby facilitating its interaction with AMPK. This enhances the rate at which AMPK can respond to the metabolic needs of the cell