UC San Diego

AMP-activated protein kinase (AMPK) is a master regulator of metabolism. AMPK senses when cellular (and organismal) energy levels are low, and orchestrates adaptive changes to metabolic pathways, cellular processes, and gene expression programs to restore energy balance in response. Due its central homeostatic function, AMPK represents an important signaling mode in many contexts of health and disease, and an intriguing target for therapeutic intervention in many metabolic diseases.Elucidating specific downstream effects of AMPK and its molecular effectors is key for understanding the role of AMPK activation in different health contexts. While many functions of AMPK have been identified, particularly cytosolically, AMPK’s function in the nuclear compartment has remained elusive. With growing evidence that AMPK has important effects on gene expression, this work aims to illuminate nuclear AMPK activity: Here we examine its nuclear localization, nuclear targets, and transcription factor effectors of AMPK activation. Using a nuclear proteomics approach, we found that AMPKα1 shuttles into the nucleus following activation. We evaluated the upstream signals of this effect and found that an LKB1-dependent pathway is involved in response to both allosteric activation and mitochondrial stress. We performed a bioinformatics screen of transcription factors to identify potential direct targets of AMPK in the nucleus and validated the novel substrate SRF and several AMPK-SRF-dependent genes. Further, we optimized a biochemical approach for the identification of additional nuclear substrates. In addition to these protein-level studies, we perform transcript-level analyses. Major known effectors of AMPK-dependent transcription are the transcription factors TFEB and TFE3 (TFEB/3). Previous work has shown that transcription factors induced by TFEB/3 can be critical adaptive mediators of transcription downstream of AMPK activation. We searched for other possible key secondary effectors of transcriptional cascades downstream of AMPK-TFEB/3 by gene expression analysis and identified five transcription factors of interest which behave as AMPK-dependent TFEB/3 target genes: CREBRF, CREBL2, KLF9, KLF11, and KLF15. This combination of biochemical approaches, proteomic strategies, gene editing, RNA-sequencing, and bioinformatics analysis has culminated in the characterization of multiple aspects of nuclear AMPK activity, the identification of a novel nuclear substrate, and insights into AMPK-mediated transcriptional rewiring.