UC San Diego

Liver kinase B1 (LKB1), encoded by the gene STK11, is a serine/threonine kinase that directly phosphorylates and activates a family of 14 downstream kinases known as the AMP-activated protein kinase (AMPK)-related protein kinases (AMPKRs). LKB1 is a tumor suppressor gene inactivated in the cancer predisposition disorder Peutz-Jeghers syndrome as well as in 20% of all human lung cancers. LKB1 plays critical roles in a number of developmental, physiological, and pathogenic processes through regulation of metabolism, cell growth, cell polarity and autophagy. LKB1 accomplishes these functions through its activation of the AMPKRs and thus delineation of which AMPKRs are critical for particular processes is an area of active interest and study.

After decades of connections to cancer and metabolic diseases, our initial studies into LKB1 and the AMPKRs uncovered an unexpected role for AMPK, a metabolic regulator in cells, in lineage specification during embryogenesis. Embryonic stem cells lacking AMPK are incapable of forming normal chimeric embryoid bodies (EBs) upon differentiation. These AMPK-null EBs expressed lower levels of the transcription factor Tfeb, a master transcriptional regulator of lysosomes, resulting in diminished endolysosomal function and a defective endoderm. These results collectively uncovered a mechanism by which LKB1 through AMPK acts as a novel regulator of cell fate determination during differentiation.

In parallel efforts to explore LKB1-dependent functions of AMPK and its related AMPKRs, we discovered that detachment of cells from their substratum is a universal promoter of LKB1-driven AMPKR activation and downstream substrate phosphorylation. Using an enrichment technique based on antibodies that capture a phosphorylated substrate motif targeted by AMPKRs for phosphorylation, we performed quantitative phosphoproteomic screens on a panel of detached mammalian cells that expressed LKB1, but were null for pairs of AMPKRs. We identified several known substrates of AMPKRs which underwent detachment-induced, LKB1-dependent increases in phosphorylation, thus validating our approach. We additionally identified several novel candidate proteins containing an optimal AMPKR motif which underwent an LKB1-dependent increase in phosphorylation. We validated six of these candidate proteins, including RASAL2, RalGAPα, SIPA1-Like proteins, MISP, BAIAP2L1 and MTSS2 as novel AMPKR substrates. Collectively, these results reveal unexpected effectors of LKB1 function and shed light on specific roles these AMPKRs play in cells.