Investigation of mTOR-independent regulation of macroautophagy

Richard Ian Odle

Macroautophagy is a critical catabolic response to cellular stress, enabling lysosomal mediated breakdown of cytosolic cargo. The nutrient-responsive mTORC1 kinase complex has been described as a master regulator of cellular metabolism. Indeed, mTORC1 inhibits autophagy via repressive phosphorylation of the key autophagy regulators ATG13, ULK1, ATG14 and TFEB. Consequently, mTORC1 has become a candidate therapeutic target in neurodegeneration and cancer; however, its essential role in other cellular programs has prompted the investigation of mTORC1-independent regulation of autophagy. This thesis explores the role of CMGC kinase family members ERK1/2 and CCNB1-CDK1 in the regulation of autophagy.

The ERK1/2 signalling cascade is activated in a high proportion of cancers. ERK2 has been proposed as a regulatory kinase of TFEB; however, we found little evidence to suggest that ERK1/2 was a direct kinase responsible for TFEB phosphorylation, including at the putative site S142. Furthermore, whilst we observed that hyperactivation of the ERK1/2 pathway did lead to increases in total TFEB protein levels in HEK293, this appeared to be a cell line specific finding. We therefore concluded ERK1/2 was not likely to be a critical regulator of TFEB.

It has been proposed that autophagy must be repressed during mitosis, otherwise nuclear envelope breakdown will expose the genome to the cytosolic autophagy machinery. Here we show that autophagy initiation, as measured by markers of the omegasome, is indeed repressed throughout mitosis. Furthermore, autophagy regulators undergo mitotic hyperphosphorylation, including at known repressive sites, in a manner dependent on CDK1 but not mTORC1. Indeed, we find mTORC1 is likely inactive as a result of CDK1-dependent hyperphosphorylation of RAPTOR. Thus, we conclude that mTORC1 is substituted by CDK1, as the master repressor of autophagy during mitosis. These results suggest that autophagy regulation is uncoupled from nutrient status during nuclear envelope breakdown as a mechanism to prevent genomic instability, a hallmark of cancer.