Studies on the regulatory mechanism of the ULK1 complex in the induction of autophagy

Abstract

Autophagy, an evolutionarily-conserved cellular process through which organelles and macromolecules are degraded in the lysosome, is induced under nutrient starvation or other unfavorable growth conditions. Unc51-like kinase 1 (ULK1) is a serine/threonine protein kinase that plays a key role in the autophagy induction process, but how ULK1 is regulated by cellular signals for induction of autophagy and how ULK1 regulates the downstream processes in autophagy remain poorly understood. ULK1 interacts with Atg13, focal adhesion kinase family interacting protein of 200 kD (FIP200) and Atg101 to form a large protein complex involved in early steps of the autophagy induction process. To better understand the function of the ULK1 complex, my thesis work has sought to identify binding proteins of the complex. Through a yeast two hybrid screen using a human fetal brain cDNA library with Atg13 as bait, a protein named MCF.2 cell line derived transforming sequence-like 2 (MCF2L2) was identified. Through co-immunoprecipitation and in vitro binding assay, MCF2L2 was determined to directly interact with Atg13 via its N-terminal region independently of ULK1. Knockdown of MCF2L2 inhibited the formation of autophagosome and autophagy flux and led to accumulation of p62/sequestosome-1, a protein degraded through autophagy. Knockdown of MCF2L2 also suppressed the aggregation of WD-repeat protein interacting with phosphoinositides-1, an autophagic isolation membrane marker. MCF2L2 contains a putative Rho-guanine nucleotide exchange factor (GEF) domain in the middle and has a sequence similarity to MCF2L and MCF2, the well-known Rho-GEFs. MCF2L2 overexpression induced a moderate increase in the active forms of Rho GTPases and MCF2L2 colocalized with actin related protein 3, the actin nucleation factor that is regulated by Rho GTPases, implying that MCF2L2 potentially contains GEF activity. MCF2L2 knockdown partially suppressed the distribution of Atg9 from trans-golgi network to the cytoplasm in response to starvation, a process that may depend on actin cytoskeleton. Combined, these results suggest that MCF2L2, as a component of the ULK1 complex, might play an important role in mediating signal transduction between the actin cytoskeleton and autophagy induction.