Abstract
The mTOR signaling controls essential biological functions including proliferation, growth, metabolism, autophagy, ageing, and others. Hyperactivation of mTOR signaling leads to a plethora of human disorders; thus, mTOR is an attractive drug target. The discovery of mTOR signaling started from isolation of rapamycin in 1975 and cloning of TOR genes in 1993. In the past 27 years, numerous research groups have contributed significantly to advancing our understanding of mTOR signaling and mTOR biology. Notably, a variety of experimental approaches have been employed in these studies to identify key mTOR pathway members that shape up the mTOR signaling we know today. Technique development drives mTOR research, while canonical biochemical and yeast genetics lay the foundation for mTOR studies. Here in this review, we summarize major experimental approaches used in the past in delineating mTOR signaling, including biochemical immunoprecipitation approaches, genetic approaches, immunofluorescence microscopic approaches, hypothesis-driven studies, protein sequence or motif search driven approaches, and bioinformatic approaches. We hope that revisiting these distinct types of experimental approaches will provide a blueprint for major techniques driving mTOR research. More importantly, we hope that thinking and reasonings behind these experimental designs will inspire future mTOR research as well as studies of other protein kinases beyond mTOR.
Highlights
As one of the most abundant gene families in human, protein kinases tightly control cell signaling and cell function through protein phosphorylation
Substrates showed distinct responses to immunoprecipitated mammalian targets of rapamycin (mTOR)-mediated phosphorylation in vitro washed with detergents—4E-BP1 phosphorylation was significantly reduced upon mTOR precipitates washed with 1% NP40 or 1% CHAPS [27], while S6K phosphorylation was enhanced by precipitated mTOR washed by either 1% NP40 or 1% Triton X-100 [28]; and (2) a 35 KD protein was copurified with mTOR [19]; and (3) a gel filtration chromatography experiment showed that yeast TOR1 or TOR2 migrated at ~2 MD molecular weight far larger than TOR itself [29]
Given that the pentameric GATOR2 complex is required for amino acid-induced mTORC1 activation [51] and Sestrins (Sestrin 1, 2 and 3) were previously identified as a GATOR2 binding partner that suppresses mTORC1 activation with unknown mechanism(s) [53], Wolfson et al examined if Sestrins binding to GATOR2 was regulated by amino acids and found that only leucine, but not arginine, depletion in HEK293T cells led to increased Sestrin 2 binding to GATOR2 [88]
Summary
As one of the most abundant gene families in human, protein kinases tightly control cell signaling and cell function through protein phosphorylation. Identification of TOR Genes by Yeast Genetics and Biochemical Purification Approaches as Rapamycin Effectors Distinct from kinases such as p44MAPK [11], whose discovery was initiated by cloning of the kinase gene, the journey of mTOR signaling starts from isolation of an antifungal compound rapamycin from soil on Easter Island in 1975 by Suren N. In early 2000s, presence of mTOR was observed broadly in other organisms including fission yeast ([23], based on sequence homology search as homologs to budding yeast TOR1 and TOR2), C. elegans ([24], based on BLAST homologous search for PIK proteins), and Drosophila ([25], through a tissue-specific genetic screen for recessive mutations regulating compound eye development or [26] through a Drosophila cDNA library screening coupled RACE (rapid amplification of cDNA ends) analysis)
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