mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy

Abstract

Growth factors and nutrients enhance protein synthesis and suppress overall protein degradation by activating the protein kinase mammalian target of rapamycin (mTOR). Conversely, nutrient or serum deprivation inhibits mTOR and stimulates protein breakdown by inducing autophagy, which provides the starved cells with amino acids for protein synthesis and energy production. However, it is unclear whether proteolysis by the ubiquitin proteasome system (UPS), which catalyzes most protein degradation in mammalian cells, also increases when mTOR activity decreases. Here we show that inhibiting mTOR with rapamycin or Torin1 rapidly increases the degradation of long-lived cell proteins, but not short-lived ones, by stimulating proteolysis by proteasomes, in addition to autophagy. This enhanced proteasomal degradation required protein ubiquitination, and within 30 min after mTOR inhibition, the cellular content of K48-linked ubiquitinated proteins increased without any change in proteasome content or activity. This rapid increase in UPS-mediated proteolysis continued for many hours and resulted primarily from inhibition of mTORC1 (not mTORC2), but did not require new protein synthesis or key mTOR targets: S6Ks, 4E-BPs, or Ulks. These findings do not support the recent report that mTORC1 inhibition reduces proteolysis by suppressing proteasome expression [Zhang Y, et al. (2014) Nature 513(7518):440-443]. Several growth-related proteins were identified that were ubiquitinated and degraded more rapidly after mTOR inhibition, including HMG-CoA synthase, whose enhanced degradation probably limits cholesterol biosynthesis upon insulin deficiency. Thus, mTOR inhibition coordinately activates the UPS and autophagy, which provide essential amino acids and, together with the enhanced ubiquitination of anabolic proteins, help slow growth. mTOR | proteasome | ubiquitination | autophagy

Coordinate regulation of autophagy and the ubiquitin proteasome system by MTOR

“…When degradation of short-lived proteins was measured in a "pulse-chase protocol" involving a 20 min pulse of 3 H-phenyalanine, this process was not affected by the inhibitors of MTOR, rapamycin or Torin 1. 6 In contrast, in all cell lines tested (e.g., HEK293, C2C12, and MEFs), the breakdown of the long-lived proteins (which were selectively labeled for 20 h with 3 H-phenyalanine and then chased for 2 h), increased rapidly upon MTOR inhibition. 6 Using selective inhibitors of proteasomes (i.e., bortezomib) or lysosomal acidification (i.e., concanamycin A) to measure substrate flux through each proteolytic pathway, we found that MTOR inhibition rapidly enhances the degradation of long-lived cell proteins by both proteasomes and lysosomes.…”

“…6 In contrast, in all cell lines tested (e.g., HEK293, C2C12, and MEFs), the breakdown of the long-lived proteins (which were selectively labeled for 20 h with 3 H-phenyalanine and then chased for 2 h), increased rapidly upon MTOR inhibition. 6 Using selective inhibitors of proteasomes (i.e., bortezomib) or lysosomal acidification (i.e., concanamycin A) to measure substrate flux through each proteolytic pathway, we found that MTOR inhibition rapidly enhances the degradation of long-lived cell proteins by both proteasomes and lysosomes. 6 Accordingly, in MEF cells lacking the key autophagy gene Atg5, proteolysis by lysosomes does not increase upon MTOR inhibition, but proteasomal degradation does.…”

“…Because this generation of amino acids is important for cell survival in nutrient-poor environments, there has been extensive interest in this rapid activation of autophagy. However, in mammalian cells under normal conditions more than 2 thirds of the protein breakdown occurs by the UPS, [4][5][6] which thus continually provides a large fraction of the amino acids in intracellular pools. Therefore, we tested the possibility that overall proteolysis by the UPS also increases when MTOR is inhibited in order to provide the cells with an additional supply of amino acids.…”

“…Therefore, we tested the possibility that overall proteolysis by the UPS also increases when MTOR is inhibited in order to provide the cells with an additional supply of amino acids. 6 The UPS not only degrades newly synthesized short-lived cell proteins, which are mainly regulatory or misfolded proteins, but also catalyzes the slower breakdown of long-lived cell proteins, which comprise the great majority of cell constituents. 6 These general classes of proteins can be differentially labeled by exposing cells to pulses of radioactive amino acids of different durations.…”

“…6 The UPS not only degrades newly synthesized short-lived cell proteins, which are mainly regulatory or misfolded proteins, but also catalyzes the slower breakdown of long-lived cell proteins, which comprise the great majority of cell constituents. 6 These general classes of proteins can be differentially labeled by exposing cells to pulses of radioactive amino acids of different durations. When degradation of short-lived proteins was measured in a "pulse-chase protocol" involving a 20 min pulse of 3 H-phenyalanine, this process was not affected by the inhibitors of MTOR, rapamycin or Torin 1.…”