The cross talk of two family members of β-TrCP in the regulation of cell autophagy and growth

Abstract

β-transducin repeat-containing protein (β-TrCP), one of the best-characterized substrate recognition components of the SKP1-CUL1-F-box (SCF) E3 ligase, has two distinct paralogs, β-TrCP1 and β-TrCP2, expressed in mammals. Through governing the ubiquitination and degradation of numerous key regulators, β-TrCP1/2 regulates various cellular physiological and pathological processes. However, whether and how these two proteins cross talk and whether they regulate cell autophagy and proliferation in different manners is completely unknown. Herein, we report that β-TrCP1 and β-TrCP2 are the physiological substrates of SCF E3 ligase and target each other for degradation that is dependent on their β-TrCP degron sequences. Furthermore, glucose deprivation activates AMPK kinase to phosphorylate β-TrCP1 and promotes the subsequent ubiquitination and degradation of β-TrCP1 by β-TrCP2, but does not promote β-TrCP2 degradation by β-TrCP1. Finally, we found that β-TrCP2, not β-TrCP1, preferentially degrades DEPTOR and REDD1, the inhibitors of mTORC1, to activate mTORC1, leading to autophagy inhibition and cell growth. Thus, our study demonstrates that β-TrCP1 and β-TrCP2 mutually target each other for degradation and that β-TrCP2 acts as a dominant paralog in the regulation of cell autophagy and growth, which might be a promising anticancer target.

DEPTOR is a direct p53 target that suppresses cell growth and chemosensitivity

“…Cells were harvested, lysed, and subjected to immunoblotting, as previously described 32 . About 30–60 μg of protein were loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis gels, and the bands with appropriate exposure time are presented.…”

Inactivation of ribosomal protein S27-like impairs DNA interstrand cross-link repair by destabilization of FANCD2 and FANCI

“…Cells were harvested following various treatments, lysed in lysis buffer with phosphatase inhibitors (Roche), and subjected to western blotting analysis and immunoprecipitation as described in ref. 43 , using various antibodies as follows: FANCD2 (Santa Cruz, sc-20022), FANCI (Bethyl, A300-212A), FANCI (Proteintech, 20789-1-AP), FANCL polyclonal rabbit antibody (gift from Dr. Weidong Wang), FLAG (Sigma-Aldrich, F1804), β-actin (Sigma-Aldrich, A5441), p62 (MBL Life science, PM045), p21 (Cell Signaling Technology, 2947S), and Beclin 1 (Cell Signaling Technology, 4122S). RPS27L and RPS27 polyclonal rabbit antibodies were raised and purified as described in ref.…”

Neddylation modification of ribosomal protein RPS27L or RPS27 by MDM2 or NEDP1 regulates cancer cell survival

“…Cells were harvested, lysed, and subjected to direct immunoblotting (IB) as previously described. 13 Briefly, cells were lysed in lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM NaF, 1 mM Na 3 VO 4 , 1 mM DTT] with protease inhibitors (11873580001; Roche) and phosphatase inhibitors (4906837001; Roche). After incubation on ice for 30 minutes, the supernatants were harvested by centrifugation at 14 000 rpm for 25 minutes at 4°C.…”