Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The VSports app下载. gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure V体育官网. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. .

. 2011 Oct 21;44(2):317-24.
doi: 10.1016/j.molcel.2011.09.005.

mTOR generates an auto-amplification loop by triggering the βTrCP- and CK1α-dependent degradation of DEPTOR

Shanshan Duan  1 , Jeffrey R Skaar, Shafi Kuchay, Alfredo Toschi, Naama Kanarek, Yinon Ben-Neriah, Michele Pagano
Affiliations

mTOR generates an auto-amplification loop by triggering the βTrCP- and CK1α-dependent degradation of DEPTOR

Shanshan Duan et al. Mol Cell. .

Abstract

DEPTOR is a recently identified inhibitor of the mTOR kinase that is highly regulated at the posttranslational level. In response to mitogens, we found that DEPTOR was rapidly phosphorylated on three serines in a conserved degron, facilitating binding and ubiquitylation by the F box protein βTrCP, with consequent proteasomal degradation of DEPTOR VSports手机版. Phosphorylation of the βTrCP degron in DEPTOR is executed by CK1α after a priming phosphorylation event mediated by either the mTORC1 or mTORC2 complexes. Blocking the βTrCP-dependent degradation of DEPTOR via βTrCP knockdown or expression of a stable DEPTOR mutant that is unable to bind βTrCP results in mTOR inhibition. Our findings reveal that mTOR cooperates with CK1α and βTrCP to generate an auto-amplification loop to promote its own full activation. Moreover, our results suggest that pharmacologic inhibition of CK1 may be a viable therapeutic option for the treatment of cancers characterized by activation of mTOR-signaling pathways. .

PubMed Disclaimer

Figures

Figure 1
Figure 1. DEPTOR is a serum-dependent substrate of βTrCP
A) DEPTOR binds βTrCP1 and βTrCP2. HEK293T cells were transfected with the indicated FLAG-tagged proteins. Forty-eight hours post-transfection, after a 16-hour serum starvation, cells were re-stimulated with media containing serum and MG132 for three hours, prior to harvesting for immunoprecipitations and immunoblotting as indicated. Asterisks indicate the position of exogenously expressed proteins. B) HEK293T cells were transfected with an empty vector (EV) or FLAG-tagged DEPTOR. Forty-eight hours post-transfection, after a 24-hour serum starvation, cells were pretreated with the indicated drugs for two hours and then stimulated with serum-containing media (SR) for three hours, prior to harvesting for immunoprecipitations and immunoblotting as indicated. (WCL, whole cell lysate). C) During a 72-hour serum starvation, T98G cells were transfected with siRNAs targeting either LacZ or βTrCP1 and βTrCP2. Cells were subsequently re-stimulated with media containing serum and cycloheximide (SR+CHX), and samples were harvested at the indicated time points for immunoblotting.
Figure 2
Figure 2. The DEPTOR degron is controlled by phosphorylation
A) Ser286, Ser287, and Ser291 are required for the interaction of DEPTOR with βTrCP. HEK293T cells were transfected with an empty vector (EV) or the indicated HA-DEPTOR constructs. Forty-eight hours post-transfection, after a 24-hour serum starvation, cells were re-stimulated with media containing serum and MG132 for three hours, prior to harvesting for HA immunoprecipitation and western blotting as indicated. (WCL, whole cell lysate). B) The DEPTOR degron requires phosphorylation to bind βTrCP1. HEK293T lysates were used in binding reactions with beads coupled to a peptide containing the sequence SSGYFS (lane 2) or the phospho-motif SpSpSGYFpS (lane 3). Beads were washed with lysis buffer, and bound proteins were eluted and subjected to SDS-PAGE and immunoblotting. C) In vivo phosphorylation of DEPTOR on Ser286/287/291/299 is induced by mitogens. HEK293T cells were transfected with the indicated HA-tagged DEPTOR constructs. Following serum deprivation (SD) for 24 hours, cells were stimulated with serum (SR) for three hours, in the presence or absence of PP242 or D4476, as indicated. Whole cell lysates (WCL) were immunoprecipitated and immunoblotted as indicated. D) Ser293 and Ser299 are required for the interaction of DEPTOR with βTrCP. HEK293T cells were transfected with FLAG-βTrCP1 and either an empty vector (EV) or the indicated HA-DEPTOR constructs. Forty-eight hours post-transfection, after a 24-hour serum starvation, cells were re-stimulated with media containing serum and MG132 for three hours, prior to harvesting for immunoprecipitations and immunoblotting as indicated. (WCL, whole cell lysate).
Figure 3
Figure 3. mTOR and CK1α are required for DEPTOR degradation
A) Inhibition of TORC1 and TORC2 blocks DEPTOR degradation. During a 72-hour serum starvation, T98G cells were transfected with siRNAs targeting LacZ, RAPTOR, RICTOR, or mTOR. Cells were subsequently re-stimulated with media containing serum (SR), and samples were harvested at the indicated time points for immunoblotting. B) Silencing of CK1α, but not CK1δ or CK1ε, induces DEPTOR accumulation. HeLa cells were infected with an empty lentivirus (EV) or lentiviruses containing shRNA targeting either CK1α, CK1δ, or CK1ε. Seven days post-infection, samples were harvested at the indicated time points for immunoblotting. C) Differential sensitivity of wild type DEPTOR and DEPTOR mutants to inhibition of CK1 and mTOR. HEK293T cells were transfected with the indicated HA-DEPTOR constructs. Forty-eight hours post-transfection, after a 24-hour serum starvation, cells were re-stimulated with media containing serum and MG132 for three hours, in the presence or absence of PP242 or D4476, as indicated. Cell lysates were immunoprecipitated and immunoblotted as indicated. (WCL, whole cell lysate). D) In vitro ubiquitylation assays of recombinant DEPTOR [WT or DEPTOR(S287/287/291A),] were conducted in the presence of the indicated proteins. Samples were incubated at 30°C for 90 minutes. The bracket on the left side of the top panel marks a ladder of bands corresponding to polyubiquitylated DEPTOR. E) In vitro phosphorylation of DEPTOR by mTOR promotes DEPTOR-CK1α interaction. Recombinant, purified GST-DEPTOR, GST-DEPTOR(S293/299A), or GST alone were incubated with ATP in the presence or absence of purified, recombinant mTOR. Reaction products were then diluted, incubated with FLAG-tagged CK1α, purified with GST sepharose 4B beads, and immunobloted as indicated.
Figure 4
Figure 4. Failure to degrade DEPTOR results in mTOR activation defects
A) During a 72-hour serum starvation, T98G cells were transfected with siRNAs targeting either LacZ or βTrCP1 and βTrCP2. Cells were subsequently re-stimulated with media containing serum (SR), and samples were harvested at the indicated time points for immunoblotting as indicated. B) T98G cells were infected with either an empty virus (EV), a retrovirus expressing wild type DEPTOR, or DEPTOR(S286/287/291A). After 72 hours of serum deprivation, cells were re-stimulated with serum (SR) for the indicated times, harvested, and analyzed by immunoblotting. C) The experiment was performed as in (B), and cell size was determined by FACS (forward scatter) in cells deprived of serum (SD) and 24 hours after serum addition (SR).
See this image and copyright information in PMC

Comment in

References (VSports app下载)

    1. Cardozo T, Pagano M. The SCF ubiquitin ligase: insights into a molecular machine. Nat Rev Mol Cell Biol. 2004;5:739–751. - PubMed
    1. Carrasco DR, Tonon G, Huang Y, Zhang Y, Sinha R, Feng B, Stewart JP, Zhan F, Khatry D, Protopopova M, et al. High-resolution genomic profiles define distinct clinico-pathogenetic subgroups of multiple myeloma patients. Cancer Cell. 2006;9:313–325. - PubMed
    1. Chin SF, Wang Y, Thorne NP, Teschendorff AE, Pinder SE, Vias M, Naderi A, Roberts I, Barbosa-Morais NL, Garcia MJ, et al. Using array-comparative genomic hybridization to define molecular portraits of primary breast cancers. Oncogene. 2007;26:1959–1970. - PubMed
    1. Dancey J. mTOR signaling and drug development in cancer. Nat Rev Clin Oncol. 2010;7:209–219. - PubMed
    1. Dehan E, Bassermann F, Guardavaccaro D, Vasiliver-Shamis G, Cohen M, Lowes KN, Dustin M, Huang DC, Taunton J, Pagano M. betaTrCP- and Rsk1/2-mediated degradation of BimEL inhibits apoptosis. Mol Cell. 2009;33:109–116. - VSports - PMC - PubMed

Publication types

VSports - MeSH terms

"VSports手机版" Substances