The expression of Cul1(1-252), a dominant negative Cul1 mutant that binds Skp1 and F-box proteins, but cannot recruit an E2 ubiquitin conjugating enzyme, results in the accumulation of SCF substrates (Piva et al., 2002
; Yen and Elledge, 2008
). To identify new SCF substrates, we transiently transfected Cul1(1-252) into HeLa cells and analyzed cell extracts for the levels of several regulators of cell proliferation by immunoblotting. The level of DEPTOR was increased compared to mock transfected controls (Fig. S1A
), suggesting that DEPTOR is an SCF substrate. Therefore, we investigated which F-box protein targets DEPTOR to the SCF using a library of F-box protein cDNAs. Screening of the FBXW (F-box proteins with WD40 repeats) family proteins, as well as Cdc20 and Cdh1 (WD40 domain-containing subunits of an SCF-like ubiquitin ligase), revealed that endogenous DEPTOR specifically interacts with βTrCP1 and βTrCP2 (), paralogous F-box proteins that share identical biochemical properties and substrates. (βTrCP will refer to both, unless specified.)
DEPTOR is a serum-dependent substrate of βTrCP
Significantly, the binding of DEPTOR to transiently-expressed βTrCP was dependent on the substrate binding domain, as demonstrated by the inability of a previously-established substrate binding point mutant, βTrCP2(R434A), or a WD40 repeat deletion mutant, βTrCP2(ΔWD40) (Suzuki et al., 2000
; Wu et al., 2003
), to bind endogenous DEPTOR (Fig. S1B
). Serum starvation of HeLa cells induces accumulation of DEPTOR, whereas serum stimulation results in DEPTOR degradation (Peterson et al., 2009
). After confirming these results in T98G cells (Fig. S1C
), we found that serum stimulation induced a significant increase in the binding of DEPTOR to endogenous βTrCP1 ().
Next, we mapped the βTrCP binding motif in human DEPTOR. Using deletion mutants, the binding motif was mapped to a region of DEPTOR between amino acids 241 and 340 (Fig. S2A
). βTrCP binds substrates via phosphorylated residues in conserved degradation motifs (degrons), typically including the consensus sequence DpSGXXpS or similar variants, such as pS/TpSGXXpS (Fig. S2B
). The βTrCP binding region of DEPTOR contains a conserved 286
motif, matching other βTrCP substrate degrons. To investigate whether DEPTOR binds βTrCP via this motif, we generated serine to alanine mutants and tested their binding to endogenous βTrCP1. Single mutations of Ser286, Ser287, and Ser291 to Ala or a triple mutation of Ser286/287/291 to Ala inhibited the interaction between DEPTOR and βTrCP1, although the mutations did not affect DEPTOR binding to endogenous mTOR ().
The DEPTOR degron is controlled by phosphorylation
To confirm the role of phosphorylation in the interaction of DEPTOR with βTrCP, we used immobilized, synthetic peptides containing the candidate degron sequence to test binding to βTrCP1. While a peptide containing phosphorylated Ser286, Ser287, and Ser291 efficiently bound βTrCP1 (but not Fbxw5 or Fbxw9), a corresponding, non-phosphorylated peptide was unable to bind βTrCP1 (). Accordingly, λ-phosphatase treatment of βTrCP1 immunoprecipitates abolished the interaction with DEPTOR (Fig. S2C
). These results, together with the analysis of point mutants (), the crystal structure of the βTrCP1-β-catenin complex (Wu et al., 2003
), and the modeling of the βTrCP1-BimEL degron interaction (the BimEL and DEPTOR degrons are identical, as shown in Figure S2B
) (Dehan et al., 2009
) indicate that phosphorylation of all three serine residues in the DEPTOR degron (Ser286, Ser287, and Ser291) is necessary for - and directly mediates - the interaction with βTrCP.
To further investigate DEPTOR phosphorylation, we used a phospho-specific antibody against the pSpSGYFpS degron motif. This antibody recognized wild type DEPTOR, but not a DEPTOR(S286/287/291A) mutant (). Additionally, DEPTOR point mutants displayed decreasing levels of detection, suggesting that all three serines are phosphorylated and contribute to recognition by this antibody. Significantly, we found that DEPTOR was phosphorylated on its degron in HEK293T cells in response to stimulation with serum, but it was poorly phosphorylated in serum-starved HEK293T cells ().
Several βTrCP substrates, such as β-catenin, Cdc25A, Emi1, Snail, Wee1, and YAP, are phosphorylated on their degrons only after an initial phosphorylation event that either allows binding to or exposure of a previously masked site for a second kinase (Frescas and Pagano, 2008
; Hunter, 2007
). To investigate whether a similar mechanism controls phosphorylation of the DEPTOR degron, we mutated a number of residues flanking the degron. Mutation of Ser279, Ser280, Ser292, Thr295, Ser297, and Ser298 to Ala, singly or in combination, did not inhibit DEPTOR binding to βTrCP1 ( and S2D
). In contrast, mutation of Ser293, Ser299, or both strongly reduced the interaction between βTrCP and DEPTOR, even in serum-stimulated cells ( and Fig. S2D
). Additionally, a DEPTOR phospho-mimic mutant, in which Ser286, Ser287, and Ser291 in the degron are mutated to Asp [DEPTOR(S286/287/291D)], retains the ability to bind βTrCP1 even when Ser293 and Ser299 are mutated to Ala (). The ability of the phospho-mimic degron mutant of DEPTOR to bind βTrCP, together with the phospho-peptide experiment in , demonstrates that Ser293 and Ser299 are dispensable for binding a pre-phosphorylated degron and suggest that phosphorylation of Ser293 and Ser299 may function to prime the phosphorylation of Ser286, Ser287, and Ser291.
We also used a phospho-specific antibody generated against a DEPTOR peptide C-terminal to the degron, with Ser299 phosphorylated. This antibody recognized wild type DEPTOR, but not DEPTOR(S299A) or DEPTOR(S293/299A) ( and data not shown). We found that DEPTOR was phosphorylated on Ser299 in HEK293T cells stimulated with serum, but this site was poorly phosphorylated in serum-starved cells (). Interestingly, of the five serines in DEPTOR that are important for binding to βTrCP, four (Ser286, Ser287, Ser293, and Ser 299) have been previously identified as phosphorylation sites (Peterson et al., 2009
; Villen et al., 2007
). Additionally, a different study also identified these four serines as sites of phosphorylation that are enriched after proteasome inhibition (Gao et al. 2011
To identify the kinase(s) involved in the phosphorylation and degradation of DEPTOR, we performed a candidate search using pharmacologic inhibition. We found that D4476 (a CK1 inhibitor) and PP242 (an mTOR inhibitor) counteracted the destabilizing effect of serum on DEPTOR, whereas GSK3i IX (a GSK3 inhibitor), U0126 (a MEK inhibitor), and API-2 (an Akt inhibitor) had no effect (Fig. S3A-B
). Importantly, D4476 and PP242, but not U0126 and GSK3i IX, inhibited the interaction between DEPTOR and βTrCP and the phosphorylation of the DEPTOR degron (, , and data not shown). In agreement with the involvement of mTOR in DEPTOR degradation, we observed that low doses of rapamycin (an mTORC1 inhibitor) and high doses of wortmannin (a PI3K inhibitor that, at high concentrations, inhibits mTOR) induced DEPTOR stabilization (Fig. S3B and S3C
). We also found that knockdown of mTOR or CK1α (but not CK1δ or CK1ε) resulted in accumulation of DEPTOR (). Furthermore, silencing of either RAPTOR or RICTOR inhibited the serum-dependent destabilization of DEPTOR, although to a lesser extent than mTOR depletion (), indicating that both mTORC1 and mTORC2 control DEPTOR turnover.
mTOR and CK1α are required for DEPTOR degradation
We then used phospho-mimic mutants of DEPTOR to study the hierarchy of mTOR- and CK1α-mediated phosphorylation of DEPTOR. The binding of wild type DEPTOR to endogenous βTrCP is inhibited by either PP242 or D4476 ( and ), but DEPTOR phosho-mimic mutants are differentially responsive to these inhibitors. The binding of DEPTOR (S286/287/291D) to βTrCP is not inhibited by either D4476 or PP242 (). In contrast, the binding of DEPTOR (S293/299D) to βTrCP is not inhibited by PP242, but is still inhibited by D4476 (). These findings suggest that mTOR phosphorylates Ser293 and Ser299 to promote degron phosphorylation by CK1α. Accordingly, while PP242 inhibited the phosphorylation of DEPTOR on both Ser299 and the degron, D4476 was able to inhibit the phosphorylation of degron, but not of Ser299 (). Finally, CK1α-mediated stimulation of the DEPTOR-βTrCP interaction was inhibited by PP242 (Fig. S3D
To test whether CK1 and mTOR can phosphorylate DEPTOR on its degron, we performed in vitro
kinase assays using recombinant, bacterially-expressed, purified DEPTOR and kinases. CK1 phosphorylated the degron of DEPTOR, as shown by western blotting with the phospho-specific antibody (Fig. S3E-F
). In contrast, mTOR alone was unable to induce phosphorylation of DEPTOR on Ser286, Ser287, and Ser291. Importantly, incubation with mTOR enhanced the CK1-dependent phosphorylation of DEPTOR, likely due to mTOR-dependent phosphorylation of Ser293 and Ser299, since no mTOR-dependent enhancement of phosphorylation was observed with DEPTOR(S293/299A). Finally, mTOR, but not CK1, was able to phosphorylate DEPTOR on Ser299 in vitro
(). Accordingly, in vivo
phosphorylation of Ser293 and Ser299 is inhibited by torin (a highly specific mTOR inhibitor; Peterson et al., 2009
), mTOR phosphorylates Ser293 and Ser299 in vitro,
and pre-phosphorylation of DEPTOR by mTOR enhances its CK1-dependent in vitro
phosphorylation (Gao et al., 2011
Finally, we reconstituted the ubiquitylation of DEPTOR in vitro
. Wild type DEPTOR, but not DEPTOR(S286/287/291A) or DEPTOR(S293/299A), was ubiquitylated only when both βTrCP1 and CK1 were present in the reaction ( and Fig. S3G
). Moreover, in agreement with the phosphorylation data, mTOR enhanced the βTrCP1- and CK1-dependent ubiquitylation of DEPTOR.
The above results indicate that mTOR promotes phosphorylation of the DEPTOR degron by CK1α, so we further investigated potential molecular mechanisms. Fig. S3D
shows that CK1α and DEPTOR bind and that treatment of HEK293T cells with PP242 inhibits this binding. Additionally, purified, recombinant mTOR strongly stimulates the in vitro
binding of CK1α to wild type DEPTOR, but not to DEPTOR(S293/299A) (), suggesting that phoshorylation of Ser293 and Ser299 in DEPTOR by mTOR generates a binding site for CK1α, promoting DEPTOR phosphorylation by CK1α.
Altogether, these data strongly support a model in which, in response to mitogenic stimulation, mTOR phosphorylates DEPTOR on Ser293 and Ser299, promoting the CK1α-mediated phosphorylation of DEPTOR on Ser286, Ser287, and Ser291, facilitating βTrCP binding, SCFβTrCP
-mediated ubiquitylation, and consequent degradation. Therefore, inhibition of βTrCP-mediated degradation of DEPTOR should lead to increased DEPTOR levels and decreased mTOR activity. This hypothesis was tested in three ways. First, T98G cells were transfected with siRNAs against LacZ or βTrCP and were synchronized in G0/G1 by serum starvation (). Following re-stimulation with serum, DEPTOR levels rapidly decreased in the siLacZ transfected cells, but they decreased much less in the siβTrCP cells. In accordance with the increased DEPTOR levels in the βTrCP knockdown samples, the induction of phosphorylated S6K1 (Thr389) was severely blunted, demonstrating a decrease in mTOR activation. Second, to confirm that the observed effect of βTrCP knockdown on mTOR activity was mediated through increased DEPTOR levels, we transiently transfected either wild type DEPTOR or DEPTOR(S286/287/291A) into HeLa cells, which were subsequently serum starved for 24 hours, before re-stimulation with serum. As predicted, in contrast to wild type DEPTOR, DEPTOR(S286/287/291A) was not degraded when cells were exposed to serum, and the mTOR-mediated phosphorylation of S6K1 in response to serum was strongly inhibited (Fig. S4A
). Virtually identical results were obtained using retroviruses that expresses DEPTOR (S286/287/291A) at near physiological levels in T98G cells (). Significantly, after serum stimulation, cells expressing DEPTOR(S286/287/291A) displayed reduced cell size relative to cells expressing wild-type DEPTOR or containing an empty virus ( and Fig. S4B
Failure to degrade DEPTOR results in mTOR activation defects