While a peptide containing phosphorylated Ser286, Ser287, and Ser291 efficiently bound TrCP1 (but not Fbxw5 or Fbxw9), a corresponding, non-phosphorylated peptide was unable to bind TrCP1 (Fig. 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. Introduction The mammalian Target Of Rapamycin (mTOR) kinase controls many aspects of the response to growth factor and nutrient signaling (Laplante and Sabatini, 2009; Zoncu et al., 2011). mTOR Complex 1 (TORC1) and TORC2 share the mTOR and mLST8/GL proteins, but the complexes also feature distinct subunits, with RAPTOR and PRAS40 in TORC1 and RICTOR, PROTOR, and mSin1 in TORC2 (Laplante and Sabatini, 2009). In general, TORC1 controls mRNA translation, ribosome biogenesis, cell growth, and autophagy Nedd4l through substrates such as S6K1 and 4E-BP1, while TORC2 controls cell proliferation, cell survival, and the cytoskeleton through substrates such as Akt, SGK1, and PGC (Dancey, 2010; Guertin and Sabatini, 2007; Sengupta et al. 2010) The pathways controlled by TORC1 and TORC2 are frequently activated in tumors by mutations in upstream signaling factors (growth factor receptors, PI3K regulators, or PTEN) and mTOR inhibitors have been used successfully in the treatment of several cancers (Dancey, 2010; Hay, 2005). However, direct activating mutations of mTOR have not been observed in cancer, and in some settings, mTOR has been shown to possess tumor suppressive properties, likely due to unfavorable feedback loops that control the TORC1 or TORC2 pathways (Laplante and Sabatini, 2009). Both mTOR complexes are directly inhibited by DEPTOR, which binds and inhibits mTOR through a PDZ domain name (Peterson et al., 2009). DEPTOR is usually downregulated in many tumors, suggesting a tumor suppressor function, which is usually consistent with the activation Bupropion of mTOR in many tumors. However, DEPTOR is usually overexpressed in multiple myeloma via transcription or copy number amplifications, and this overexpression is necessary for Akt activation and cell survival, which is likely mediated through the feedback inhibition of PI3K (Carrasco et al., 2006; Peterson et al., 2009). Notably, despite a general downregulation of DEPTOR across other tumor types, amplification of the genomic region made up of the DEPTOR locus is an indicator of poor prognosis or tumor progression in tumor subsets from multiple cancers, including breast malignancy, prostate cancer, lung cancer, and CML, and DEPTOR is usually overexpressed in many of these tumors (Chin et al., 2007; Joos et al., 2003; van Duin et al., 2005a; van Duin et al., 2005b). The impact of DEPTOR in cancer makes it vital to understand the regulation of DEPTOR. Bupropion DEPTOR activity appears to be regulated largely through the control of DEPTOR levels, which are tightly controlled both transcriptionally and posttranslationally in response to growth factor signaling (Peterson et al., 2009). While DEPTOR levels are high in the absence of serum, in response to serum, transcription of decreases and DEPTOR protein is usually rapidly phosphorylated on as many as 13 sites. Many of these phosphorylations are mTOR-dependent, and non-phosphorylated mutants of DEPTOR bind mTOR more efficiently than wild type DEPTOR, indicating that phosphorylation of DEPTOR inhibits binding to mTOR. mTOR activity also correlates with DEPTOR degradation, suggesting that these two processes are linked. However, the precise mechanisms for this regulation remain unclear. Skp1/Cul1/F-box protein (SCF) ubiquitin ligase complexes control the degradation of many important regulatory proteins (Cardozo and Pagano, 2004). In mammals, there are 69 SCF ligases, each characterized by a different F-box substrate targeting subunit (Jin et al., 2004). In this study, we identify SCFTrCP as the ubiquitin ligase for DEPTOR and demonstrate that SCFTrCP mediates the mTOR- and CK1-dependent degradation of DEPTOR. Results The expression of Cul1(1-252), a dominant unfavorable Cul1 mutant Bupropion that binds Skp1 and F-box proteins, but cannot recruit an E2 ubiquitin conjugating enzyme, results in the accumulation of SCF substrates (Piva.