Nuclear run on assays were performed as previously described (18). activities. Our findings support a role for the AKT-dependent regulation of AIP4/Itch activity in mediating the differential cyclin D1 and c-MYC transcriptional responses to rapamycin. Keywords:AKT, mTOR, rapamycin, cyclin D1, c-MYC, JUNB, AIP4, transcriptional regulation == Introduction == Inhibitors targeting the mTOR kinase are in clinical development as potential chemotherapeutic agents. MTOR is a regulatory kinase which integrates signals linking the ability of cells to undergo cell cycle transit to the availability of nutrients (1,2). Treatment with mTOR inhibitors typically results in a starvation response and cell cycle arrest, however cells of hematopoeitic origins undergo apoptotic death following exposure (3). The mTOR kinase is a member of the serine/threonine phosphatidyl inositol 3-like family of kinases and is found in at least two separate complexes (4). The mammalian target of rapamycin complex 1 (mTORC1) is composed of Raptor, mLST8, PRAS40, Deptor and the catalytic subunit mTOR. MTORC2 components include Rictor, mSIN1, Protor, Deptor, as well as mLST8 and mTOR. MTORC1 is sensitive to rapamycin and is known to regulate several cell functions including growth, size, cap-dependent mRNA translation and ribosomal component biogenesis (5). MTORC2 has signaling outputs regulating cell growth and motility and is generally assumed to be insensitive to rapamycin, but the macrolide does seem to inhibit mTORC2 assembly in some cell lines following prolonged exposure (6,7). The signaling effectors of mTORC1 include the p70S6 kinase, the translational repressor 4EBP1 and the recently described activation of SGK (8). MTORC2 is the major kinase responsible for full activation of AKT via phosphorylation of serine 473 (9). Several studies have described the hypersensitivity of different tumor types to mTOR inhibitors in cells whose AKT activity is elevated due to gene amplification or loss of function mutations in the tumor suppressorPTEN(10-12). We have demonstrated that differential sensitivity can be explained, in part, by the differential regulation of cyclin D1 and c-MYC gene expression at the levels of mRNA Mutant IDH1-IN-4 translation initiation and stability (13,14). Continued internal ribosome entry site (IRES)-dependent translation initiation and enhanced mRNA stability of cyclin D1 and c-MYC mRNAs is sufficient to overcome rapamycin-induced G1arrest. Our data however, also suggested coordinate regulation of cyclin D1 and c-MYC transcription in addition to the post-transcriptional control exerted by AKT in the face of mTOR inhibition (12). How AKT activity may regulate the transcriptional responses of cells to mTOR inhibitors is unknown. In the current study, we have extended our previous analysis of AKT-dependent cyclin D1 and c-MYC post-transcriptional regulation to try and understand the mechanisms controlling gene transcription of these determinants following rapamycin exposure. Tumor cells containing active AKT were found to repress transcription Rabbit polyclonal to ACTL8 of cyclin D1 and c-MYC, while in cells with relatively quiescent AKT activity transcription was induced. Subsequent deletion and mutational analysis of cyclin D1 and c-MYC promoter constructs identified rapamycin responsive promoter elements containing AP-1 transcription factor binding sites. JUNB binding to these promoter elements correlated with transcriptional repression of cyclin D1 and c-MYC promoter activity, whereas phosphorylated c-JUN binding strongly activated these promoters Mutant IDH1-IN-4 in an AKT-dependent manner upon rapamycin Mutant IDH1-IN-4 treatment. Moreover, the AKT-dependent regulation of promoter activity correlated with alterations in E3 ubiquitin ligase AIP4/Itch-mediated JUNB ubiquitination. These data support the involvement of differential AIP4/Itch-mediated JUNB degradation in regulating the transcriptional responses of cyclin D1 and c-MYC to mTOR inhibition in a manner dependent on cellular AKT activity. == Materials and methods == == Cell Lines and Transfections == The isogenic cell lines pairs used in this study differ significantly in their relative AKT activities by virtue of either their PTEN status or forced expression of an activated allele of AKT1. These lines were kindly provided by Ingo Mellinghoff and Charles Sawyers and have been described previously (13). The isogenic Pten+/+and Pten/MEF cells were kindly provided by Hong Wu and have also been described (15). Transient luciferase reporter transfections were performed using FUGENE 6 (Roche) as recommended by the manufacturer. To generate the JUNB and JunB-ER expressing lines cells were transfected similarly using FUGENE 6, and clones.