mTORC2 is a multimeric kinase composed of the mammalian target of rapamycin kinase (mTOR), mLST8, mSin1, and rictor. The complex is insensitive to acute rapamycin exposure and has shown functions in controlling cell growth and actin cytoskeletal assembly. mTORC2 has recently been shown to phosphorylate and activate Akt. Because f70% of gliomas harbor high levels of activated Akt, we investigated whether mTORC2 activity was elevated in gliomas. In this study, we found that mTORC2 activity was elevated in glioma cell lines as well as in primary tumor cells as compared with normal brain tissue (P < 0.05). Moreover, we found that rictor protein and mRNA levels were also elevated and correlated with increased mTORC2 activity. Overexpression of rictor in cell lines led to increased mTORC2 assembly and activity. These lines exhibited increased anchorage-independent growth in soft agar, increased S-phase cell cycle distribution, increased motility, and elevated integrin B 1 and B 3 expression. In contrast, small interfering RNAmediated knockdown of rictor inhibited these oncogenic activities. Protein kinase CA (PKCA) activity was shown to be elevated in rictor-overexpressing lines but reduced in rictor-knockdown clones, consistent with the known regulation of actin organization by mTORC2 via PKCA. Xenograft studies using these cell lines also supported a role for increased mTORC2 activity in tumorigenesis and enhanced tumor growth. In summary, these data suggest that mTORC2 is hyperactivated in gliomas and functions in promoting tumor cell proliferation and invasive potential due to increased complex formation as a result of the overexpression of rictor. [Cancer Res 2007;67(24):11712-20]

The translation of the cyclin D1 and c-myc mRNAs occurs via internal ribosome entry site (IRES)-mediated initiation under conditions of reduced eIF-4F complex formation and Akt activity. Here we identify hnRNP A1 as an IRES trans-acting factor that regulates cyclin D1 and c-myc IRES activity, depending on the Akt status of the cell. hnRNP A1 binds both IRESs in vitro and in intact cells and enhances in vitro IRES-dependent reporter expression. Akt regulates this IRES activity by inducing phosphorylation of hnRNP A1 on serine 199. Serine 199-phosphorylated hnRNP A1 binds to the IRESs normally but is unable to support IRES activity in vitro. Reducing expression levels of hnRNP A1 or overexpressing a dominant negative version of the protein markedly inhibits rapamycin-stimulated IRES activity in cells and correlated with redistribution of cyclin D1 and c-myc transcripts from heavy polysomes to monosomes. Importantly, knockdown of hnRNP A1 also renders quiescent Aktcontaining cells sensitive to rapamycin-induced G 1 arrest. These results support a role for hnRNP A1 in mediating rapamycin-induced alterations of cyclin D1 and c-myc IRES activity in an Akt-dependent manner and provide the first direct link between Akt and the regulation of IRES activity.A majority of eukaryotic mRNAs contain 5Ј-UTRs 2 that are relatively unstructured and typically less than 100 nucleotides in length, which allows for efficient cap-dependent translation initiation. However, the leaders of some cellular mRNAs are relatively long and highly structured and can contain multiple upstream AUG or CUG codons such that scanning ribosomes are unlikely to efficiently initiate translation. In a number of these mRNAs, translation initiation is mediated by cap-independent mechanisms via an internal ribosome entry site (1). IRES-mediated translation initiation can occur during a variety of physiological conditions and has been reported to promote initiation for several mRNAs during cell cycle progression, differentiation, and apoptosis and during stress responses (2-6). IRESs are thought to directly recruit the ribosome to within close proximity to the start codon, thus bypassing the need for cap binding and ribosome scanning (7). Our previous data have demonstrated that both the cyclin D1 and c-myc mRNAs contain IRESs whose function is markedly enhanced following the inhibition of cap-dependent initiation by rapamycin in a manner dependent on Akt activity (8). In cells containing quiescent Akt, the IRESs of the cyclin D1 and c-myc mRNAs are constitutively active and are stimulated following rapamycin treatment; however, in cells containing active Akt cyclin D1 and c-myc, IRES activity is repressed and is not induced following rapamycin exposure.Several proteins that regulate IRES activity, collectively termed IRES trans-acting factors (ITAFs), have been described (7). These ITAFs function by associating with the IRES and either facilitate direct ribosome binding with the mRNA or alter the structure of the IRES. For instance, the ITAFs PTB, Unr, and h...

The differential expression of the critical cell cycle control proteins cyclin D1 and c-myc has been shown to result in Akt-dependent hypersensitivity of tumor cells to mTOR inhibitors. We have previously demonstrated that the differential utilization of internal ribosome entry sites within the mRNAs of these transcripts allows maintenance of protein synthesis in the face of rapamycin (rapa) exposure in an Akt-dependent manner. Here, we demonstrate that in addition to this mechanism, cyclin D1 and c-myc mRNA stability is also coordinately regulated following rapa treatment depending on Akt activity status. We identify A/U-rich response elements within the 3 0 untranslated regions (UTRs) of these transcripts, which confer the observed differential stabilities and show that the RNA-binding protein, tristetraprolin (TTP), interacts with these elements. We also present evidence that TTP accumulates in response to rapa exposure, binds to the cis-acting elements within the cyclin D1 and c-myc 3 0 UTRs and is differentially serine phosphorylated in an Akt-dependent manner. Furthermore, the differential phosphorylation status of TTP results in its sequestration by 14-3-3 proteins in quiescent Akt-containing cells. Finally, siRNA-mediated knockdown of TTP expression or inhibiting a known regulator of TTP phosphorylation, p38 MAP kinase, abolishes the effects on cyclin D1 and c-myc mRNA stability. We assume that the differential control of cyclin D1 and c-myc mRNA stability and translational efficiency constitutes a coordinate response to rapa contributing to the maintenance of expression of these determinants in rapa-resistant quiescent Aktcontaining cells following exposure.

One mechanism by which AKT kinase-dependent hypersensitivity to mammalian target of rapamycin (mTOR) inhibitors is controlled is by the differential expression of cyclin D1 and c-MYC. Regulation of post-transcriptional processes has been demonstrated to be crucial in governing expression of these determinants in response to rapamycin. Our previous data suggested that cyclin D1 and c-MYC expression might additionally be coordinately regulated in an AKT-dependent manner at the level of transcription. Under conditions of relatively quiescent AKT activity, treatment of cells with rapamycin resulted in upregulation of cyclin D1 and c-MYC nascent transcription, while in cells containing active AKT, exposure repressed transcription. Promoter analysis identified AKT-dependent rapamycin responsive elements containing AP-1 transactivation sites. Phosphorylated c-JUN binding to these promoters correlated with activation of transcription while JUNB occupancy was associated with promoter repression. Forced overexpression of JunB or a conditionally active JunB-ER allele repressed cyclin D1 and c-MYC promoter activity in quiescent AKT-containing cells following rapamycin exposure. AIP4/Itch-dependent JUNB protein degradation was found to be markedly reduced in active AKT-containing cells compared to cells harboring quiescent AKT. Moreover, silencing AIP4/Itch expression or inhibiting JNK mediated AIP4 activity abrogated the rapamycin-induced effects on cyclin D1 and c-MYC promoter 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.

mTORC2 is a multiprotein kinase composed of mTOR, mLST8, PRR5, mSIN1 and Rictor. The complex is insensitive to rapamycin and has demonstrated functions controlling cell growth, motility, invasion and cytoskeletal assembly. mTORC2 is the major hydrophobic domain kinase which renders Akt fully active via phosphorylation on serine 473. We isolated Hsp70 as a putative Rictor interacting protein in a yeast two-hybrid assay and confirmed this interaction via co-immunoprecipitation and colocalization experiments. In cells expressing an antisense RNA targeting Hsp70, mTORC2 formation and activity were impaired. Moreover, in cells lacking Hsp70 expression, mTORC2 activity was inhibited following heat shock while controls demonstrated increased mTORC2 activity. These differential effects on mTORC2 activity were specific, in that mTORC1 did not demonstrate Hsp70-dependent alterations under these conditions. These data suggest that Hsp70 is a component of mTORC2 and is required for proper assembly and activity of the kinase both constitutively and following heat shock.