Predominant Nuclear Localization of Mammalian Target of Rapamycin in Normal and Malignant Cells in Culture

Abstract

Mammalian target of rapamycin (mTOR) controls initiation of translation through regulation of ribosomal p70S6 kinase (S6K1) and eukaryotic translation initiation factor-4E (eIF4E) binding protein (4E-BP). mTOR is considered to be located predominantly in cytosolic or membrane fractions and may shuttle between the cytoplasm and nucleus. In most previous studies a single cell line, E1A-immortalized human embryonic kidney cells (HEK293), has been used. Here we show that in human malignant cell lines, human fibroblasts, and murine myoblasts mTOR is predominantly nuclear. In contrast, mTOR is largely excluded from the nucleus in HEK293 cells. Hybrids between HEK293 and Rh30 rhabdomyosarcoma cells generated cells co-expressing markers unique to HEK293 (E1A) and Rh30 (MyoD). mTOR distribution was mainly nuclear with detectable levels in the cytoplasm. mTOR isolated from Rh30 nuclei phosphorylated recombinant GST-4E-BP1 (Thr-46) in vitro and thus has kinase activity. We next investigated the cellular distribution of mTOR substrates 4E-BP, S6K1, and eIF4E. 4E-BP was exclusively detected in cytoplasmic fractions in all cell lines. S6K1 was localized in the cytoplasm in colon carcinoma, HEK293 cells, and IMR90 fibroblasts. S6K1 was readily detected in all cellular fractions derived from rhabdomyosarcoma cells. eIF4E was detected in all fractions derived from rhabdomyosarcoma cells but was not detectable in nuclear fractions from colon carcinoma HEK293 or IMR90 cells.

Relation between outcomes and localisation of p-mTOR expression in gastric cancer

“…S6K1 drives translation of 5 0 TOP (terminal oligopyrimidine tract) mRNAs, and 4E-BP1 inhibits the mRNA cap-binding protein elF4E (Schmelzle and Hall, 2000). 4E-BP1 and S6K1 were detected exclusively in the cytoplasm of cancer cells (Zhang et al, 2002). We suggest that increased cytoplasmic mTORC1 complex may activate signalling to cytoplasmic S6K1 and 4E-BP1 and contribute to tumour progression, although 4E-BP1 and S6K1 were not investigated in this study.…”

“…This finding suggests that changes in the localisation of p-mTOR may be involved in tumour progression. Other investigators reported that m-TOR is mainly localised in the cytoplasm (Janus et al, 2005;Bachmann et al, 2006), although a small fraction of mTOR is found at a steady state in the nucleus in both normal and malignant cells (Zhang et al, 2002). mTOR is part of two distinct complexes: mTORC1 containing raptor and a mammalian orthologue of yeast Lst8p (mLST8; also known as GbL), and mTORC2 containing rictor, mLST8, and sin1 (also known as mitogen-activated protein-kinase-associated protein 1) (Sarbassov et al, 2005;Petroulakis et al, 2006;Rosner and Hengstschläger, 2008).…”

“…We suggest that increased cytoplasmic mTORC1 complex may activate signalling to cytoplasmic S6K1 and 4E-BP1 and contribute to tumour progression, although 4E-BP1 and S6K1 were not investigated in this study. In addition, mTOR shuttles between the nucleus and the cytoplasm (Zhang et al, 2002;Bachmann et al, 2006). The nuclear import of mTOR has an important role in activating its cytoplasmic signalling (Bachmann et al, 2006).…”

Constitutively active Rheb induces oncogenic transformation

“…The subcellular localization of TOR is likely signal dependent and cells observed under different conditions yield different results. Although a nuclear localization has been reported for TOR (Zhang et al, 2002), TOR has also been found in the cytoplasm and might shuttle between cytoplasm and nucleus (Sabatini et al, 1999;Kim and Chen, 2000). Drenan et al demonstrate a predominantly perinuclear distribution of TOR and show a close association with ER and the Golgi apparatus (Drenan et al, 2004).…”

mTOR Signaling in Lymphangioleiomyomatosis

“…1); constitutive nuclear localization of mTOR is observed in several tumor cell lines. 22,23 A distinct set of residues in the HEAT repeat region determines mTOR localization to endoplasmic reticulum (ER) and=or Golgi membranes. 24 The ER may provide a physical platform for mTOR metabolic sensing functions, control of the ER stress response, 25 and coordination of mTOR signaling to the translational and transcriptional machinery.…”