Lili Shu

Sustained Activation of the JNK Cascade and Rapamycin-Induced Apoptosis Are Suppressed by p53/p21Cip1

Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces apoptosis of cells lacking functional p53. Cells expressing wild-type p53 or p21(Cip1)arrest in G1 and remain viable. In cells lacking functional p53, rapamycin or amino acid deprivation induces rapid and sustained activation of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase, and elevation of phosphorylated c-Jun that results in apoptosis. This stress response depends on expression of eukaryotic initiation factor 4E binding protein 1 and is suppressed by p21(Cip1) independent of cell cycle arrest. Rapamycin induces p21(Cip1) binding to ASK1, suppressing kinase activity and attenuating cellular stress. These results suggest that inhibition of mTOR triggers a potentially lethal response that is prevented only in cells expressing p21(Cip1).

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

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.

p53 mutation and MDM2 amplification frequency in pediatric rhabdomyosarcoma tumors and cell lines

BACKGROUND The p53 tumor suppressor gene is the most commonly mutated gene in human cancer, and mutations arise in a wide variety of tumor types. Wild-type p53 functions as a regulator of apoptosis, so mutations in the p53 gene are generally associated with aggressive tumors and a poor prognosis. PROCEDURE We have investigated the p53 mutation and MDM2 amplification frequencies in biopsies from pediatric rhabdomyosarcoma (RMS) tumors and cell lines by SSCP and Southern analyses. RESULTS A mutation was detected in only 1 of 20 tumor specimens (5%), whereas the frequency in established RMS cell lines was significantly higher (6/10, 60%). p53 Mutations were more common in cell lines derived from tumors previously exposed to chemotherapy compared to those derived from tumors at di-agnosis, and it is likely that these mutations enhanced the probability of successful long-term culture. The frequency of MDM2 gene amplification in patient biopsies was also low (2/20, 10%). Interestingly, complete responses to treatment were obtained in the two patients with tumors that demonstrated amplification of MDM2. The response to treatment of patients with tumors wild-type for p53 and without MDM2 amplification was quite varied, indicating that expression of a wild-type p53 gene at diagnosis cannot always facilitate a favorable outcome. CONCLUSIONS p53 mutation and MDM2 gene amplification frequencies are extremely low in RMS tumors, but a wild-type p53 genotype is not always associated with a favorable prognosis.

Myogenic Differentiation Is Dependent on Both the Kinase Function and the N-terminal Sequence of Mammalian Target of Rapamycin

The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase known to control initiation of translation through two downstream pathways: eukaryotic initiation factor 4E-binding protein 1 (4E-BP1)/eukaryotic initiation factor 4E and ribosomal p70 S6 kinase (S6K1). We previously showed in C2C12 murine myoblasts that rapamycin arrests cells in G1phase and completely inhibits terminal myogenesis. To elucidate the pathways that regulate myogenesis, we established stable C2C12 cell lines that express rapamycin-resistant mTOR mutants (mTORrr; S2035I) that have N-terminal deletions (Δ10 or Δ91) or are full-length kinase-dead mTORrr proteins. Additional clones expressing a constitutively active S6K1 were also studied. Our results show that Δ10mTORrr signals 4E-BP1 and permits rapamycin-treated myoblasts to differentiate, confirming the mTOR dependence of the inhibition of myogenesis by rapamycin. C2C12 cells expressing either Δ91mTORrr or kinase-dead mTORrr(D2338A) could not phosphorylate 4E-BP1 in the presence of rapamycin and could not abrogate the inhibition of myogenesis. Taken together, our results indicate that both the kinase function of mTOR and the N terminus (residues 11–91, containing part of the first HEAT domain) are essential for myogenic differentiation. In contrast, constitutive activation of S6K1 does not abrogate rapamycin inhibition of either proliferation or myogenic differentiation.

The mTORC2 Complex Regulates Terminal Differentiation of C2C12 Myoblasts

ABSTRACT Rapamycin, a selective inhibitor of mTORC1 signaling, blocks terminal myoblast differentiation. We found that downregulation of rictor, a component of the mTORC2 complex, but not downregulation of raptor, a component of the mTORC1 complex, prevented terminal differentiation (fusion) of C2C12 myoblasts. Both rapamycin and rictor downregulation suppressed the phosphorylation of AKT(S473), and rapamycin treatment of C2C12 myoblasts disrupted the mTORC2 complex. Importantly, downregulation of rictor inhibited TORC2 signaling without inhibiting mTORC1 signaling, suggesting that inhibition of mTORC1 by rapamycin may not be the cause of arrested differentiation. In support of this, expression of a phosphomimetic mutant AKT(S473D) in rictor-deficient cells rescued myoblast fusion even in the presence of rapamycin. mTORC2 signaling to AKT appears necessary for downregulation of the Rho-associated kinase (ROCK1) that occurs during myogenic differentiation. Rapamycin treatment prevented ROCK1 inactivation during differentiation, while suppression of ROCK1 activity during differentiation and myoblast fusion was restored through expression of AKT(S473D), even in the presence of rapamycin. Further, the ROCK inhibitor Y-27632 restored terminal differentiation in rapamycin-treated myoblasts. These results provide the first evidence of a specific role for mTORC2 signaling in terminal myogenic differentiation.

mTORC1 Signaling Can Regulate Growth Factor Activation of p44/42 Mitogen-activated Protein Kinases through Protein Phosphatase 2A

The mTORC1 complex (mammalian target of rapamycin (mTOR)-raptor) is modulated by mitogen-activated protein (p44/42 MAP) kinases (p44/42) through phosphorylation and inactivation of the tuberous sclerosis complex. However, a role for mTORC1 signaling in modulating activation of p44/42 has not been reported. We show that in two cancer cell lines regulation of the p44/42 MAPKs is mTORC1-dependent. In Rh1 cells rapamycin inhibited insulin-like growth factor-I (IGF-I)-stimulated phosphorylation of Thr202 but not Tyr204 and suppressed activation of p44/42 kinase activity. Down-regulation of raptor, which inhibits mTORC1 signaling, had a similar effect to rapamycin in blocking IGF-I-stimulated Tyr204 phosphorylation. Rapamycin did not block maximal phosphorylation of Tyr204 but retarded the rate of dephosphorylation of Tyr204 following IGF-I stimulation. IGF-I stimulation of MEK1 phosphorylation (Ser217/221) was not inhibited by rapamycin. Higher concentrations of rapamycin (≥100 ng/ml) were required to inhibit epidermal growth factor (EGF)-induced phosphorylation of p44/42 (Thr202). Rapamycin-induced inhibition of p44/42 (Thr202) phosphorylation by IGF-I was reversed by low concentrations of okadaic acid, suggesting involvement of protein phosphatase 2A (PP2A). Both IGF-I and EGF caused dissociation of PP2A catalytic subunit (PP2Ac) from p42. Whereas low concentrations of rapamycin (1 ng/ml) inhibited dissociation of PP2Ac after IGF-I stimulation, it required higher concentrations (≥100 ng/ml) to block EGF-induced dissociation, consistent with the ability for rapamycin to attenuate growth factor-induced activation of p44/42. The effect of rapamycin on IGF-I or insulin activation of p44/42 was recapitulated by amino acid deprivation. Rapamycin effects altering the kinetics of p44/42 phosphorylation were completely abrogated in Rh1mTORrr cells that express a rapamycin-resistant mTOR, whereas the effects of amino acid deprivation were similar in Rh1 and Rh1mTORrr cells. These results indicate complex regulation of p44/42 by phosphatases downstream of mTORC1. This suggests a model in which mTORC1 modulates the phosphorylation of Thr202 on p44/42 MAPKs through direct or indirect regulation of PP2Ac.