Marina K. Holz

mTOR and S6K1 Mediate Assembly of the Translation Preinitiation Complex through Dynamic Protein Interchange and Ordered Phosphorylation Events

In response to nutrients, energy sufficiency, hormones, and mitogenic agents, S6K1 phosphorylates several targets linked to translation. However, the molecular mechanisms whereby S6K1 is activated, encounters substrate, and contributes to translation initiation are poorly understood. We show that mTOR and S6K1 maneuver on and off the eukaryotic initiation factor 3 (eIF3) translation initiation complex in a signal-dependent, choreographed fashion. When inactive, S6K1 associates with the eIF3 complex, while the S6K1 activator mTOR/raptor does not. Cell stimulation promotes mTOR/raptor binding to the eIF3 complex and phosphorylation of S6K1 at its hydrophobic motif. Phosphorylation results in S6K1 dissociation, activation, and subsequent phosphorylation of its translational targets, including eIF4B, which is then recruited into the complex in a phosphorylation-dependent manner. Thus, the eIF3 preinitiation complex acts as a scaffold to coordinate a dynamic sequence of events in response to stimuli that promote efficient protein synthesis.

RAS/ERK Signaling Promotes Site-specific Ribosomal Protein S6 Phosphorylation via RSK and Stimulates Cap-dependent Translation

Converging signals from the mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) pathways are well established to modulate translation initiation. Less is known regarding the molecular basis of protein synthesis regulated by other inputs, such as agonists of the Ras/extracellular signal-regulated kinase (ERK) signaling cascade. Ribosomal protein (rp) S6 is a component of the 40S ribosomal subunit that becomes phosphorylated at several serine residues upon mitogen stimulation, but the exact molecular mechanisms regulating its phosphorylation and the function of phosphorylated rpS6 is poorly understood. Here, we provide evidence that activation of the p90 ribosomal S6 kinases (RSKs) by serum, growth factors, tumor promoting phorbol esters, and oncogenic Ras is required for rpS6 phosphorylation downstream of the Ras/ERK signaling cascade. We demonstrate that while ribosomal S6 kinase 1 (S6K1) phosphorylates rpS6 at all sites, RSK exclusively phosphorylates rpS6 at Ser235/236 in vitro and in vivo using an mTOR-independent mechanism. Mutation of rpS6 at Ser235/236 reveals that phosphorylation of these sites promotes its recruitment to the 7-methylguanosine cap complex, suggesting that Ras/ERK signaling regulates assembly of the translation preinitiation complex. These data demonstrate that RSK provides an mTOR-independent pathway linking the Ras/ERK signaling cascade to the translational machinery.

S6 Kinase 1 Regulates Estrogen Receptor α in Control of Breast Cancer Cell Proliferation

The 40 S ribosomal S6 kinase 1 (S6K1) acts downstream of mTOR (mammalian target of rapamycin) and is sensitive to inhibition by rapamycin. The chromosomal region 17q23 containing the RPS6KB1 gene is frequently amplified in breast cancer cells, leading to S6K1 overexpression. The role of S6K1 in disease development and progression is supported by the observation that S6K1 overexpression is associated with poor prognosis in breast cancer patients. However, the identity of mammary cell-specific S6K1 targets is not well understood. In this study, we report that overexpression of S6K1 endows breast cancer cells with a proliferative advantage in low serum conditions and enhanced sensitivity to rapamycin. We investigate the molecular mechanism behind this observation to show that S6K1 regulates estrogen receptor α (ERα) by phosphorylating it on serine 167, leading to transcriptional activation of ERα. By contributing to the activation of ERα, S6K1 promotes ERα-mediated cell proliferation and may be a target of therapeutic intervention in breast cancer.

mTOR/S6K1 and MAPK/RSK signaling pathways coordinately regulate estrogen receptor α serine 167 phosphorylation

Resistance to anti‐estrogen therapy is a major clinical concern in treatment of breast cancer. Estrogen‐independent phosphorylation of estrogen receptor α, specifically on Ser167, is one of the contributing causes to development of resistance, and a prognostic marker for the disease. Here, we dissect the signaling pathways responsible for Ser167 phosphorylation. We report that the mTOR/S6K1 and MAPK/RSK contribute non‐overlapping inputs into ERα activation via Ser167 phosphorylation. This cooperation may be targeted in breast cancer treatment by a combination of mTOR and MAPK inhibitors.

mTOR signaling for biological control and cancer.

Mammalian target of rapamycin (mTOR) is a major intersection that connects signals from the extracellular milieu to corresponding changes in intracellular processes. When abnormally regulated, the mTOR signaling pathway is implicated in a wide spectrum of cancers, neurological diseases, and proliferative disorders. Therefore, pharmacological agents that restore the regulatory balance of the mTOR pathway could be beneficial for a great number of diseases. This review summarizes current understanding of mTOR signaling and some unanswered questions in the field. We describe the composition of the mTOR complexes, upstream signals that activate mTOR, and physiological processes that mTOR regulates. We also discuss the role of mTOR and its downstream effectors in cancer, obesity and diabetes, and autism. J. Cell. Physiol. 228: 1658–1664, 2013.

The combination of rapamycin and resveratrol blocks autophagy and induces apoptosis in breast cancer cells.

Hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) is a frequent event in breast cancer and current efforts are aimed at targeting the mTORC1 signaling pathway in combination with other targeted therapies. However, patients often develop drug resistance in part due to activation of the oncogenic Akt signaling and upregulation of autophagy, which protects cancer cells from apoptosis. In the present study we investigated the effects of combination therapy of rapamycin (an allosteric mTORC1 inhibitor) together with resveratrol (a phytoestrogen that inhibits autophagy). Our results show that combination of these drugs maintains inhibition of mTORC1 signaling, while preventing upregulation of Akt activation and autophagy, causing apoptosis. Additionally, this combination was effective in estrogen receptor positive and negative breast cancer cells, underscoring its versatility. J. Cell. Biochem. 116: 450–457, 2015.

Estrogenic regulation of S6K1 expression creates a positive regulatory loop in control of breast cancer cell proliferation

The 40S ribosomal S6 kinase 1 (S6K1) is an important regulator of cell growth. Expression of S6K1 is often elevated in breast cancer cells. However, the transcriptional mechanism of S6K1 overexpression is not understood. In this report, we demonstrate that estrogen activates expression of S6K1 via estrogen receptor (ER)α in ER-positive breast cancer cells. We also show that estrogen acts on the proximal promoter of the S6K1 gene in a mechanism involving the transcriptional factor GATA-3. Finally, we provide data that support the importance of estrogenic regulation of S6K1 expression in breast cancer cell proliferation. S6K1 directly phosphorylates and regulates ligand-independent activity of ERα, while ERα upregulates S6K1 expression. This S6K1–ERα relationship creates a positive feed-forward loop in control of breast cancer cell proliferation. Furthermore, the co-dependent association between S6K1 and ERα may be exploited in the development of targeted breast cancer therapies.

Resveratrol prevents rapamycin-induced upregulation of autophagy and selectively induces apoptosis in TSC2-deficient cells.

The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway is hyperactivated in a variety of cancers and disorders, including lymphangioleiomyomatosis (LAM) and tuberous sclerosis complex (TSC), which are characterized by mutations in tumor suppressors TSC1 or TSC2. The concern with the use of mTORC1 inhibitors, such as rapamycin or its analogs (rapalogs), is that they cause upregulation of autophagy and suppress the negative feedback loop to Akt, which promotes cell survival, causing the therapy to be only partially effective, and relapse occurs upon cessation of treatment. In this study, we investigate the use of rapamycin in combination with resveratrol, a naturally occurring polyphenol, in TSC2-deficient cells. We tested whether such combination would prevent rapamycin-induced upregulation of autophagy and shift the cell fate toward apoptosis. We found that this combination treatment blocked rapamycin-induced upregulation of autophagy and restored inhibition of Akt. Interestingly, the combination of rapamycin and resveratrol selectively promoted apoptosis of TSC2-deficient cells. Thus, the addition of resveratrol to rapamycin treatment may be a promising option for selective and targeted therapy for diseases with TSC loss and mTORC1 hyperactivation.

The role of S6K1 in ER-positive breast cancer.

The 40S ribosomal S6 kinase 1 (S6K1) is a conserved serine/threonine protein kinase that belongs to the AGC family of protein kinases, which also includes Akt and many others. S6K1 is the principal kinase effector downstream of the mammalian target of rapamycin complex 1 (mTORC1). S6K1 is sensitive to a wide range of signaling inputs, including growth factors, amino acids, energy levels and hypoxia. S6K1 relays these signals to regulate a growing list of substrates and interacting proteins in control of oncogenic processes, such as cell growth and proliferation, cell survival and apoptosis and cell migration and invasion. Several lines of evidence suggest an important role for S6K1 in estrogen receptor (ER)-positive breast cancer. S6K1 directly phosphorylates and activates ERα. Furthermore, S6K1 expression is estrogenically regulated. Therefore, hyperactivation of mTORC1/S6K1 signaling may be closely related to ER-positive status in breast cancer and may be utilized as a marker for prognosis and a therapeutic target.

Phosphoproteomics reveals resveratrol-dependent inhibition of Akt/mTORC1/S6K1 signaling.

Resveratrol, a plant-derived polyphenol, regulates many cellular processes, including cell proliferation, aging and autophagy. However, the molecular mechanisms of resveratrol action in cells are not completely understood. Intriguingly, resveratrol treatment of cells growing in nutrient-rich conditions induces autophagy, while acute resveratrol treatment of cells in a serum-deprived state inhibits autophagy. In this study, we performed a phosphoproteomic analysis after applying resveratrol to serum-starved cells with the goal of identifying the acute signaling events initiated by resveratrol in a serum-deprived state. We determined that resveratrol in serum-starved conditions reduces the phosphorylation of several proteins belonging to the mTORC1 signaling pathway, most significantly, PRAS40 at T246 and S183. Under these same conditions, we also found that resveratrol altered the phosphorylation of several proteins involved in various biological processes, most notably transcriptional modulators, represented by p53, FOXA1, and AATF. Together these data provide a more comprehensive view of both the spectrum of phosphoproteins upon which resveratrol acts as well as the potential mechanisms by which it inhibits autophagy in serum-deprived cells.