Our laboratory has uncovered important biochemical mechanisms that coordinate extracellular cues, intracellular signaling pathways, and metabolic processes, such as gene expression and protein synthesis. The lab has researched the major signaling pathways, including the Ras/ERK/RSK and the PI3-K/Akt signaling circuits. Moreover, the lab has performed extensive analyses of mammalian target of rapamycin (mTOR), a checkpoint kinase that deftly integrates signals emanating from growth factors and environmental conditions (for example, nutrient status) to guide cellular decisions. We wish to understand how these pathways are regulated and integrated in normal and cancer cells, as well as in a variety of other disease states.
Mitogenic/Oncogenic Signaling via Ras and the ERK-MAP kinase/RSK pathway.
Activation of the proto-oncoprotein Ras, results in activation of the Mitogen-Activated Protein Kinases (or ERK-MAPKs) and the ERK-regulated kinases (or RSKs). When improperly regulated, ERK and RSK contribute to a variety of human diseases, including cancer. We are utilizing a variety of biochemical, cell biological and RNAi-based approaches to identify new ERK and RSK targets and are determining how they contribute to the regulation of immediate-early gene expression, cell motility, cell proliferation, differentiation, and/or cell survival. We are also examining how temporal regulation and spatial distribution of ERK and RSK contributes to cell context-specific responses and how the Ras signaling system communicates with the G1 cell cycle machinery.
Mitogenic/Oncogenic Signaling via PI3-kinase and the p70-S6 protein kinase pathway.
Phosphatidylinositol 3-kinase (PI3K) signaling regulates protein translation, cell size/growth, G1 cell cycle progression, and cell survival. PI3K is activated by a variety of growth factors and cytokines and is constitutively activated by loss of function of the tumor suppressor PTEN, which occurs in greater than 30% of human cancers. The molecular basis of S6K and eIF4E activation and signaling, two major PI3K effectors, their role in mRNA processing/translation and ribosome biogenesis, and their contribution to tumorigenesis is a focus or our work.
Integration of signaling by nutrients, energy sufficiency and growth factors.
Mitogen-regulated signaling pathways converge on nutrient/energy sensing pathways to regulate various critical biological processes. Tumor suppressors mutated in diseases such as tuberous sclerosis (TSC1/2) or Peutz-Jeghers syndrome (LKB1) regulate signaling to the nutrient/energy sensor mTOR. mTOR is specifically inhibited by the drug rapamycin, an FDA-approved immunosuppressant and inhibitor of restenosis. Rapamycin is also currently in clinical trials for a variety of cancers. How nutrients, energy sufficiency or cell stress signal to mTOR, and how mitogenic signals and nutrient/energy signals collaborate to regulate S6 kinases and eIF4E is under investigation.
Identification and characterization of novel kinases/phosphatases involved in carcinogenesis.
Evasion from apoptosis is a hallmark of cancer. We have utilized RNA interference (RNAi) to systematically screen the kinase and phosphatase component of the human genome. We have identified several new survival kinases. phosphatases and associated regulatory subunits that promote cell survival. We also identified novel phosphatases with tumor suppressor-like activity. Finally, RNAi targeting of specific kinases sensitizes resistant cells to chemotherapeutic agents. We are currently characterizing many of these novel enzymes.
Some current focus points.
- Study biochemical aspects of the predominant signal transduction circuits, the Ras, PI3K, and mTOR pathways, the integration of their signals (that is, “cross-talk”), and their role in sensing environmental fluctuations.
- Investigate how these signaling pathways modulate and coordinate cellular metabolic processes such as gene expression, pre-mRNA splicing and protein synthesis.
- Capitalize on the lab's in-depth knowledge of intracellular signaling to identify new biomarkers, as well as novel therapeutic targets for cancer, aging, neurodegenerative diseases, diabetes, and obesity.
- Study the biochemical mechanism of rapamycin inhibition of mTOR and the development of resistance.
- Explore the role of Ras/ERK/RSK signaling in cell survival, migration, and invasion during the epithelial-to-mesenchymal transition and its link to metastasis.
mTORC1 and Translation
Our current model, which is based on our work and that of others (Hershey, Sonenberg, Pagano, …); is that inactive S6 kinase (S6K1) is associated with free eIF3 multi-subunit complex in nutrient/growth factor replete cells.
Upon stimulation, the mTOR Complex 1 (mTORC1) kinase binds to the eIF3 complex (binding sites & mechanism unknown). This brings mTORC1 into proximity of S6K where it can phosphorylate it at threonine 389 (the hydrophobic motif site).
This promotes its dissociation (the S6K1-T389A mutant, which cannot be phosphorylated, does not dissociate from the eIF3 complex and the S6K1-T389D phospho-mimetic mutant does not associate well with the eIF3 complex and importantly, S6K1-T389D is found in the translation preinitiation complex (PIC), even without nutrient stimulation).
Phosphorylation at the S6K hydrophobic motif is not sufficient for activation but importantly, Thr389 phosphorylation creates a docking site for PDPK1 (phosphoinositide-dependent protein kinase 1, also referred to as PDK1), which then phosphorylates the S6K activation loop site (T229) for enzymatic activation (originally shown by D. Alessi).
Concurrently, the eIF3j subunit binds in a rapa-sensitive manner (mechanism unknown - from John Hershey’s lab). eIF3j also enhances the binding of the eIF3 complex to the 40S ribosome (T. Pestova) to which, based on other literature and our biochemistry, is associated with the other translation initiation factors shown in the movie. There is literature supporting the ability of the 40S:eIF3 complex to bind eIF4E without eIF4G, and we have biochemical evidence supporting this possibility, however, we do not rule out the possibility that weak eIF4G binding may be first or concomitant with binding of the 40S:eIF3 complex (see below).
Our biochemical analysis (cap binding assays, co-ip’s, sucrose gradient analysis, etc.) supports the hypothesis that the association of eIF3 complex to the 5’ cap structure recruits mTORC1 into proximity of 4EBP1 for phosphorylation and dissociation from the 5’ cap.
4EBP1 dissociation from eIF4E reveals the binding site shared with eIF4G (N. Sonenberg). eIF4G binds, stabilizing the formation of the eIF4F translation preinitiation complex. Again, our data support this order of events but does not exclude the possibility that 4G associates weakly first (through eIF4E-independent interactions), followed by mTORC1/eIF3 binding, mTORC1-mediated 4E-BP1 phosphorylation and strong 4G binding to stabilize and recruit additional factors into the translation preinitiation complex.
This translation preinitiation complex, which contains the helicase eIF4A, lacks optimal translation efficiency with regards to its ability to translate mRNAs with highly structured 5’ UTRs. In our model, S6K, which is now active, phosphorylates eIF4B promoting its recruitment into the 4F complex (importantly, the eIF4B-S/A mutant binds poorly and the eIF4B-S/D-mutant binds efficiently without nutrient and/or mitogenic input). The eIF4B regulatory subunit is important for eIF4A helicase activity.
However, for full activation of the eIF4 helicase, S6K also phosphorylates the eIF4A helicase inhibitor and tumor suppressor, PDCD4, and targets it for bTRCP-mediated ubiquitination and turnover (Michele Pagano). This coordination of S6K1-mediated phosphorylations is proposed to optimally activate the eIF4A helicase.
We still don't understand the function of rpS6 phosphorylation. Additionally, we have unpublished data, along with some published data from others, that mTORC1 can regulate the phosphorylation of other translation initiation factors. The role of these phosphorylation events is not clearly understood at this time.
Some of the mRNAs with highly structured 5’ UTRs encode for proteins regulating cellular bioenergetics, cell cycle progression, angio- and lymphangiogenesis (Hif1a, Glut1, c-Myc, CycD1, ODC, VEGF, Fra1, …). It is very important to note that this is not an on/off switch for translation of these mRNAs. In our model based on the data we have generated to date, this is a mechanism for increasing the efficiency of translation of these messages. It may be as simple as increasing the ability of the preinitiation complex to find the translation start site but it may also represent a mechanism for revealing hidden internal ribosome entry sites.
Our References relevant to this model include:
Holz MK, Ballif BA, Gygi SP and Blenis J. mTOR and S6K1 mediate assembly of the translation preinitiation complex through dynamic protein interchange and ordered phosphorylation events. Cell 2005; 123: 569-580. PMID: 16286006
Gu X, Yu JJ, Ilter D, Blenis N, Henske EP, Blenis J. Integration of mTOR and estrogen-ERK2 signaling in lymphangioleiomyomatosis pathogenesis. Proc Natl Acad Sci U S A. 2013; 110: 14960-14965. PMCID: PMC3773757
Csibi A*, Lee G*, Yoon SO, Tong X, Ilter D, Elia I, Fendt SM, Roberts TM, Blenis J. The mTORC1/S6K1 pathway regulates glutamine metabolism through the eIF4B-dependent control of c-Myc translation. Current Biology, 2014; in press. *co-1st authors.
This represents one potential mechanism for the selective regulation of protein synthesis by mTORC1. Others include the more global mechanisms where mTORC1 regulates the translation efficiency of mRNAs with 5’ UTR terminal oligo-pyrimidine (TOP) sequences (see studies by Myehaus and Sabatini), eIF4E/4EBP1 (studies by Sonenberg) and our work on S6K1 and splicing-dependent increased translation efficiency (Ma X, Yoon, SO, Richardson CJ, Jülich K, and Blenis J. SKAR links pre-mRNA splicing to mTOR/S6K1-mediated enhanced translation efficiency of spliced mRNAs. Cell 2008; 133: 303–313. PMID: 18423201).
Finally, as with many AGC kinases, RSK activation downstream of Ras/ERK-MAP kinase signaling can also phosphorylate eIF4B and PDCD4 (Roux, Sonenberg and Blenis).