Early Steps Discovered in Protein-making Process

This Breakdown of Translation Initiation May Illuminate Cancer Pathogenesis

Translation, the synthesis of protein from an mRNA template, has long been considered a benign sequela to transcription. After all, dysregulation of transcription causes a multitude of human disorders, including cancer and metabolic diseases. But it turns out that translation is not so innocuous. Recent evidence has shown that dysregulation of two proteins involved in translation, eukaryotic initiation factor 4E (eIF4E) and the S6 kinases, contributes to carcinogenesis, for example. For this reason alone, translational regulation may be getting less attention than it should.

Photo by Graham Ramsay

Marina Holz and John Blenis have a new interpretation of translational regulation, which helps explain the link between protein synthesis and tumor development.

Last month John Blenis, HMS professor of cell biology, and colleagues helped rectify that. In the Nov. 18 Cell, they revealed just how S6 kinase 1 (S6K1) helps to regulate the initiation of protein synthesis. The findings not only fill a huge gap in the understanding of translation, but they may also lead to new insights into the development and treatment of cancer. “We are really excited about defining the molecular basis of translation initiation, but more importantly, the findings provide the real potential for developing biomarkers that can measure inappropriate activation of this signaling pathway in cancer and other diseases,” said Blenis.

Caveat mTOR
Over the last five years or so, researchers in several laboratories, including Blenis’s, showed that eIF4E and S6K1 are regulated by the protein mTOR (for mammalian target of rapamycin). Activated by growth hormones and mitogens, mTOR contributes to cell cycle progression and, in certain conditions, carcinogenesis. In fact, though its mechanisms of action are not completely clear, rapamycin, an mTOR inhibitor, is currently in clinical trials for several types of cancer and has been used worldwide as an immunosuppressant because it inhibits the proliferation of T cells.

Though research has shown that mTOR and another kinase, phosphoinositide-dependent kinase 1 (PDK1), can phosphorylate S6K1, and that S6K1 can phosphorylate other proteins, how and if all these different modifications are synchronized to kick-start translation was a mystery. Now, graduate student Marina Holz and colleagues show that the translational initiation factor, eukaryotic initiation factor 3 (eIF3), acts as a scaffold to coordinate the kinases, regulating assembly of a preinitiation complex, an essential first step in the recruitment of the ribosome and the initiation of protein synthesis.

Using tandem affinity purification, a relatively recent and powerful method for isolating associated proteins, Holz and colleagues found that eIF3 and S6K1 are normally bound together in human cells that are starved of nutrients. But when Holz stimulated the cells with insulin or epidermal growth factor, either of which spurs protein synthesis, she could no longer copurify the proteins, suggesting that S6K1 had been released from the initiation factor. In contrast, when Holz treated the cells with rapamycin, the insulin had no effect on the partnership. The findings suggest that mTOR, in response to growth factors, drives a wedge between eIF3 and S6K1, possibly by phosphorylating the S6 kinase.

Holz and colleagues tested this hypothesis in two ways. First, they determined if mTOR also binds eIF3. They found that it does, but in the exact opposite manner to the way eIF3 binds S6K1. In the absence of growth stimuli, mTOR remains free, but when cells are stimulated with growth factors, mTOR binds to the initiation factor. Next they tested the potential role of phosphorylation. Because mTOR is known to modify S6K1 on a specific amino acid, threonine 389, Holz and colleagues transfected cells with an S6K1 mutant harboring an alanine residue at that position. This mutant bound to eIF3 even when the cells were stimulated with growth factors, indicating that S6K1 needs to be phosphorylated to be released from eIF3. All told, these experiments, for the first time, confirm a sequence of events whereby stimulation of translation by growth factors starts with activation of mTOR, which in turn phosphorylates S6K1, causing it to dissociate from a major component of the translation initiation complex, eIF3. “This is the first time that anyone has ever made a molecular connection between translational regulation and a rapamycin-sensitive cell signaling event that is regulated by growth factor binding and a variety of oncogenic proteins,” said Blenis.

Not Lost in Translation
But what happens to S6K1 once it is released—does it just drift off to be dephosphorylated or worse, degraded? In fact, Holz and colleagues found that the kinase continues to play a crucial role even after it is freed from eIF3. Importantly, the action of S6K1 dovetails with another unexplained phenomenon—the post-translational modification of yet a different initiation factor, eIF4B.

Image courtesy of Marina Holz

Scaffold building for protein synthesis. When mTOR and its partner Raptor bind to the eIF3/eIF4E complex, both S6K1 and another protein called 4E binding protein 1 (4E-BP1) are phosphorylated and released (A). Then, through the action of phosphoinositide-dependent kinase 1 (PDK1), the free S6K1 is further phosphorylated, whereupon it begins to phosphorylate two more proteins, S6 (the 40S subunit of the small ribosome) and eIF-4B (B). The entire preinitiation complex, including the 40S subunit, eIF4A, eIF4B, eIF4G, and the poly A binding protein 1 (PABP1), can then assemble at the mRNA cap (m7-GTP) and start the translation process.

No one has yet found a role for phosphorylation of eIF4B, but the researchers convincingly show that not only is it essential for the protein to be incorporated into the translational initiation complex, but that it is S6K1 that catalyzes the modification. Holz and colleagues found that eIF4B binds to eIF3, but, as in the case of mTOR, only when cells are stimulated with growth factors. What’s more, when that same S6K1 mutant that fails to dissociate from eIF3 is added to cells, eIF4B fails to bind eIF3, under any circumstances. The researchers also found that when they blocked the site on eIF4B that is phosphorylated by S6K1, then eIF4B also fails to bind the preinitiation complex. The results show that displacement of S6K1 by mTOR accommodates the arrival of eIF4B after it is modified by S6K1.

The role of S6K1 does not stop there. The researchers were able to show that it also phosphorylates S6, part of the small subunit of the ribosomal complex that plays an essential role in translation, helping to recruit S6 to the eIF3 scaffold. Again, this happens only after mTOR has activated and released S6K1.

Taken together these experiments outline the spatial and temporal events that lead to the initiation of translation (see model above) and identify eIF3 as a principal element, or hub, upon which the various spokes of the initiation complex are added. The findings also suggest a potentially valuable therapeutic intervention. Because activation of mTOR can be blocked by rapamycin, cancers that depend on activation of this pathway could be treated with the drug or a similar alternative. In addition, binding of mTOR to eIF3 could be used as a diagnostic tool to identify cells that have this pathway inappropriately activated.

But there are still large pieces of the puzzle that need to be solved. “No one yet knows all the upstream events that lie between growth factor signaling and mTOR activation,” said Blenis. He is now giving that pathway his undivided attention.

—Tom Fagan

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