Hypoxia-inducible Factor 1α Is Regulated by the Mammalian Target of Rapamycin (mTOR) via an mTOR Signaling Motif

Stephen C. Land,Andrew R. Tee

doi:10.1074/jbc.m611782200

Stephen C. Land, Andrew R. Tee

Open Access

https://doi.org/10.1074/jbc.m611782200

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Abstract

Tumors that form as a result of heightened mammalian target of rapamycin (mTOR) signaling are highly vascularized. This process of angiogenesis is regulated through hypoxia-inducible factor (HIF)-mediated transcription of angiogenic factors. It is recognized that inhibition of mTOR with rapamycin can diminish the process of angiogenesis. Our work shows that activation of mTOR by Ras homologue enriched in brain (Rheb) overexpression potently enhances the activity of HIF1alpha and vascular endothelial growth factor (VEGF)-A secretion during hypoxia, which is reversed with rapamycin. Mutants of Rheb, which do not bind guanine nucleotide (D60K, D60V, N119I, and D122N) and are unable to activate mTOR, inhibit the activity of HIF when overexpressed. We show that regulatory associated protein of mTOR (Raptor) interacts with HIF1alpha and requires an mTOR signaling (TOS) motif located in the N terminus of HIF1alpha. Furthermore, a mutant of HIF1alpha lacking this TOS motif dominantly impaired HIF activity during hypoxia and was unable to bind to the co-activator CBP/p300. Rapamycin treatments do not affect the stability of HIF1alpha and modulate HIF activity via a Von Hippel-Lindau (VHL)-independent mechanism. We demonstrate that the high levels of HIF activity in cells devoid of TSC2 can be reversed by treatments with rapamycin or the readdition of TSC2. Our work explains why human cancers with aberrant mTOR signaling are prone to angiogenesis and suggests that inhibition of mTOR with rapamycin might be a suitable therapeutic strategy.

Highlights

As well as vascularization, hypoxia-inducible factor (HIF) is known to control the expression of Ͼ70 genes involved in energy metabolism, apoptosis/survival, and metastasis
We show that Ras homologue enriched in brain (Rheb) potently stimulates mammalian target of rapamycin (mTOR)-dependent phosphorylation of S6 protein kinase 1 (S6K1) at Thr389 (Fig. 1B)
In DMOG-treated cells without Rheb overexpression, we observed a reduction of HIF1␣ upon treatment with rapamycin, which confirms previous reports that inhibition of mTOR reduces the levels of endogenous HIF1␣ within cells (Fig. 1B) [30]

Summary

EXPERIMENTAL PROCEDURES

Chemicals and Materials—[3H]GTP, [3H]GDP, and [35S]methionine-radiolabeled reagents were purchased from Amersham Biosciences (GE Healthcare UK Ltd.). Raptor Binding to mTOR Targets—To generate Raptor protein to be used in the overlay assays, Myc-tagged Raptor was overexpressed in HEK293 cells. These cells were lysed with 50 mM ␤-glycerophosphate, pH 7.4; 1 mM EDTA; 1 mM EGTA; 0.5 mM Na3VO4; 1 mM benzamidine hydrochloride; 1 mM dithiothreitol; 0.1 mM phenylmethane sulfonyl fluoride; 1% (v/v) Triton X-100, and 1 ␮g/ml each of pepstatin, antipain, and leupeptin. Myctagged Raptor that bound to proteins immobilized on Immobilon-P was detected by incubating the blot with anti-Myc antibodies for 2 h in 2% (w/v) bovine serum albumin Tris-buffered saline followed by anti-mouse horseradish peroxidaseconjugated secondary antibodies and ECL as described for Western blotting. A p value of Ͻ0.05 was considered to be statistically significant

RESULTS

It was postulated that mTOR signaling enhances the stability of

DISCUSSION