PerspectiveCell Signaling

Blocking Akt-ivity

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Science  28 Aug 2009:
Vol. 325, Issue 5944, pp. 1083-1084
DOI: 10.1126/science.1179972

Aberrations in cellular signaling pathways that involve the enzyme Akt (also called protein kinase B) are implicated in diverse diseases, including cancer, diabetes, and neurodegenerative disorders (1, 2). Thus, proteins involved in Akt activation and signaling are potential targets for therapeutic intervention. In fact, drugs directed against some of these targets are now in clinical trials for treating cancers, and the inhibition of Akt activation and signaling remains a major goal of drug discovery (3, 4). On page 1134 of this issue, Yang et al. (5) identify a chemical modification of Akt that controls its activation, identifying another potential means to inhibit this kinase in human cancers.

An important step in Akt activation is its translocation from the cytosol to the plasma membrane, where it becomes activated in response to the stimulation of growth factor receptors at the cell surface. However, the mechanisms that control this membrane localization are not clear. Akt possesses a PH domain, which binds to the molecule phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in the plasma membrane. Similarly, the enzyme phosphoinositide-dependent protein kinase 1 (PDK-1), which phosphorylates and thereby activates Akt, localizes to the plasma membrane by binding to PIP3. The membrane localization of PDK-1 triggers the recruitment of Akt to the membrane (6), though it is unclear precisely how. Now, Yang et al. show that this process is even more complicated (see the figure).

The authors identify Akt as a target of TRAF6, an E3 ubiquitin ligase. Ubiquitin ligases attach a small protein called ubiquitin to target proteins, which induces their degradation or promotes interactions with other proteins to transduce signals. These effects are distinguished by the attachment of single ubiquitin moieties to a protein substrate (monoubiquitination) or chains of ubiquitin proteins (polyubiquitination), as well as by the specific lysine residue that is modified. By ubiquinating Akt, TRAF6 promotes Akt translocation to the plasma membrane, where it becomes phosphorylated. In cells lacking TRAF6, ubiquitination, membrane localization, activation, and signaling of Akt were impaired in response to treatment with growth factors.

The amino acids modified by TRAF6 are lysine residues at positions 8 (K8) and 14 (K14), both of which are monoubiquitinated and lie in the PH domain. Mutation of either lysine residue to arginine impaired Akt activation. The K14R mutation specifically disrupts Akt interaction with PIP3 (7). However, Yang et al. found that the K8R mutation did not affect binding to PIP3. Nevertheless, membrane localization of this mutant was impaired in response to growth factors. Thus, by ubiquitinating Akt on two specific residues, TRAF6 promotes localization of the kinase to the plasma membrane for subsequent activation. Ubiquitination may cause a conformational change that enables Akt to interact with a protein that transports the kinase to the membrane. Ubiquitination of the protein neurotrophin receptor interacting factor (NRIF) by TRAF6 allows NRIF to associate with the protein p62. The resulting complex is then able to translocate to the nucleus (8).

To determine whether TRAF6 is an effective target for inhibiting oncogenic Akt hyperactivation, Yang et al. examined an activated, mutant form of Akt identified in tumor samples of patients with breast, colorectal, or ovarian cancer (9). In this mutant, glutamic acid at position 17 is replaced with lysine (E17K), which increases interaction of its PH domain through a conformational change with PIP3. Enhanced membrane association of the mutant form of Akt increases its activation, even in the absence of growth factors. The E17K mutant also displayed greater over-all ubiquitination—lysine residues at positions 8, 14, and 17 become modified—and its ubiquitination was further potentiated when TRAF6 was overexpressed in cells. Mutating the K8 residue in this mutant decreased Akt activation and downstream signaling. Thus, the Akt mutant uses ubiquitination to attain its hyperactive state.

Moving to the membrane.

The enzyme TRAF6 adds ubiquitin (Ub) to Akt, a modification that enhances localization to the membrane, where Akt is phosphorylated and activated. Blocking TRAF6 in tumor cells could increase the effect of mTORC1 inhibitors and cause cell death in tumors.

CREDIT: N. KEVITIYAGALA/SCIENCE

The authors extended this concept by depleting TRAF6 (by RNA interference) from a human tumor cell line that expresses the hyperactive, mutant form of Akt, and then injecting these cells into “nude” mice (animals that do not mount an immune response to foreign cells). Tumor formation by these cells was severely impaired compared to tumor cells expressing TRAF6 that were injected into animals, consistent with the potential of TRAF6 inhibition to stop tumor growth.

Could inhibiting TRAF6 be an effective clinical therapy for human cancer? Although no inhibitors of TRAF6 are currently available, blocking the function of E3 ligases has shown effective antitumor properties in preclinical studies, and such inhibitors are moving toward clinical trials (10). Studies of the E3 ligase Mdm2, which targets the tumor suppressor protein p53 for degradation, show that Mdm2 inhibition can be attained by blocking interaction with its substrate. Yang et al. show that a stable complex forms between TRAF6 and Akt, suggesting that this approach may be a good way to block TRAF6-mediated Akt activation. The potential effectiveness of this approach for tumor therapy is highlighted by the point in the signaling cascade at which TRAF6 contributes to Akt activation—downstream of common mutations observed in the clinic that affect phosphatidylinositol 3-kinase (PI3K) or the phosphatase PTEN, both of which cause hyperactivation of Akt. In support of this, the tumor cell line depleted of TRAF6 that was injected into mice by Yang et al. did not express PTEN and displayed strong Akt activation.

TRAF6 could be used to augment the effectiveness of rapamycin analogs (rapalogs), drugs that inhibit the mammalian target of rapamycin complex 1 (mTORC1). Rapalogs are approved for limited antitumor therapy because they may temporarily stabilize tumors in clinical trials but rarely elicit a full response in terms of tumor ablation. Preclinical studies indicate that rapalogs have a cytostatic effect on tumors, due at least in part to increased Akt activation, because a negative feedback loop that normally prevents PI3K signaling is lost. As Yang et al. show, cells lacking TRAF6 display increased spontaneous apoptosis (programmed cell death). Thus, TRAF6 inhibition in conjunction with rapalogs could shift the response of tumors to rapalogs from cytostatic to cytotoxic, increasing the efficacy of these drugs in cancer therapy.

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