This study contributes to our understanding of the link between Akt and NFκB. The Akt pathway is actively involved in the regulation of NFκB, and NFκB activity is essential for oncogenic transformation by PI3K and Akt.5-7, 9, 22
In cells with a gain of function in Akt, the transcriptional activity of NFκB is upregulated, and inhibition of Akt interferes with this upregulation of NFκB. In CEF transformed by myristylated Akt, there is greatly enhanced degradation of the IκB protein and increased phosphorylation of the p65 NFκB subunit. Our data rule out direct phosphorylation of the p65 or p50 NFκB subunits by Akt. Neither protein contains an Akt phosphorylation consensus sequence, and Akt fails to phosphorylate p65 in an in vitro
kinase assay. Rather, our observations show that Akt phosphorylates IKKα, in accordance with published studies.9
The IKK complex then not only targets the IκB inhibitor protein, but, in agreement with previous observations, also phosphorylates the p65 NFκB subunit.22, 34, 37, 38
Thus the IKK complex targets both the IκB and NFκB p65 proteins and functions as the intermediary between Akt and NFκB. NFκB p65 and IκB are located in the same cellular compartment and are phosphorylated in a similar time-dependent pattern.34
The putative IKK phosphorylation site of p65, S534, matches the IKKβ consensus sequence, is located in the COOH-terminal transactivation domain 1, and is conserved in human, mouse, chicken and Xenopus. Phosphorylation of this residue eliminates a hydrogen bond to the nearby D531 residue in p65, and results in an efficient association of p65 with TAFII31, a component of the basal transcriptional machinery. At the same time phosphorylation reduces the affinity of p65 for the transcriptional corepressor AES (Amino-terminal Enhancer of Split). This change of affinities suggests a possible mechanism for the activation of NFκB by phosphorylation.39
In more general terms, a phosphate group, providing additional negative charge is likely to enhance the transcriptional activity of the acidic activator domain of NFκB.40
Our data document the functional significance of the phosphorylation cascade that originates with Akt and progresses through T23 of IKKα to S534 of p65.
Signaling between Akt and NFκB is complex, and the published data contain several seemingly contradictory observations. At least some of these discrepancies may reflect the use of different cell types and signaling conditions. For instance, Sizemore et al.
showed that PI3K/Akt was necessary for the phosphorylation and activation of p65 in response to TNF and IL-1, and that Akt-mediated NFκB activation required IKK activity.6, 41
In contrast, Yang et al.
reported that in mouse macrophages, LPS-induced p65 phosphorylation at S534 was unaffected by LY294002, an inhibitor of PI3K.42
Ozes and associates9
found that Akt was an essential mediator of the TNFα-induced activation of NFκB, operating through the phosphorylation of IKKα at T23, but this finding has been challenged by Delhase and coworkers.43
Studies on cells with genetic inactivation of components of Akt signaling have produced some surprising results. Deletion of glycogen synthase kinase 3β (GSK3β) interferes with the TNFα-induced activation of NFκB, and, surprisingly, even with the TNFα-triggered activation of Akt.44
Cells that are deficient in tuberous sclerosis complex 1/2 proteins (TSC1/TSC2) are also defective in the TNFα-induced activation of NFκB, and this defect can be eliminated by rapamycin, suggesting that in this constellation, mTOR is a negative regulator of the TNFα- NFκB signal.45
In contrast, PTEN -negative cells depend on the mTOR-Rictor complex for the TNFα-induced activation of NFκB.46, 47
These divergent observations suggest that the activation of NFκB by extracellular stimuli can be affected by numerous signaling components and that these effects are dependent on the physiological conditions and genetic make-up of the cells. NFκB is an important factor in cancer development and progression, in addition to being a central coordinator of immune responses.27, 48, 49
The viral protein v-rel
(homolog of c-rel
) was originally identified as retroviral oncogene.50, 51
Many tumors show constitutively elevated levels of NFκB activity caused by genetic changes, including loss-of-function mutations in the IκB gene or activation of upstream regulators such as IKKs.27
Blocking NFκB activity decreases tumorigenicity.52-54
Our results show that suppression of NFκB activity by IκBSR induces a strong and selective resistance to P3K- or Akt-induced oncogenic transformation, suggesting an essential role for NFκB in the transforming mechanisms induced by these oncoproteins. The relative importance of IκB degradation versus p65 phosphorylation in mediating transformation cannot be determined from the available data. However, the essentialness of NFκB activity for PI3K /Akt oncogenicity is particularly significant in view of the fact that the PI3K pathway is dysregulated in many human cancers. PI3K and Akt are considered promising cancer targets, and the dependence of these oncoproteins on NFκB needs to be considered in the search for therapeutically effective inhibitors.
In Akt-transformed CEF as well as in BT20 human breast cancer cells, signaling through Akt is upregulated, and the downstream targets GSK-3β, ribosomal protein S6 kinase, and eukaryotic translation initiation factor 4E-binding protein are all phosphorylated.16, 25
The constitutive activity of Akt in these cells defines a signaling landscape that is shared with other cells harboring a gain of function in the PI3K-Akt-TOR pathway. These types of cells will probably show the direct signal from activated Akt via the IKK complex to p65 that is suggested by the data presented here. Whether this pathway also applies to all external stimulus-induced activations of NFκB will have to be decided by future investigations.