The regulation and control of IKK continues to be a widely studied area. Given the cardinal role IKK plays in coordinating stress-induced immune and inflammatory responses the identification of substrates has been mostly limited to modulators of NF-κB activity (Häcker and Karin, 2006
). The discovery that IKK is required for mammalian autophagy, independent of NF-κB activation, however, suggests that IKK influences cellular function beyond simply controlling inflammatory transcription networks (Criollo et al., 2010
; Comb et al., 2010
). These findings also highlight the need to identify novel IKK substrates involved in crosstalk with signaling networks that regulate cellular metabolism.
The mechanism by which cellular starvation activates and promotes IKK-dependent changes in metabolism are not well-understood but this stress often induces cessation of signaling through the PI3K/Akt pathway. Cells lacking IKK subunits or treated with an IKK-specific inhibitor display persistent Akt activation under periods of starvation, suggesting that both IKKα and IKKβ play important roles in controlling PI3K-dependent inhibition following nutrient deprivation (). Interestingly, loss of IKKβ in vivo appears to have a greater effect in response to fasting than it does in immortalized null MEFs (data not shown).
Starvation-induced IKK activity has not been well-studied. Increased IKK activity is observed following just 15 minutes of nutrient depletion (), consistent with our previous findings that starvation-induced NF-κB DNA binding is maximal at 30 minutes, and data reported by Criollo, et al. demonstrating that IKK plays an early role in the initiation of autophagy (Criollo et al., 2010
; Comb et al., 2010
) . Induction of IKK activity corresponds well with the kinetics of PI3K/Akt feedback in response to starvation in cultured cells (), indicating that the effect of IKK on PI3K feedback is likely direct. The mechanism by which IKK is rapidly activated in response to nutrient deprivation is an important question and a topic of current investigation. Our data indicate that core components of the IKK holoenzyme (IKKα, IKKβ, NEMO) are required for starvation-induced Akt inhibition, but that the IKK activating kinase Tak1 is not. These data therefore suggest the interesting possibility that activation of IKK by nutrient deprivation differs from activation by inflammatory stimuli such as TNFα (Supplemental Figure S6
The PI3K regulatory subunit p85α contains a strong consensus IKK phosphorylation motif in the C-terminal SH2 domain, and we show that p85 Ser690 is indeed phosphorylated in vitro and in vivo in response to cellular starvation ( and ). In addition, p85 is phosphorylated in animals in response to whole-body fasting (). Starvation-induced p85 Ser690 phosphorylation inversely correlates with markers of PI3K/AKT pathway activation (such as pAKT and pS6) () and mutation of Ser690 to alanine abrogates IKK-mediated, or starvation-induced, PI3K feedback inhibition (). Taken together these data confirm that p85α is a bona fide IKK substrate.
In order to elucidate the mechanism by which IKK promotes PI3K feedback inhibition we analyzed the crystal structure of a p85 SH2 domain bound to a phosphotyrosine peptide. Serine 690 resides in a conserved region of the second α-helix that stabilizes the β-sheets that form the phospho-tyrosine binding pocket. The hydroxyl side-chain, which makes direct contact with the +3 acidic residue, faces the outside surface of the phosphotyrosine pocket, and is thus accessible (Booker et al., 1992
; Hoedemaeker et al., 1999
). This structure suggests that S690 phosphorylation may disrupt the alpha helix causing destabilization of the beta-sheets and loss of phosphotyrosine binding. Consistent with this hypothesis, data in show that a phosphorylated p85α cSH2 domain fails to bind tyrosine-phosphorylated proteins as efficiently as an unphosphorylated SH2 domain, and this is dependent on Serine690. We also find that interaction between Gab1 and WT p85, but not p85 S690A, is decreased in Cos7 cells overexpressing IKK (). Additionally, IKK-deficient cells displayed prolonged pTyr binding following starvation compared to WT cells ( and S4B
), which is consistent with the inability of IKK deficient cells to induce p85 phosphorylation and attenuate Akt signaling in response to cellular starvation ( and ). Our data therefore indicate that phosphorylation of p85 disrupts GF-dependent interactions with the cSH2 domain. Interestingly, a recent report demonstrated that PKC family members phosphorylate p85-cSH2 domain in response to phorbol ester stimulation and this also negatively regulates pTyr binding (Lee et al., 2011
). Intriguingly, phorbol esters are known activators of IKK/NF-κB signaling. It would therefore be interesting to determine whether PKD- and IKK-dependent cSH2 phosphorylation events are coordinated.
Identification of the PI3K regulatory subunit p85 as an important molecule for starvation-induced PI3K feedback inhibition is consistent with a number of previous studies that have also defined roles for p85 subunits in negatively regulating PI3K activity. For example, mutations in p110 catalytic subunits are thought to induce cell transformation via abrogating the ability of p85 to negatively regulate PI3K activity (Wu et al., 2009
), and monomeric p85 can form sequestration complexes in response to insulin stimulation to restrict phospho-tyrosine from PI3K p85/p110 dimers (Luo et al., 2005
). In addition, p85 has been shown to bind to and promote the activity of PTEN following stimulus-induced activation of PI3K as a means of feedback inhibition (Taniguchi et al., 2006
). Mammalian cells have also evolved further mechanisms to allow precise regulation of PI3K activity, including an important leucine-dependent feedback loop involving S6K phosphorylation and inhibition of IRS proteins (Harrington et al., 2004
; Shah et al., 2004
). We demonstrate that Akt reactivation in response to leucine deprivation is restricted by IKK. Furthermore, we show that AA-withdrawal is both necessary and sufficient to induce IKK kinase activity and phosphorylation of p85 S690 (). These studies reveal an IKK-dependent compensatory feedback loop that prevents activation of PI3K in amino acid-deprived environments.
While the ancient mTOR pathway regulates cell size and growth in all eukaryotes, multi-system organisms also possess components of the PI3K/Akt pathway to allow the additional layer of regulation provided by hormones and GFs. It is therefore exciting, though perhaps not surprising, that inflammatory signaling networks converge on metabolic pathways in higher eukaryotes to influence growth in response to stress. Indeed, TNFα and LPS, well-known inflammatory signals and IKK stimuli, induce phosphorylation of p85 at S690 (Supplemental Figure S2
). TNF also activates mTOR in an IKKβ-dependent manner involving phosphorylation and inhibition of the upstream inhibitor TSC1 to control growth and angiogenic potential in a breast cancer model (Lee et al., 2007
). Moreover, our lab has demonstrated that constitutive activation of Akt drives an interaction between IKKα and mTORC1 in PTEN-null prostate cancer cells which is important for control of mTOR phosphorylation of substrates S6K and 4EBP1 (Dan et al., 2007
). Thus, under oncogenic conditions, where Akt is constitutively activated, IKKα can function to promote mTORC1 activaiton. Conversely, TNFα and IKK family members are essential inflammatory mediators that promote insulin resistance in response to high fat diet (Hotamisligil et al., 1993
; Yuan et al., 2001
; Chiang et al., 2009
; Baker et al., 2011
). These studies indicate that under pathological conditions chronic inflammatory signaling pathways converge with and modulate GF responsive pathways that control metabolism. Further understanding the mechanistic underpinnings of inflammatory and metabolic pathway intersections will be important to understand how cell stress influences metabolic activity.