Jason A. Gustin

NF-κB activation by tumour necrosis factor requires the Akt serine–threonine kinase

Activation of the nuclear transcription factor NF-κB by inflammatory cytokines requires the successive action of NF-κB-inducing kinase (NIK) and an IκB-kinase (IKK) complex composed of IKKα and IKKβ. Here we show that the Akt serine–threonine kinase is involved in the activation of NF-κB by tumour necrosis factor (TNF). TNF activates phosphatidylinositol-3-OH kinase (PI(3)K) and its downstream target Akt (protein kinase B). Wortmannin (a PI(3)K inhibitor), dominant-negative PI(3)K or kinase-dead Akt inhibits TNF-mediated NF-κB activation. Constitutively active Akt induces NF-κB activity and this effect is blocked by dominant-negative NIK. Conversely, NIK activates NF-κB and this is blocked by kinase-dead Akt. Thus, both Akt and NIK are necessary for TNF activation of NF-κB. Akt mediates IKKα phosphorylation at threonine 23. Mutation of this amino acid blocks phosphorylation by Akt or TNF and activation of NF-κB. These findings indicate that Akt is part of a signalling pathway that is necessary for inducing key immune and inflammatory responses.

A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1

Tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) by the insulin receptor permits this docking protein to interact with signaling proteins that promote insulin action. Serine phosphorylation uncouples IRS-1 from the insulin receptor, thereby inhibiting its tyrosine phosphorylation and insulin signaling. For this reason, there is great interest in identifying serine/threonine kinases for which IRS-1 is a substrate. Tumor necrosis factor (TNF) inhibited insulin-promoted tyrosine phosphorylation of IRS-1 and activated the Akt/protein kinase B serine-threonine kinase, a downstream target for phosphatidylinositol 3-kinase (PI 3-kinase). The effect of TNF on insulin-promoted tyrosine phosphorylation of IRS-1 was blocked by inhibition of PI 3-kinase and the PTEN tumor suppessor, which dephosphorylates the lipids that mediate PI 3-kinase functions, whereas constitutively active Akt impaired insulin-promoted IRS-1 tyrosine phosphorylation. Conversely, TNF inhibition of IRS-1 tyrosine phosphorylation was blocked by kinase dead Akt. Inhibition of IRS-1 tyrosine phosphorylation by TNF was blocked by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a downstream target of Akt. mTOR induced the serine phosphorylation of IRS-1 (Ser-636/639), and such phosphorylation was inhibited by rapamycin. These results suggest that TNF impairs insulin signaling through IRS-1 by activation of a PI 3-kinase/Akt/mTOR pathway, which is antagonized by PTEN.

Cell Type-specific Expression of the IκB Kinases Determines the Significance of Phosphatidylinositol 3-Kinase/Akt Signaling to NF-κB Activation

Phosphatidylinositol (PI) 3-kinase/Akt signaling activates NF-κB through pleiotropic, cell type-specific mechanisms. This study investigated the significance of PI 3-kinase/Akt signaling to tumor necrosis factor (TNF)-induced NF-κB activation in transformed, immortalized, and primary cells. Pharmacological inhibition of PI 3-kinase blocked TNF-induced NF-κB DNA binding in the 293 line of embryonic kidney cells, partially affected binding in MCF-7 breast cancer cells, HeLa and ME-180 cervical carcinoma cells, and NIH 3T3 cells but was without significant effect in H1299 and human umbilical vein endothelial cells, cell types in which TNF activated Akt. NF-κB is retained in the cytoplasm by inhibitory proteins, IκBs, which are phosphorylated and targeted for degradation by IκB kinases (IKKα and IKKβ). Expression and the ratios of IKKα and IKKβ, which homo- and heterodimerize, varied among cell types. Cells with a high proportion of IKKα (the IKK kinase activated by Akt) to IKKβ were most sensitive to PI 3-kinase inhibitors. Consequently, transient expression of IKKβ diminished the capacity of the inhibitors to block NF-κB DNA binding in 293 cells. Also, inhibitors of PI 3-kinase blocked NF-κB DNA binding in Ikkβ–/– but not Ikkα–/– or wild-type cells in which the ratio of IKKα to IKKβ is low. Thus, noncoordinate expression of IκB kinases plays a role in determining the cell type-specific role of Akt in NF-κB activation.

Cell type specific expression of the IkB kinases determines the significance of PI 3-kinase/Akt signaling to NF-kB activation

Phosphatidylinositol (PI) 3-kinase/Akt signaling activates NF-κB through pleiotropic, cell type-specific mechanisms. This study investigated the significance of PI 3-kinase/Akt signaling to tumor necrosis factor (TNF)-induced NF-κB activation in transformed, immortalized, and primary cells. Pharmacological inhibition of PI 3-kinase blocked TNF-induced NF-κB DNA binding in the 293 line of embryonic kidney cells, partially affected binding in MCF-7 breast cancer cells, HeLa and ME-180 cervical carcinoma cells, and NIH 3T3 cells but was without significant effect in H1299 and human umbilical vein endothelial cells, cell types in which TNF activated Akt. NF-κB is retained in the cytoplasm by inhibitory proteins, IκBs, which are phosphorylated and targeted for degradation by IκB kinases (IKKα and IKKβ). Expression and the ratios of IKKα and IKKβ, which homo- and heterodimerize, varied among cell types. Cells with a high proportion of IKKα (the IKK kinase activated by Akt) to IKKβ were most sensitive to PI 3-kinase inhibitors. Consequently, transient expression of IKKβ diminished the capacity of the inhibitors to block NF-κB DNA binding in 293 cells. Also, inhibitors of PI 3-kinase blocked NF-κB DNA binding in Ikkβ–/– but not Ikkα–/– or wild-type cells in which the ratio of IKKα to IKKβ is low. Thus, noncoordinate expression of IκB kinases plays a role in determining the cell type-specific role of Akt in NF-κB activation.

Sir-two-homolog 2 (Sirt2) modulates peripheral myelination through polarity protein Par-3/atypical protein kinase C (aPKC) signaling

The formation of myelin by Schwann cells (SCs) occurs via a series of orchestrated molecular events. We previously used global expression profiling to examine peripheral nerve myelination and identified the NAD+-dependent deacetylase Sir-two-homolog 2 (Sirt2) as a protein likely to be involved in myelination. Here, we show that Sirt2 expression in SCs is correlated with that of structural myelin components during both developmental myelination and remyelination after nerve injury. Transgenic mice lacking or overexpressing Sirt2 specifically in SCs show delays in myelin formation. In SCs, we found that Sirt2 deacetylates Par-3, a master regulator of cell polarity. The deacetylation of Par-3 by Sirt2 decreases the activity of the polarity complex signaling component aPKC, thereby regulating myelin formation. These results demonstrate that Sirt2 controls an essential polarity pathway in SCs during myelin assembly and provide insights into the association between intracellular metabolism and SC plasticity.

Akt Regulates Basal and Induced Processing of NF-κB2 (p100) to p52

NF-κB is a family of transcription factors important for innate and adaptive immunity. NF-κB is restricted to the cytoplasm by inhibitory proteins that are degraded when specifically phosphorylated, permitting NF-κB to enter the nucleus and activate target genes. Phosphorylation of the inhibitory proteins is mediated by an IκB kinase (IKK) complex, which can be composed of two subunits with enzymatic activity, IKKα and IKKβ. The preferred substrate for IKKβ is IκBα, degradation of which liberates p65 (RelA) to enter the nucleus where it induces genes important to innate immunity. IKKα activates a non-canonical NF-κB pathway in which p100 (NF-κB2) is processed to p52. Once produced, p52 can enter the nucleus and induce genes important to adaptive immunity. This study shows that Akt binds to and increases the activity of IKKα and thereby increases p52 production in cells. Constitutively active Akt augments non-canonical NF-κB activity, whereas kinase dead Akt or inhibition of phosphatidylinositol 3-kinase have the opposite effect. Basal and ligand-induced p52 production is reduced in mouse embryo fibroblasts deficient in Akt1 and Akt2 compared with parental cells. These observations show that Akt plays a role in activation of basal and induced non-canonical NF-κB activity.

Misexpression of Pou3f1 Results in Peripheral Nerve Hypomyelination and Axonal Loss

Pou3f1/SCIP/Oct-6 is a POU-domain transcription factor that is an important regulator of peripheral nerve myelination by Schwann cells. Pou3f1-deficient mice experience a developmental delay in myelination indicating that transient induction of Pou3f1 is required for normal development of peripheral myelin. The mechanism by which Pou3f1 regulates myelination is unclear, because it can both increase expression of Egr2, a transcription factor that promotes the myelination program, and also repress the promoters of specific myelin genes such as myelin protein zero (MPZ) and myelin basic protein (MBP). Therefore, to investigate the effects of persistent Pou3f1 expression on peripheral nerve myelination, we created a conditional transgenic mouse [condPou3f1:MPZ(Cre)] that constitutively expresses Pou3f1 specifically in peripheral glia. Examination of sciatic nerves from condPou3f1:MPZ(Cre) mice revealed persistent hypomyelination and eventual axonal loss but no evidence of demyelination/remyelination processes or impaired Schwann cell proliferation. Nerves from these mice had normal levels of Egr2 mRNA but decreased levels of MPZ, MBP, and Pmp22 mRNA. Thus, unlike the Pou3f1 null mice, the condPou3f1:MPZ(Cre) mice exhibit persistent hypomyelination, indicating that strict control of Pou3f1 expression is critical to proper myelination. Our findings establish the importance of identifying factor(s) responsible for Pou3f1 downregulation during myelination, because they may play important roles in the development of peripheral neuropathies.

reply: Kinase regulation in inflammatory response

Ozes et al. replyDelhase et al. take issue with our claim that Akt induces activation of NF-κB by phosphorylating IKKα, contending that IKKα plays no role in the activation by TNF of NF-κB, and consequently that Akt could not affect NF-κB through IKKα. They point out that Hu et al. have shown that cells deficient in IKKα have normal TNF-induced NF-κB activity, but this has been refuted by Li et al., who reported significant reduction of TNF-induced NF-κB in IKKα-deficient cells. Indeed, the observations of Hu et al. show that degradation of IκBα is diminished in cells from IKKα-deficient mice and are therefore not consistent with the conclusion that IKKα plays no role in TNF induction of NF-κB. Furthermore, deficiency of IKKβ only partially impairs TNF-induced NF-κB activation, which reserves a role for IKKα in this pathway. Others have shown that activation of the IKK complex is dependent on the kinase activity of IKKα to activate IKKβ. Thus, strong evidence supports a role for IKKα in TNF induction of NF-κB.

Tumor Necrosis Factor-α/Receptor Signaling Through the Akt Kinase

Tumor necrosis factor (TNF) is a pleiotropic cytokine that can affect the growth, differentiation, and metabolism of virtually every nucleated cell type in the body. TNF promotes immunity, but its expression is also associated with pathologies, such as rheumatoid arthritis, type II diabetes, and cachexia. Two distinct cell-surface receptors bind TNF, the type I receptor (TNFR1), which contains a conserved motif called a “death domain“ in its C-terminus, and the type II receptor. Binding of TNF to TNFR1 brings the death domains of TNFR1 into physical proximity, thereby promoting their interactions with cytoplasmic proteins that also contain death domains. Thus, a signal transduction cascade is initiated that coincidentally activates caspases that promote cell death and, additionally, anti-apoptotic events. The balance between these arms of the TNFR1 signaling cascade determines whether cells live or die.