B-cell CLL/Lymphoma 10 (BCL10) Is Required for NF-��B Production by Both Canonical and Noncanonical Pathways and for NF-��B-inducing Kinase (NIK) Phosphorylation

B-cell CLL/lymphoma 10 (BCL10), the caspase recruitment domain (CARD)-containing protein involved in the etiology of the mucosa-associated lymphoid tissue (MALT) lymphomas, has been implicated in inflammatory processes in epithelial cells, as well as in immune cells. Experiments in this report indicate that BCL10 is required for activation of nuclear factor (NF)-κB by both canonical and noncanonical pathways, following stimulation by the sulfated polysaccharide carrageenan (CGN). In wild type and IκB-kinase (IKK)α^−/− mouse embryonic fibroblasts, increases in phospho-IκBα, nuclear NF-κB p65 (RelA) and p50, and KC, the mouse analog of human interleukin-8, were markedly reduced by silencing BCL10 or by exposure to the free radical scavenger Tempol. In IKKβ^−/− cells, BCL10 silencing, but not Tempol, reduced the CGN-induced increases in KC, phospho-NF-κB-inducing kinase (NIK), cytoplasmic NF-κB p100, and nuclear NF-κB p52 and RelB, suggesting a BCL10 requirement for activation of the noncanonical pathway. In NCM460 cells, derived from normal, human colonic epithelium, the CGN-induced increases in NF-κB family members, p65, p50, p52, and RelB, were inhibited by BCL10 silencing. Although enzyme-linked immunosorbent assay and confocal images demonstrated no change in total NIK following CGN, increases in phospho-NIK in the wild type, IKKβ^−/− and IKKα^−/− cells were inhibited by silencing BCL10. These findings indicate an upstream signaling role for BCL10, in addition to its effects on IKKγ, the regulatory component of the IKK signalosome, and a requirement for BCL10 in both canonical and noncanonical pathways of NF-κB activation. Also, the commonly used food additive carrageenan can be added to the short list of known activators of both pathways.     

Akt-dependent Activation of mTORC1 Complex Involves Phosphorylation of mTOR (Mammalian Target of Rapamycin) by IκB Kinase α (IKKα)

The serine/threonine protein kinase Akt promotes cell survival, growth, and proliferation through phosphorylation of different downstream substrates. A key effector of Akt is the mammalian target of rapamycin (mTOR). Akt is known to stimulate mTORC1 activity through phosphorylation of tuberous sclerosis complex 2 (TSC2) and PRAS40, both negative regulators of mTOR activity. We previously reported that IκB kinase α (IKKα), a component of the kinase complex that leads to NF-κB activation, plays an important role in promoting mTORC1 activity downstream of activated Akt. Here, we demonstrate IKKα-dependent regulation of mTORC1 using multiple PTEN null cancer cell lines and an animal model with deletion of IKKα. Importantly, IKKα is shown to phosphorylate mTOR at serine 1415 in a manner dependent on Akt to promote mTORC1 activity. These results demonstrate that IKKα is an effector of Akt in promoting mTORC1 activity.     

Lysine 63-linked Polyubiquitination of TAK1 at Lysine 158 Is Required for Tumor Necrosis Factor ��- and Interleukin-1��-induced IKK/NF-��B and JNK/AP-1 Activation

Transforming growth factor-β-activated kinase 1 (TAK1) plays an essential role in the tumor necrosis factor α (TNFα)- and interleukin-1β (IL-1β)-induced IκB kinase (IKK)/nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK)/activator protein 1 (AP-1) activation. Here we report that TNFα and IL-1β induce Lys⁶³-linked TAK1 polyubiquitination at the Lys¹⁵⁸ residue within the kinase domain. Tumor necrosis factor receptor-associated factors 2 and 6 (TRAF2 and -6) act as the ubiquitin E3 ligases to mediate Lys⁶³-linked TAK1 polyubiquitination at the Lys¹⁵⁸ residue in vivo and in vitro. Lys⁶³-linked TAK1 polyubiquitination at the Lys¹⁵⁸ residue is required for TAK1-mediated IKK complex recruitment. Reconstitution of TAK1-deficient mouse embryo fibroblast cells with TAK1 wild type or a TAK1 mutant containing a K158R mutation revealed the importance of this site in TNFα and IL-1β-mediated IKK/NF-κB and JNK/AP-1 activation as well as IL-6 gene expression. Our findings demonstrate that Lys⁶³-linked polyubiquitination of TAK1 at Lys¹⁵⁸ is essential for its own kinase activation and its ability to mediate its downstream signal transduction pathways in response to TNFα and IL-1β stimulation.     

Phosphorylation of Serine 399 in LKB1 Protein Short Form by Protein Kinase Cζ Is Required for Its Nucleocytoplasmic Transport and Consequent AMP-activated Protein Kinase (AMPK) Activation

Two splice variants of LKB1 exist: LKB1 long form (LKB1_L) and LKB1 short form (LKB1_S). In a previous study, we demonstrated that phosphorylation of Ser-428/431 (in LKB1_L) by protein kinase Cζ (PKCζ) was essential for LKB1-mediated activation of AMP-activated protein kinase (AMPK) in response to oxidants or metformin. Paradoxically, LKB1_S also activates AMPK although it lacks Ser-428/431. Thus, we hypothesized that LKB1_S contained additional phosphorylation sites important in AMPK activation. Truncation analysis and site-directed mutagenesis were used to identify putative PKCζ phosphorylation sites in LKB1_S. Substitution of Ser-399 to alanine did not alter the activity of LKB1_S, but abolished peroxynitrite- and metformin-induced activation of AMPK. Furthermore, the phosphomimetic mutation (S399D) increased the phosphorylation of AMPK and its downstream target phospho-acetyl-coenzyme A carboxylase (ACC). PKCζ-dependent phosphorylation of Ser-399 triggered nucleocytoplasmic translocation of LKB1_S in response to metformin or peroxynitrite treatment. This effect was ablated by pharmacological and genetic inhibition of PKCζ, by inhibition of CRM1 activity and by substituting Ser-399 with alanine (S399A). Overexpression of PKCζ up-regulated metformin-mediated phosphorylation of both AMPK (Thr-172) and ACC (Ser-79), but the effect was ablated in the S399A mutant. We conclude that, similar to Ser-428/431 (in LKB1_L), Ser-399 (in LKB1_S) is a PKCζ-dependent phosphorylation site essential for nucleocytoplasmic export of LKB1_S and consequent AMPK activation.