Gregory W. Peet

Novel NEMO/IκB Kinase and NF-κB Target Genes at the Pre-B to Immature B Cell Transition

The IκB kinase (IKK) signaling complex is responsible for activating NF-κB-dependent gene expression programs. Even though NF-κB-responsive genes are known to orchestrate stress-like responses, critical gaps in our knowledge remain about the global effects of NF-κB activation on cellular physiology. DNA microarrays were used to compare gene expression programs in a model system of 70Z/3 murine pre-B cellsversus their IKK signaling-defective 1.3E2 variant with lipopolysaccharide (LPS), interleukin-1 (IL-1), or a combination of LPS + phorbol 12-myristate 13-acetate under brief (2 h) or long term (12 h) stimulation. 70Z/3-1.3E2 cells lack expression of NEMO/IKKγ/IKKAP-1/FIP-3, an essential positive effector of the IKK complex. Some stimulated hits were known NF-κB target genes, but remarkably, the vast majority of the up-modulated genes and an unexpected class of repressed genes were all novel targets of this signaling pathway, encoding transcription factors, receptors, extracellular ligands, and intracellular signaling factors. Thirteen stimulated (B-ATF, Pim-2, MyD118,Pea-15/MAT1, CD82, CD40L,Wnt10a, Notch 1, R-ras,Rgs-16, PAC-1, ISG15, andCD36) and five repressed (CCR2,VpreB, λ5, SLPI, andCMAP/Cystatin7) genes, respectively, were bona fide NF-κB targets by virtue of their response to a transdominant IκBαSR (super repressor). MyD118 andISG15, although directly induced by LPS stimulation, were unaffected by IL-1, revealing the existence of direct NF-κB target genes, which are not co-induced by the LPS and IL-1 Toll-like receptors.

Novel NEMO/IKKγ and NF-κB target genes at the Pre-B to immature B cell transition

The IκB kinase (IKK) signaling complex is responsible for activating NF-κB-dependent gene expression programs. Even though NF-κB-responsive genes are known to orchestrate stress-like responses, critical gaps in our knowledge remain about the global effects of NF-κB activation on cellular physiology. DNA microarrays were used to compare gene expression programs in a model system of 70Z/3 murine pre-B cellsversus their IKK signaling-defective 1.3E2 variant with lipopolysaccharide (LPS), interleukin-1 (IL-1), or a combination of LPS + phorbol 12-myristate 13-acetate under brief (2 h) or long term (12 h) stimulation. 70Z/3-1.3E2 cells lack expression of NEMO/IKKγ/IKKAP-1/FIP-3, an essential positive effector of the IKK complex. Some stimulated hits were known NF-κB target genes, but remarkably, the vast majority of the up-modulated genes and an unexpected class of repressed genes were all novel targets of this signaling pathway, encoding transcription factors, receptors, extracellular ligands, and intracellular signaling factors. Thirteen stimulated (B-ATF, Pim-2, MyD118,Pea-15/MAT1, CD82, CD40L,Wnt10a, Notch 1, R-ras,Rgs-16, PAC-1, ISG15, andCD36) and five repressed (CCR2,VpreB, λ5, SLPI, andCMAP/Cystatin7) genes, respectively, were bona fide NF-κB targets by virtue of their response to a transdominant IκBαSR (super repressor). MyD118 andISG15, although directly induced by LPS stimulation, were unaffected by IL-1, revealing the existence of direct NF-κB target genes, which are not co-induced by the LPS and IL-1 Toll-like receptors.

Recombinant IκB Kinases α and β Are Direct Kinases of IκBα

Activation of the transcription factor NF-κB is regulated by the phosphorylation and subsequent degradation of its inhibitory subunit, IκB. A large multiprotein complex, the IκB kinase (IKK), catalyzes the phosphorylation of IκB. The two kinase components of the IKK complex, IKKα and IKKβ, were overexpressed in insect cells and purified to homogeneity. Both purified IKKα and IKKβ specifically catalyzed the phosphorylation of the regulatory serine residues of IκBα. Hence, IKKα and IKKβ were functional catalytic subunits of the IKK complex. Purified IKKα and IKKβ also preferentially phosphorylated serine as opposed to threonine residues of IκBα, consistent with the substrate preference of the IKK complex. Kinetic analysis of purified IKKα and IKKβ revealed that the kinase activity of IKKβ on IκBα is 50–60-fold higher than that of IKKα. The primary difference between the two activities is the K m for IκBα. The kinetics of both IKKα and IKKβ followed a sequential Bi Bi mechanism. No synergistic effects on IκBα phosphorylation were detected between IKKα and IKKβ. Thus, in vitro, IKKα and IKKβ are two independent kinases of IκBα.