NF-κB-Inducing Kinase Is Involved in the Activation of the CD28 Responsive Element through Phosphorylation of c-Rel and Regulation of Its Transactivating Activity

Abstract

Previous evidence suggested that NF-κB-inducing kinase (NIK) might regulate IL-2 synthesis. However, the molecular mechanism is not understood. In this study, we show that NIK is involved in CD3 plus CD28 activation of IL-2 transcription. Splenic T cells from aly/aly mice (that have a defective NIK protein) have a severe impairment in IL-2 and GM-CSF but not TNF secretion in response to CD3/CD28. This effect takes place at the transcriptional level as overexpression of alyNIK inhibits IL-2 promoter transcription. NIK activates the CD28 responsive element (CD28RE) of the IL-2 promoter and strongly synergizes with c-Rel in this activity. We found that NIK interacts with the N-terminal domain of c-Rel, mapping this interaction to aa 771–947 of NIK. Moreover, NIK phosphorylates the c-Rel C-terminal transactivation domain (TAD) and induces Gal4-c-Rel-transactivating activity. Anti-CD28 activated Gal4-c-Rel transactivation activity, and this effect was inhibited by a NIK-defective mutant. Deletion studies mapped the region of c-Rel responsive to NIK in aa 456–540. Mutation of several serines, including Ser471, in the TAD of c-Rel abrogated the NIK-enhancing activity of its transactivating activity. Interestingly, a Jurkat mutant cell line that expresses one of the mutations of c-Rel (Ser471Asn) has a severe defect in IL-2 and CD28RE-dependent transcription in response to CD3/CD28 or to NIK. Our results support that NIK may be controlling CD28RE-dependent transcription and T cell activation by modulating c-Rel phosphorylation of the TAD. This leads to more efficient transactivation of genes which are dependent on CD28RE sites where c-Rel binds such as the IL-2 promoter.

Mutation of an IKK phosphorylation site within the transactivation domain of REL in two patients with B-cell lymphoma enhances REL's in vitro transforming activity

“…IKK-and NIK-associated kinases have been shown to phosphorylate sites within residues 422-540 of REL in vitro (Lawrence et al, 2005;Harris et al, 2006;Sa´nchez-Valdepen˜as et al, 2006), and we show that both IKKa and IKKb can phosphorylate Ser525 of REL in vitro. However, Ser525 appears to be only one of multiple sites in the C-terminal, half of REL that can serve as in vitro phosphorylation sites for IKK, given that a fragment containing the entire C-terminal REL transactivation domain with the S525P mutation has only a slightly reduced ability to be phosphorylated by IKKa and IKKb in vitro (Figure 6b and e).…”

Post-translational modifications regulating the activity and function of the nuclear factor kappa B pathway

“…c-Rel-mediated transactivation is stimulated by phosphorylation within its transactivation domain at Ser-471 by PKCz in response to TNF and possibly also by NIK (Martin and Fresno, 2000;Martin et al, 2001;Sa´nchez-Valdepen˜as et al, 2006) (Figure 3; Table 4). Other residues in the c-Rel transactivation domain are also phosphorylated (Martin and Fresno, 2000), and mutation of Ser-454 and Ser-460 has been shown to affect c-Rel function (Starczynowski et al, 2005).…”

Nuclear factor- B inducing kinase is required for graft-versus-host disease

“…2 Several groups have previously shown that NIK has an important role in IL-2 production by T cells. 15,16 IL-2 enhances the expansion of T lymphocytes and is crucial for correct CD8 effector function. 42 NIK regulates the production of IL-17, 27 another cytokine with a role in GVHD.…”

“…This may be related to the lower IL-2 concentration found in aly/aly mice and the poor ability to release IL-2 shown in this study and previously described. 16 T-cell apoptosis linked to T-cell activation is a process known as activation-induced cell death. 30,31 T lymphocytes are resistant to activation-induced cell death in the earliest period after activation.…”

“…We recently found that NIK has a role in T lymphocyte activation, regulating the production of IL-2 through the activation of the CD28 responsive element (CD28RE) of the IL-2 promoter. 16 This effect takes place through the phosphorylation of the cRel C-terminal transactivation domain by NIK. In the light of the crucial role of IL-2 in GVHD, 2 we addressed the role of NIK in GVHD, using primary human samples and a murine model of the disease.…”