NF-kappaB-inducing kinase is involved in the activation of the CD28 responsive element through phosphorylation of c-Rel and regulation of its transactivating activity.
Previous evidence suggested that NF-kappaB-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.
Sánchez-Valdepeñas
Carmen
C
Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain.
Martín
Angel G
AG
Ramakrishnan
Parameswaran
P
Wallach
David
D
Fresno
Manuel
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D018106:CD28 Antigens / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020573:Genes, rel; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis; D019169:Jurkat Cells; D051379:Mice; D008817:Mice, Mutant Strains; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000276:immunology / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D020569:Proto-Oncogene Proteins c-rel; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D000097782:NF-kappaB-Inducing Kinase
D018106:CD28 Antigens; D004268:DNA-Binding Proteins; D007376:Interleukin-2; D016328:NF-kappa B; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D020569:Proto-Oncogene Proteins c-rel; D015534:Trans-Activators; D000097782:NF-kappaB-Inducing Kinase; C068059:REL protein, human