Structural and functional analysis of interferon regulatory factor 3: Localization of the transactivation and autoinhibitory domains

Citation
Rt. Lin et al., Structural and functional analysis of interferon regulatory factor 3: Localization of the transactivation and autoinhibitory domains, MOL CELL B, 19(4), 1999, pp. 2465-2474
Citations number
36
Categorie Soggetti
Molecular Biology & Genetics
Journal title
MOLECULAR AND CELLULAR BIOLOGY
ISSN journal
02707306 → ACNP
Volume
19
Issue
4
Year of publication
1999
Pages
2465 - 2474
Database
ISI
SICI code
0270-7306(199904)19:4<2465:SAFAOI>2.0.ZU;2-X
Abstract
The interferon regulatory factor 3 (IRF-3) gene encodes a 55-kDa protein wh ich is expressed constitutively in all tissues. In unstimulated cells, IRF- 3 is present in an inactive cytoplasmic form; following Sendai virus infect ion, IRF3 is posttranslationally modified by protein phosphorylation at mul tiple serine and threonine residues located in the carboxy terminus. Virus- induced phosphorylation of IRF3 leads to cytoplasmic to nuclear translocati on of phosphorylated IRF3, association with the transcriptional coactivator CBP/p300, and stimulation of DNA binding and transcriptional activities of virus-inducible genes, Using yeast and mammalian one-hybrid analysis, we n ow demonstrate that an extended, atypical transactivation domain is located in the C terminus of IRF3 between amino acids (aa) 134 and 394, We also sh ow that the C-terminal domain of IRF-3 located between aa 380 and 427 parti cipates in the autoinhibition of IRF3 activity via an intramolecular associ ation with the N-terminal region between aa 98 and 240, After Sendai virus infection, an intermolecular association between IRF-3 proteins is detected , demonstrating a virus-dependent formation of IRF3 homodimers; this intera ction is also observed in the absence of virus infection with a constitutiv ely activated form of IRF3. Substitution of the C-terminal Ser-Thr phosphor ylation sites with the phosphomimetic Asp in the region ISNSHPLSLTSDQ betwe en amino acids 395 and 407 [IRF-3(5D)], but not the adjacent S385 and S386 residues, generates a constitutively activated DNA binding form of IRF3. In contrast, substitution of S385 and S386 with either Ala or Asp inhibits bo th DNA binding and transactivation activities of the IRF-3(5D) protein. The se studies thus define the transactivation domain of IRF-3, two domains tha t participate in the autoinhibition of IRF3 activity, and the regulatory ph osphorylation sites controlling IRF-3 dimer formation, DNA binding activity , and association with the CBP/p300 coactivator.