Replacement of the C-terminal tetrapeptide (314PAPV317 to 314SSSM317) in interferon regulatory factor-2 alters its N-terminal DNA-binding activity

Abstract

Interferon regulatory factor-2 (IRF-2) is an important transcription factor involved in cell growth regulation, immune response and cancer. IRF-2 can function as a transcriptional repressor and activator depending on its DNA-binding activity and protein-protein interactions. We compared the amino acid sequences of IRF-2 and found a C-terminal tetrapeptide (314PAPV317) of mouse IRF-2 to be different (314SSSM317) from human IRF-2. Recombinant GST-IRF-2 with 314PAPV317 (wild type) and 314SSSM317 (mutant) expressed in Escherichia coli were assessed for DNA-binding activity with 32P-(GAAAGT) 4 by electrophoretic mobility shift assay (EMSA). Wild type- and mutant GST-IRF-2 showed similar expression patterns and immunoreactivities but different DNA-binding activities. Mutant (mt) IRF-2 formed higher-molecular-mass, more and stronger DNA-protein complexes in comparison to wild type (wt) IRF-2. Anti-IRF-2 antibody stabilized the DNA-protein complexes formed by both wt IRF-2 and mt IRF-2, resolving the differences. This suggests that PAPV and SSSM sequences at 314-317 in the C-terminal region of mouse and human IRF-2 contribute to conformation of IRF-2 and influence DNA-binding activity of the N-terminal region, indicating intramolecular interactions. Thus, evolution of IRF-2 from murine to human genome has resulted in subtle differences in C-terminal amino acid motifs, which may contribute to qualitative changes in IRF-2-dependent DNA-binding activity and gene expression.

HCFC2 is needed for IRF1- and IRF2-dependent Tlr3 transcription and for survival during viral infections

“…We hypothesize that the interaction between HCFC2 and either IRF1 or IRF2 may generate a conformational change in the IRF that favors DNA binding. An effect of amino acid substitutions or of protein interactions with the IRF2 C terminus, to which HCFC2 binds, on DNA binding by the N terminus has previously been reported ( Childs and Goodbourn, 2003 ; Prakash and Rath, 2010 ) and supports this hypothesis. Moreover, a function similar to the one we propose for HCFC2 has been suggested for HCFC1 in its interaction with VP16, an HSV protein that directs the formation of a DNA-binding complex necessary for viral immediate early gene transcription ( Kristie and Sharp, 1990 ).…”