e IL12p40 reporter gene. Notably the aspartic acid substitution of all six modified residues showed maximal activity indicating a modest contribution of other residues. RIP2 activation of the IRF5 aspartic acid substitutions showed a similar positive effect of S451D and S462D function in this assay. Activity levels are not due to differences in protein expression. Considered together, the substitution mutations indicate two carboxyl terminal serine residues S451 and S462, particularly S462, are critical for transcriptional activity of IRF5. The T10, S158, S309, S317 appear to play an auxiliary role. To purchase BGJ 398 evaluate the biological significance of the critical phosphorylation sites in IRF5, we tested the response of IRF5 to activation by endogenous NOD2 pattern recognition receptors. NOD2 receptors are activated by muramyl dipeptide which is a component of the peptidoglycan cell wall of both Gram positive and Gram negative bacteria. We tested NOD2 receptors since RIP2 is a critical downstream effector of NOD2, and RIP2 was found to be a potent stimulator of IRF5 transcriptional activity. Macrophage cells expressing the IL12p40 reporter with either wt IRF5 or the IRF5 alanine substitution SS451,462AA were treated with either of the NOD2 natural ligands, MDP or PGN. Results clearly show that in response to endogenous NOD2 signaling, IRF5 wt induced the transcriptional activity of the IL12p40 gene. However, the IRF5 mutant that lacks the serines 451 and 462 was not able to induce gene expression. The results demonstrate natural ligand activation of NOD2 stimulates IRF5 activity only if the serine residues 451/ 462 are available for phosphorylation, confirming the relevance of the phosphoserines 451 and 462 identified with mass spectrometry. Phosphorylation regulates IRF5 nuclear trafficking In a 
static image latent IRF5 appears in the cytoplasm, however it dynamically shuttles in and out of the nucleus. This can be demonstrated by inhibiting IRF5 nuclear export with the antibiotic leptomycin B . Treatment of cells with LMB results in nuclear accumulation of latent IRF5, and this result indicates IRF5 has a constitutive nuclear localization signal IRF5 Activation interactive domain, the nuclear localization signals, and the nuclear export signal. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22187495 The amino acid phosphorylation sites of IRF5 identified by mass spectrometry are noted . b) The IL12p40 luciferase reporter assay was used to evaluate the effect of loss-of-function alanine substitutions in IRF5 in HEK293 cells. Single mutations or double mutations were introduced in IRF5 and their effect on the IL12p40 luciferase reporter was measured alone or with RIP2. Luciferase activity is expressed relative to Renilla control and values are means of three to six independent experiments. P,0.0001 c) Gain-of-function mutations were evaluated with aspartic acid substitutions for the phosphorylated amino acids. Effect of aspartic acid mutations on basal activity of IRF5 was measured with the IL12p40 luciferase reporter assay as in. P,0.0001 d) Effect of IRF5 aspartic acid activating mutations in the absence or presence of RIP2 with the IL12p40 luciferase reporter assay as in. doi:10.1371/journal.pone.0033098.g003 as well as a constitutive dominant nuclear export signal. The location of two identified NLSs and one NES have been reported and are shown in 4 IRF5 Activation had no notable effect on IRF5 localization, but S451D resulted in detectable nuclear IRF5 in 2030% of the cells. The reason for the se
