In the progress of seeking for new partners of IRAK4 - a critical kinase of theIL-1R/TLR signaling pathways - using the yeast two-hybrid screening, AUP1(Ancient Ubiquitous Protein 1) was identified to interact with IRAK4. AUP1 is aprotein which is highly conserved in evolution and is ubiquitously expressed intissues of mouse and human. However, the functions of AUP1 in cell biology havenot been clearly defined yet.The interaction of AUP1 was confirmed for the interaction with IRAK4 and IRAK1in HEK293RI cells. Interestingly, AUP1 was only found to interact with kinaseinactive IRAK4 and IRAK1 but not with the wild type molecules, indicating that thekinase activity of IRAK molecules interferes with their association with AUP1. Inaddition, AUP1 can be phosphorylated by IRAK4 and possibly IRAK1 as shown inthe forced co-immunoprecipitation and in vitro kinase assays. Besides IRAK4 andIRAK1, AUP1 interacts with other components in the IL-1 signaling machinerysuch as IL-1RI, MyD88, Tollip, TRAF6, suggesting the involvement of AUP1 in theIL-1 pathway.Indeed, overexpression of AUP1 in HEK293RI and KeratinoRI-/- cells reduces IL-1beta- and LPS-induced activation of NF-kappaB as well as chemokine/cytokineproduction, respectively. However, the regulatory effect of AUP1 overexpression isnot specific for IL-1 or LPS signaling as TNFalpha signaling is also impaired by AUP1overexpression. AUP1 was also found to associate with TNFR1, TRADD andTRAF2 which are components of TNFalpha; pathway. Interestingly, althoughoverexpression of AUP1 does not affect STAT-mediated IL-6 signaling, AUP1interacts with the IL-6R alpha chain but not with the signaling transducer gp130. Incontrast, silencing of AUP1 via siRNA enhances the IL-1beta-, TNFalpha-stimulatedactivation of NF-kappaB as well as IL-6-stimulated activation of STAT3, indicating thatAUP1 is a common negative regulator of the cytokine signaling pathwaysinvestigated.As overexpression of AUP1 interferes with IL-1beta/LPS signaling transductionupstream of IkappaB and the JNK/p38 MAPK and AUP1 was found to associate with allthree of the cytokine receptors IL-1RI, TNFR1 and IL-6R alpha, the role of AUP1 maybe to down-regulate the response of cells to outside stimuli via the impairment ofthe expression and/or performance of these receptors on the cell surface.
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