tyrosine phosphorylation of the e3 ubiquitin ligase trim21 positively regulates interaction with irf3 and hence trim21 activity酪氨酸磷酸化的e3泛素连接酶trim21积极调节与irf3因此trim21互动活动.pdfVIP

Tyrosine Phosphorylation of the E3 Ubiquitin Ligase TRIM21 Positively Regulates Interaction with IRF3 and Hence TRIM21 Activity 1 1,2 1 Kevin B. Stacey , Eamon Breen , Caroline A. Jefferies * 1 Department Molecular and Cellular Therapeutics, Royal College Surgeons in Ireland Research Institute, Royal College of Surgeons in Ireland, Dublin, Ireland, 2 Centenary Institute and Arthur William (AW) Morrow Gastroenterology and Liver Centre, Camperdown, Australia, Abstract Patients suffering from Systemic Lupus Erythematous (SLE) have elevated type I interferon (IFN) levels which correlate with disease activity and severity. TRIM21, an autoantigen associated with SLE, has been identified as an ubiquitin E3 ligase that targets the transcription factor IRF3 in order to turn off and limit type I IFN production following detection of viral and bacterial infection by Toll Like Receptors (TLRs). However, how the activity of TRIM21 is regulated downstream of TLRs is unknown. In this study we demonstrate that TRIM21 is tyrosine phosphorylated following TLR3 and TLR4 stimulation, suggesting that its activity is potentially regulated by tyrosine phosphorylation. Using Netphos, we have identified three key tyrosines that are strongly predicted to be phosphorylated, two of which are conserved between the human and murine forms of TRIM21, at residues 343, 388, and 393, all of which have been mutated from tyrosine to phenylalanine (Y343F, Y388F, and Y393F). We have observed that tyrosine phosphorylation of TRIM21 only occurs in the substrate binding PRY/ SPRY domain, and that Y393, and to a lesser extent, Y388 are required for TRIM21 to function as a negative regulator of IFN- b promoter activity. Further studies revealed that mutating Y393 to phenylalanine inhibits the ability of TRIM21 to i