title: Identification of the phosphorylation sites on the E3 ubiquitin ligase Pellino that are critical for activation by IRAK1 and IRAK4.
abstract: The E3 ubiquitin ligase Pellino can be activated by phosphorylation in vitro, catalyzed by IL-1 receptor-associated kinase 1 (IRAK1) or IRAK4. Here, we show that phosphorylation enhances the E3 ligase activity of Pellino 1 similarly with any of several E2-conjugating enzymes (Ubc13-Uev1a, UbcH4, or UbcH5a/5b) and identify 7 amino acid residues in Pellino 1 whose phosphorylation is critical for activation. Five of these sites are clustered between residues 76 and 86 (Ser-76, Ser-78, Thr-80, Ser-82, and Thr-86) and decorate a region of antiparallel beta-sheet, termed the "wing," which is an appendage of the forkhead-associated domain that is thought to interact with IRAK1. The other 2 sites are located at Thr-288 and Ser-293, just N-terminal to the RING-like domain that carries the E3 ligase activity. Unusually, the full activation of Pellino 1 can be achieved by phosphorylating any one of several different sites (Ser-76, Thr-86, Thr-288, or Ser-293) or a combination of other sites (Ser-78, Thr-80, and Ser-82). These observations imply that dephosphorylation of multiple sites is required to inactivate Pellino 1, which could be a device for prolonging Pellino's E3 ubiquitin ligase activity in vivo.
authors.affiliation: Medical Research Council Protein Phosphorylation Unit, The Sir James Black Centre, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland.
authors.forename: Hilary
authors.initials: H
authors.lastname: Smith
authors.forename: Mark
authors.initials: M
authors.lastname: Peggie
authors.forename: David G
authors.initials: DG
authors.lastname: Campbell
authors.forename: Franck
authors.initials: F
authors.lastname: Vandermoere
authors.forename: Emma
authors.initials: E
authors.lastname: Carrick
authors.forename: Philip
authors.initials: P
authors.lastname: Cohen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D002853:Chromatography, Liquid; D004789:Enzyme Activation; D007313:Insecta; D053592:Interleukin-1 Receptor-Associated Kinases / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D017433:Protein Structure, Secondary; D020539:Sequence Analysis, Protein; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D050505:Mutant Proteins; D010455:Peptides; D010769:Phosphothreonine; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D053592:Interleukin-1 Receptor-Associated Kinases