Identification of the phosphorylation sites on the E3 ubiquitin ligase Pellino that are critical for activation by IRAK1 and IRAK4.
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.
Smith
Hilary
H
Medical Research Council Protein Phosphorylation Unit, The Sir James Black Centre, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland.
Peggie
Mark
M
Campbell
David G
DG
Vandermoere
Franck
F
Carrick
Emma
E
Cohen
Philip
P
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
D050505:Mutant Proteins; D010455:Peptides; D010769:Phosphothreonine; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D053592:Interleukin-1 Receptor-Associated Kinases