Identification of a regulatory autophosphorylation site in the serine-threonine kinase RIP2.
Receptor-interacting protein 2 (RIP2) is a serine-threonine kinase that mediates signaling for many receptors of the innate and adaptive immune systems. Toll like receptors (TLR) are an important component of the innate immune response. Stimulation of RIP2-deficient cells with ligands for TLR 2, 3 and 4 results in impaired cytokine production and decreased activation of NF-kB and MAP kinases compared to wild-type cells. Stimulation of TLR 4 with its ligand lipopolysaccaride (LPS) leads to the activation of RIP2 kinase activity and its autophosphorylation. Here we identify serine residue 176 as a site of autophosphorylation using a combination of mass spectrometry and mutational analysis. Mutation of S176 to alanine not only abolishes autophosphorylation of RIP2 but also significantly decreases its catalytic activity. A phospho-specific anti-S176 antibody detects wild-type RIP2 but not kinase-dead RIP2 or the RIP2 S176A mutant. Endogenous RIP2 in THP-1 cells and mouse bone marrow derived macrophages can be detected by the phospho-RIP2 (S176) antibody only after stimulation with LPS suggesting that the antibody recognizes activated RIP2. In summary, our results indicate that S176 is a regulatory autophosphorylation site for RIP2 and that S176 phosphorylation can be used to monitor the activation state of RIP2.
Dorsch
Marion
M
Millennium Pharmaceuticals, Inc, 35 Landsdowne St., Cambridge, MA 02130, USA. marion.dorsch@novartis.com
Wang
Anlai
A
Cheng
Hong
H
Lu
Chafen
C
Bielecki
Andrew
A
Charron
Kathryn
K
Clauser
Karl
K
Ren
Hong
H
Polakiewicz
Roberto D
RD
Parsons
Tom
T
Li
Ping
P
Ocain
Tim
T
Xu
Yajun
Y
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity / Q000276:immunology; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002853:Chromatography, Liquid; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D020539:Sequence Analysis, Protein / Q000379:methods; D018411:Spodoptera
D011994:Recombinant Proteins; C506253:RIPK2 protein, human; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2