[The attachment manner of ubiquitin to proinflammatory molecule discoidin domain receptor 2].
OBJECTIVE
To understand which lysine (K) residue in ubiquitin (Ub) is used to form a poly-Ubs chain on discoidin domain receptor 2 (DDR2).


METHODS
The constructs encoding human DDR2 and Cbl-b were transiently transfected into HEK293T cells together with the Ub mutants with lysine mutation at different sites, and the transfected cells were stimulated with collagens in order to induce the activation of DDR2. Immunoprecipitation was performed to isolate DDR2, and the immunoprecipitates were then subjected to immunoblot analysis for the conjugation of DDR2 with Ub.


RESULTS
Ub K27-only mutant displayed the strongest poly-Ub chain formation on DDR2, while Ub K33-only mutant exhibited weak ability to be conjugated with DDR2. These findings were further confirmed by Ub K27R and K33R mutants, which reduced DDR2 polyubiquitination.


CONCLUSIONS
DDR2 is linked to a polyUb chain predominantly through K27 conjugation and slightly through K33.
Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China.
Jiangtian
J
Yu
Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China.
Xin
X
Bu
Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China.
Hu
H
Zhao
Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China.
Xia
X
Li
Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China.
Jin
J
Su
D001665:Binding Sites / Q000235:genetics; D000070818:Discoidin Domain Receptors; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D025821:Polyubiquitin / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011975:Receptors, Mitogen / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*
D011975:Receptors, Mitogen; D025801:Ubiquitin; D025821:Polyubiquitin; D000070818:Discoidin Domain Receptors; D020794:Receptor Protein-Tyrosine Kinases; D008239:Lysine