부산대학교 도서관

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.bbrc.2004.09.097 Byline: Joan Sayos (a), Agueda Martinez-Barriocanal (a), Friederike Kitzig (a), Teresa Bellon (b), Miguel Lopez-Botet (a) Keywords: Inhibitory receptors; CD85j; Csk; SHP-1; Src kinases Abstract: The CD85j inhibitory receptor (also termed ILT2 or LIR-1) is a type-I transmembrane protein that belongs to the Ig superfamily and is expressed by different leukocyte lineages. The extracellular region of CD85j binds HLA class I molecules and its cytoplasmic domain displays four immunoreceptor tyrosine-based inhibition motifs (ITIM). Upon tyrosine phosphorylation CD85j recruits the SHP-1 tyrosine phosphatase, involved in negative signaling. In order to identify other molecules to which CD85j might interact with in a phosphotyrosine-dependent manner, a cDNA B-cell library was screened in a three-hybrid system in yeast using the CD85j cytoplasmic tail as bait in the presence of the Src-kinase c-fyn.sub.420, 531Y-F, 176R-Q mutant. In this system, the C-terminal Src kinase (Csk) was shown to interact with CD85j. Phosphorylation-dependent recruitment of Csk to the CD85j cytoplasmic tail was confirmed in CD85j-transfected mammalian cells by immunoprecipitation and Western blot analysis. Mutational analyses and phospho-peptide mapping suggested that the SH2 domain of Csk may preferentially bind to ITIM Y562 of CD85j; yet, mutation to phenylalanine of Y533, Y614, and Y644 also significantly reduced Csk recruitment by CD85j. Even though CD85j was detected in both anti-SHP1 and CSK immunoprecipitates, these two molecules did not co-precipitate together with CD85j. Our data support the possibility that Csk regulates the function of CD85j. Author Affiliation: (a) Molecular Immunopathology Unit, DCEXS, Universitat Pompeu Fabra, Barcelona, Spain (b) Servicio de Alergia, Hospital Universitario la Paz, Madrid, Spain Article History: Received 2 September 2004 Article Note: (footnote) [star] Abbreviations: Csk, C-terminal Src kinase; ITIM, immunoreceptor tyrosine-based inhibition motif; ONPG, o-nitrophenyl-d-galactopyranoside; SHP-1, SH2 domain-containing protein tyrosine; Cbp, Csk binding protein.