Recruitment of C-terminal Src kinase by the leukocyte inhibitory receptor CD85j.
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-fyn420, 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.
Sayós
Joan
J
Molecular Immunopathology Unit, DCEXS, Universitat Pompeu Fabra, Barcelona, Spain. joan.sayos@upf.edu
Martínez-Barriocanal
Agueda
A
Kitzig
Friederike
F
Bellón
Teresa
T
López-Botet
Miguel
M
D020816:Amino Acid Motifs; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism / Q000502:physiology*; D001402:B-Lymphocytes / Q000378:metabolism; D015153:Blotting, Western; D000081247:CSK Tyrosine-Protein Kinase; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D019169:Jurkat Cells; D000075362:Leukocyte Immunoglobulin-like Receptor B1; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000378:metabolism / Q000502:physiology*; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000737:chemistry; D014638:Vanadates / Q000737:chemistry; D001616:beta-Galactosidase / Q000378:metabolism; D019061:src-Family Kinases
D015703:Antigens, CD; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; C105299:LILRB1 protein, human; D000075362:Leukocyte Immunoglobulin-like Receptor B1; D010455:Peptides; D011518:Proto-Oncogene Proteins; D011971:Receptors, Immunologic; C076715:pervanadate; D014638:Vanadates; D014443:Tyrosine; D004247:DNA; D010770:Phosphotransferases; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; C509249:PTPN6 protein, human; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D001616:beta-Galactosidase