title: Determination of the tyrosine phosphorylation sites in the T cell transmembrane glycoprotein CD5.
abstract: Studies of CD5-deficient mice indicate that the transmembrane glycoprotein CD5 negatively regulates antigen receptor-mediated signals in thymocytes, lymph node T cells and B1a cells. CD5 contains four tyrosine residues in its cytoplasmic domain and is phosphorylated on tyrosine residues following antigen receptor ligation. Recently it has been proposed that CD5 function is dependent on the recruitment of the tyrosine phosphatase SHP-1 to tyrosine-phosphorylated CD5 and subsequent dephosphorylation of signaling molecules. In this study we investigated the requirements for, and sites of, CD5 tyrosine phosphorylation. Using a T cell line deficient in the tyrosine kinase p56(lck) and the same cell line reconstituted with this kinase, we show that p56(lck) expression is required for efficient CD5 tyrosine phosphorylation. Using tyrosine-phosphorylated peptides corresponding to CD5 cytoplasmic sequences we also show that the Src homology 2 (SH2) domain of p56(lck) binds prominently to pY429SQP, with 30-fold less affinity to pY463DLQ and not to pY441PAL. A number of murine CD5 Y --> F and deletion mutants were expressed in Jurkat T cells. The Y441F mutant was tyrosine phosphorylated at levels comparable to wild-type, but the Y429F and Y463F mutants were phosphorylated at lower levels. Two deletion mutants, which contain only one tyrosine residue (Y378) located at the interface of the transmembrane and cytoplasmic domains, were not tyrosine phosphorylated, suggesting that Y378 is not readily available for phosphorylation. Taken together these results suggest that both Y429 and Y463 can recruit p56(lck), and that these residues are the only prominent sites for CD5 tyrosine phosphorylation.
authors.affiliation: Department of Medical Biochemistry and MRC Centre for Molecular and Cellular Biology, University of Stellenbosch, Tygerberg, 7505, South Africa.
authors.forename: K M
authors.initials: KM
authors.lastname: Dennehy
authors.forename: W F
authors.initials: WF
authors.lastname: Ferris
authors.forename: H
authors.initials: H
authors.lastname: Veenstra
authors.forename: L A
authors.initials: LA
authors.lastname: Zuckerman
authors.forename: N
authors.initials: N
authors.lastname: Killeen
authors.forename: A D
authors.initials: AD
authors.lastname: Beyers
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018956:CD5 Antigens / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D017384:Sequence Deletion; D013601:T-Lymphocytes / Q000201:enzymology / Q000378:metabolism*; D018909:src Homology Domains / Q000235:genetics / Q000276:immunology
chemical_list: D018956:CD5 Antigens; D008562:Membrane Glycoproteins; D010748:Phosphopeptides; D019000:Phosphotyrosine; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)