title: Visualization of Syk-antigen receptor interactions using green fluorescent protein: differential roles for Syk and Lyn in the regulation of receptor capping and internalization.
abstract: The cross-linking of the B cell Ag receptor (BCR) is coupled to the stimulation of multiple intracellular signal transduction cascades via receptor-associated, protein tyrosine kinases of both the Src and Syk families. To monitor changes in the subcellular distribution of Syk in B cells responding to BCR cross-linking, we expressed in Syk-deficient DT40 B cells a fusion protein consisting of Syk coupled to green fluorescent protein. Treatment of these cells with anti-IgM Abs leads to the recruitment of the kinase from cytoplasmic and nuclear compartments to the site of the cross-linked receptor at the plasma membrane. The Syk-receptor complexes aggregate into membrane patches that redistribute to form a cap at one pole of the cell. Syk is not demonstrably associated with the internalized receptor. Catalytically active Syk promotes and stabilizes the formation of tightly capped BCR complexes at the plasma membrane. Lyn is not required for the recruitment of Syk to the cross-linked receptor, but is required for the internalization of the clustered BCR complexes. In the absence of Lyn, receptor-Syk complexes at the plasma membrane are long lived, and the receptor-mediated activation of the NF-AT transcription factor is enhanced. Thus, Lyn appears to function to negatively regulate aspects of BCR-dependent signaling by stimulating receptor internalization and down-regulation.
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: H
authors.initials: H
authors.lastname: Ma
authors.forename: T M
authors.initials: TM
authors.lastname: Yankee
authors.forename: J
authors.initials: J
authors.lastname: Hu
authors.forename: D J
authors.initials: DJ
authors.lastname: Asai
authors.forename: M L
authors.initials: ML
authors.lastname: Harrison
authors.forename: R L
authors.initials: RL
authors.lastname: Geahlen
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000201:enzymology / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D002645:Chickens; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation / Q000276:immunology; D004792:Enzyme Precursors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D007152:Immunologic Capping / Q000235:genetics; D007424:Intracellular Fluid / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011505:Protein-Tyrosine Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011947:Receptors, Antigen, B-Cell / Q000276:immunology / Q000378:metabolism*; D000072377:Syk Kinase; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D019061:src-Family Kinases / Q000172:deficiency / Q000235:genetics / Q000502:physiology*
chemical_list: D004268:DNA-Binding Proteins; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011947:Receptors, Antigen, B-Cell; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases