Systematic analysis of the role of CD19 cytoplasmic tyrosines in enhancement of activation in Daudi human B cells: clustering of phospholipase C and Vav and of Grb2 and Sos with different CD19 tyrosines.
CD19 is a coreceptor on B cells that enhances the increase in cytoplasmic calcium and ERK2 activation when coligated with the B cell Ag receptor. Constructs containing point mutations and truncations were expressed in Daudi human B lymphoblastoid cells to systematically determine the requirement for individual CD19 cytoplasmic tyrosines in these responses. Evidence for activity was found for Y330, Y360, and Y421 as well as that previously published for Y391. Precipitates formed with phosphopeptides consisting of CD19 sequences flanking these residues were used to screen for cytoplasmic proteins that mediate signaling. Phosphopeptide Y330 precipitated Grb2 and Sos, whereas phosphopeptides Y391 and Y421 both precipitated Vav and phospholipase C-gamma2. These molecules also were found associated with native CD19. In mapping studies with altered constructs, CD19 Y330 and/or Y360 were necessary for binding Grb2 and Sos. Vav associated with CD19 constitutively in unstimulated cells by a tyrosine-independent mechanism requiring the portion of CD19 encoded by exons 9-12. After B cell Ag receptor stimulation, Vav association was tyrosine-dependent, but binding was influenced by multiple residues. However, when maximally phosphorylated by pervanadate, Y391 and, to a lesser extent, Y421 were sufficient. CD19 Y391 was also both necessary and sufficient for binding phospholipase C-gamma2. Thus, different tyrosines along the CD19 cytoplasmic domain provide scaffolding for the formation of complexes of different signaling molecules.
Brooks
S R
SR
Departments of Medicine and Microbiology, University of Alabama, Birmingham, AL 35294, USA.
Li
X
X
Volanakis
E J
EJ
Carter
R H
RH
D048868:Adaptor Proteins, Signal Transducing; D000276:Adjuvants, Immunologic / Q000378:metabolism / Q000502:physiology; D018941:Antigens, CD19 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001402:B-Lymphocytes / Q000187:drug effects / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism; D020013:Calcium Signaling / Q000276:immunology; D018797:Cell Cycle Proteins; D003593:Cytoplasm / Q000276:immunology / Q000378:metabolism; D005091:Exons; D051380:GRB2 Adaptor Protein; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008213:Lymphocyte Activation / Q000187:drug effects / Q000276:immunology*; D020935:MAP Kinase Signaling System / Q000276:immunology; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D051966:Phospholipase C gamma; D010748:Phosphopeptides / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051567:Proto-Oncogene Proteins c-vav; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism / Q000502:physiology; D020839:Son of Sevenless Protein, Drosophila / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014638:Vanadates / Q000494:pharmacology
D048868:Adaptor Proteins, Signal Transducing; D000276:Adjuvants, Immunologic; D018941:Antigens, CD19; D018797:Cell Cycle Proteins; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D007527:Isoenzymes; D010748:Phosphopeptides; D011506:Proteins; D011518:Proto-Oncogene Proteins; D051567:Proto-Oncogene Proteins c-vav; D011947:Receptors, Antigen, B-Cell; D020839:Son of Sevenless Protein, Drosophila; C496618:VAV1 protein, human; C076715:pervanadate; D014638:Vanadates; D014443:Tyrosine; D019950:Mitogen-Activated Protein Kinase 1; D010738:Type C Phospholipases; D051966:Phospholipase C gamma