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

Abstract

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-γ2. 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-γ2. Thus, different tyrosines along the CD19 cytoplasmic domain provide scaffolding for the formation of complexes of different signaling molecules.

A CD19-Dependent Signaling Pathway Regulates Autoimmunity in Lyn-Deficient Mice

“…Before and following BCR ligation, immunoprecipitated CD19 complexes include Lyn, Fyn, and Lck (30 -32). CD19 also interacts with effector molecules downstream of BCR signaling and Lyn amplification, such as phosphatidylinositol 3-kinase and Vav (33)(34)(35)(36)(37)(38). CD19 amplification of Lyn kinase activity facilitates the processive phosphorylation of CD19 and the recruitment and phosphorylation of Vav (16).…”

Critical role of SHP2 (PTPN11) signaling in germinal center-derived lymphoma

“…25 These results are consistent with results of published studies showing that SHP2 acts upstream of the SRC and ERK MAPK pathway, 26,27 possibly by recruiting the GRB2/SOS complex. 39 We also demonstrated that SHP2 could induce the activation of AP-1 after CD19 stimulation through the recruitment and activation of ERK and SRC. 26,27 Given the known requirement of AP-1 activation for progression of Burkitt lymphoma, and ERK1/2 signaling for the activation of AP-1 transcription factors, the involvement of AP-1 in the induction of genes that encode cyclin proteins and in the suppression of p27 kip1 gene transcription following mitogenic stimulation, 50 …”

“…39,40 SHP2 has a positive effect on MAPK signaling mainly through its scaffolding adapter proteins GAB2 and GRB2 in the hematopoietic system. 24,25,38 CD19 deficiency resulted in a loss of SHP2 catalytic activity and was associated with a reduction in …”

Fc gamma receptor IIb modulates the molecular Grb2 interaction network in activated B cells

“…It appears possible that integration of positive PI3K regulators is stabilized by Grb2 in context with the co-receptor CD19 in a similar way. CD19 is known bind the SH2 domains of Grb2 and p85 [18,45] and together with Vav and BCAP it orchestrates full PI3K activation [46]. CD19 tyrosine phosphorylation has been reported to be diminished after BCR/FcγRIIb co-ligation [39].…”

“…Moreover, absence of FcγRIIb renders mice susceptible for autoimmunity [17]. Interestingly, CD19, CD22 and FcγRIIb bind to the adapter protein growth factor receptor-bound protein 2 (Grb2) [18][19][20]. It has been shown that FcγRIIb-mediated SHIP recruitment is strongly compromised in Grb2-deficient DT40 cells [19].…”