SPECIFIC BINDING OF FYN AND PHOSPHATIDYLINOSITOL 3-KINASE TO THE B-CELL SURFACE GLYCOPROTEIN CD19 THROUGH THEIR SRC HOMOLOGY-2 DOMAINS

CD19 is a B cell surface protein capable of forming non-covalent molec ular complexes with a number of other B cell surface proteins includin g the CD21/CD81/Leu-13 complex as well as with surface immunoglobulin. CD19 tyrosine phosphorylation increases after B cell activation, and is proposed to play a role in signal transduction through its cytoplas mic domain, which contains nine tyrosine residues. Several second mess enger proteins have been shown to immunoprecipitate with CD19, includi ng p59 Fyn (Fyn), p59 Lyn (Lyn) and phosphatidylinositol-3-kinase (PI- 3 kinase). These associations are predicted to occur via the src-homol ogy 2 (SH2) domains of the second messenger proteins. Two of the cytop lasmic tyrosines in the CD19 cytoplasmic region contain the consensus binding sequence for the PI-3 kinase SH2 domain (Y-PO4-X-X-M). However , the reported consensus binding sequence for the Fyn and Lyn SH2 doma ins (Y-PO4-X-X-I/L) is not found in CD19. We investigated the capacity of CD19 cytoplasmic tyrosines to bind both Fyn and PI-3 kinase SH2-do main fusion proteins. In activated B cells, both Fyn and PI-3 kinase S H2-domain fusion proteins precipitate CD19. Using synthetic tyrosine-p hosphorylated peptides comprising each of the CD19 cytoplasmic tyrosin es and surrounding amino acids, we investigated the ability of the Fyn SH2 and PI-3 kinase SH2 fusion proteins to bind to the different CD19 cytoplasmic phosphotyrosine peptides. ELISA revealed that the two CD1 9 cytoplasmic tyrosine residues contained within the Y-X-X-M sequences (Y-484 and Y-515) bound preferentially to the PI-3 kinase SH2-domain fusion proteins. Two different tyrosines (Y-405 and Y-445) bound prefe rentially to the Fyn SH2-domain fusion protein via a novel sequence, Y -E-N-D/E, different from that previously reported for the Fyn SH2 doma in. In precipitation studies, peptide Y-484 was able to compete with t yrosine phosphorylated CD19 specifically for binding to the PI-3 kinas e SH2 domain fusion proteins, while peptides Y-405 and Y-445 were able to compete specifically for binding to the Fyn SH2 domain fusion prot eins. These results indicate that CD19 may be capable of binding both Fyn and PI-3 kinase concurrently, suggesting a mechanism for CD19 sign al transduction, in which binding of PI-3 kinase to the Fyn SH3 domain results in activation of PI-3 kinase.