Among the earliest detectable events in B-cell antigen receptor-mediated signal transduction are the activation of receptor-associated Src-family tyrosine kinases and the tyrosine phosphorylation of Ig-a and Ig-,B receptor subunits.These kinases appear to interact with resting B-cell antigen receptor complexes primarily through the Ig-a chain antigen receptor homology 1 (ARH1) motif. Recent studies showed a dramatic increase in the amount of Src-family kinase p59gfY bound to Ig-a when ARH1 motif tyrosines were phosphorylated. To explore the submolecular basis of these interactions, we conducted mutational analysis to localize sites in p53/56'yn and p59f" that bind nonphosphorylated and phosphorylated Ig-a. Here we report that distinct regions within these kinases bind nonphosphorylated and phosphorylated Ig-a ARH1 motifs. The N-terminal 10 residues mediate binding to the nonphosphorylated Ig-a ARH1 motif. Association with the phosphorylated Ig-a ARH1 motif is mediated by Src homology 2 domains. These mings suggest a mechanism whereby ligandinduced Ig-a tyrosine phosphorylation initiates a change in the orientation of an associated kinase that may alter its activity and/or access to substrates and other effectors. These studies also demonstrate strong p59fyn recruitment to both Ig-a and Ig-,8 ARH1 motifs and kinase activation when the two tyrosine residues are phosphorylated.To define the region(s) within Src-family kinases that directs association with nonphosphorylated and phosphorylated Ig-a ARH1 motifs, we assessed the ability ofbacterially expressed N-terminal Lyn and Fyn peptides (11) to bind Ig-a.The N-terminal 27 residues of p53/56'yn and p59fyn were found to be sufficient to associate with nonphosphorylated Ig-a. Further studies revealed that binding to Ig-a in which Tyr'82 and Tyr'93 were phosphorylated was mediated by the Src homology 2 (SH2) domain. To verify these findings using full-length kinases expressed in eukaryotic cells, we took advantage of the inability of p60src to bind Ig-a (12) and assessed the binding activity of p59fyn/p60src chimeric proteins. Insertion of the N-terminal 10 residues of p59fY into p60src was sufficient to confer binding to nonphosphorylated Ig-a ARH1 motifs. Finally, these findings were confirmed and extended to Lyn by using domain-deletion mutants.MATERIALS AND METHODS DNA Constructions. The production of Src/Fyn/Myc chimeric proteins (13) and Lyn and Fyn fusion proteins (11) has been described. The wild-type p561yn construct was subcloned from Lyn/pZIP (generously provided by Tadashi Yamamoto, University of Tokyo). The insert was liberated with Mlu I, blunted with Klenow DNA polymerase, and then digested with BamHI. This was cloned into pCMV5 (a generous gift from Mark Stinski, University ofIowa) that was digested with HindIII, blunted with Klenow DNA polymerase, and then digested with BamHI. The Lyn deletion mutants were constructed by using the PCR splice-overlap extension technique to fuse the regions flanking the deletions (14). For all mutants, the exter...