SH2 DOMAINS OF THE PROTEIN-TYROSINE KINASES BLK, LYN, AND FYN(T) BINDDISTINCT SETS OF PHOSPHOPROTEINS FROM B-LYMPHOCYTES

Several members of the Src family, including Blk, Lyn, Fyn(T), and Lck , are expressed in B cells. These kinases associate with the antigen r eceptor complex, and the activities of Blk, Fyn(T), and Lyn increase u pon receptor engagement. Differences in the amino acid sequences and p atterns of expression of these kinases suggest that they serve distinc t functions. In this communication it is shown that the SH2 domains fr om Blk, Lyn, and Fyn(T) preferentially bind distinct sets of phosphopr oteins from the mature B cell line A20. These interactions were found to depend on recognition of phosphotyrosine. The Blk SH2 domain bound more than 10 distinct phosphoprotein species, most of which reacted wi th an antiphosphotyrosine antibody; the phosphotyrosine content of the se proteins was increased if surface immunoglobulin was cross-linked b efore extracts were made. Phosphoproteins of 72, 76, 115, and 130 kDa bound to the SH2 domains of Blk, Lyn, and Fyn(T). Phosphoamino acid an alysis of these four proteins revealed that each contained phosphoseri ne, phosphothreonine, and phosphotyrosine. Proteins of 90 kDa, 130 kDa , and 150 kDa were preferentially bound by the Blk SH2 domain, while t he Fyn(T) SH2 domain showed preferential binding to proteins of 76 and 180 kDa. The Lyn SH2 binding profile resembled that of Blk, but diffe rences in the binding specificities of these kinases were also observe d. Thus, among proteins that exhibit increased tyrosine phosphorylatio n following antigen receptor cross-linking, several have been identifi ed that bind preferentially to SH2 domains of Blk, Fyn(T), or Lyn, sug gesting that these kinases serve distinct functions. In addition, chim eric Fyn(T)-Blk SH2 domains were shown to be functional in binding ass ays and to exhibit binding specificities intermediate between those of the parent domains, consistent with the interpretation that the diffe rences we observe in phosphoprotein binding by Fyn(T) and Blk SH2 doma ins reflect differences in their native structures.