SH2 domains of the protein-tyrosine kinases Blk, Lyn, and Fyn(T) bind distinct 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 receptor complex, and the activities of Blk, Fyn(T), and Lyn increase upon receptor engagement. Differences in the amino acid sequences and patterns of expression of these kinases suggest that they serve distinct functions. In this communication it is shown that the SH2 domains from Blk, Lyn, and Fyn(T) preferentially bind distinct sets of phosphoproteins 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 with an antiphosphotyrosine antibody; the phosphotyrosine content of these proteins was increased if surface immunoglobulin was cross-linked before extracts were made. Phosphoproteins of 72, 76, 115, and 130 kDa bound to the SH2 domains of Blk, Lyn, and Fyn(T). Phosphoamino acid analysis of these four proteins revealed that each contained phosphoserine, phosphothreonine, and phosphotyrosine. Proteins of 90 kDa, 130 kDa, and 150 kDa were preferentially bound by the Blk SH2 domain, while the Fyn(T) SH2 domain showed preferential binding to proteins of 76 and 180 kDa. The Lyn SH2 binding profile resembled that of Blk, but differences in the binding specificities of these kinases were also observed. Thus, among proteins that exhibit increased tyrosine phosphorylation following antigen receptor cross-linking, several have been identified that bind preferentially to SH2 domains of Blk, Fyn(T), or Lyn, suggesting that these kinases serve distinct functions. In addition, chimeric Fyn(T)-Blk SH2 domains were shown to be functional in binding assays and to exhibit binding specificities intermediate between those of the parent domains, consistent with the interpretation that the differences we observe in phosphoprotein binding by Fyn(T) and Blk SH2 domains reflect differences in their native structures.
Malek
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Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
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D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019061:src-Family Kinases
D017931:DNA Primers; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D011947:Receptors, Antigen, B-Cell; D011993:Recombinant Fusion Proteins; C082599:protein-tyrosine kinase p55(blk); D011505:Protein-Tyrosine Kinases; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases