Abstract
Mutations in Bruton's tyrosine kinase (Btk) have been associated with immunodeficiencies in man and in the mouse. Btk and two related proteins, Itk and Tec, are members of a distinct family of tyrosine kinases. These kinases are believed to function in various receptor-mediated signaling pathways, but their specific functions are as yet undefined. Btk and its homologues share extensive sequence similarity, including a conserved region, the Tec-homology (TH) domain, that has been proposed to mediate specific intermolecular or intramolecular interactions. The TH region of Btk contains a functional SH3-binding site at residues 189-192. SH3 binding is selective: Btk is retained by the SH3 domain of Fyn but not by that of Blk, another Src-type kinase. TH-SH3 binding in vitro is abolished by specific, single amino acid substitutions within the Btk TH domain or the Fyn SH3 domain. We provide two lines of evidence that the SH3-binding site in the Btk TH domain mediates protein interactions in intact cells. First, treatment of cells with pervanadate induces an increase in the phosphotyrosine content of kinase-inactive Btk; this response is substantially reduced by a mutation that inactivates the SH3-binding site in the Btk TH domain. Second, in cell lysates Btk is found in association with an as yet unidentified 72-kDa phosphotyrosine-containing protein; this interaction requires a functional SH3-binding site in the TH domain. The TH domain may therefore interact in vivo with other proteins that regulate the phosphorylation state of Btk.
Highlights
Treatment of cells with pervanadate induces an increase in the phosphotyrosine content of kinase-inactive Bruton's tyrosine kinase (Btk); this response is substantially reduced by a mutation that inactivates the Src-homology 3 (SH3)-binding site in the Btk TH domain
I The abbreviations used are: Btk, Bruton's tyrosine kinase; PH, pleckstrin homology; TH, Tec homology; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel elechave been associated with a profound B cell immunodeficiency (X-linked agammaglobulinemia or XLA) in man [7, 8] and a heritable defect of B cell function (X-linked immunodeficiency or xid) in the CBAIN mouse strain [9, 10]
Treatment of cells with pervanadate induces an increase in the phosphotyrosine content of kinase-inactive Btk; this response is substantially reduced by a mutation that inactivates the SH3binding site in the Btk TH domain
Summary
Bruton's tyrosine kinase; PH, pleckstrin homology; TH, Tec homology; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel elechave been associated with a profound B cell immunodeficiency (X-linked agammaglobulinemia or XLA) in man [7, 8] and a heritable defect of B cell function (X-linked immunodeficiency or xid) in the CBAIN mouse strain [9, 10]. All three kinases become newly phosphorylated on tyrosine and their associated kinase activity increases in response to extracellular stimuli: Btk upon cross-linking of surface Ig on B cells [18, 19] or FCERI receptors on myeloid cells [20], Itk upon ligation of the costimulatory molecule CD28 in the human leukemic T cell line Jurkat [21], and Tec upon treatment of myeloid or B lymphoid cell lines with interleukin-3 [22] These kinases are believed to participate in various receptor-mediated signaling pathways, but their specific functions are as yet undefined. A striking feature of the TH region is the presence (Fig. 1, B and C) of one or more consensus motifs (XPPPXP, where denotes a hydrophobic residue) for binding to the Src SH3 domain, as defined by peptide selection trophoresis; PVDF, polyvinylidine difluoride; GST, glutathione S-transferase.
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