AN SH3-BINDING SITE CONSERVED IN BRUTONS TYROSINE KINASE AND RELATED TYROSINE KINASES MEDIATES SPECIFIC PROTEIN INTERACTIONS IN-VITRO AND IN-VIVO

Mutations in Bruton's tyrosine kinase (Btk) have been associated with immunodeficiencies in man and in the mouse. Btk and two related protei ns, Itk and Tee, are members of a distinct family of tyrosine kinases. These kinases are believed to function in various receptor-mediated s ignaling 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 propos ed to mediate specific intermolecular or intramolecular interactions. The TH region of Btk contains a functional SH3-binding site at residue s 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 bindin g in vitro is abolished by specific, single amino acid substitutions w ithin 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 prot ein interactions in intact cells. First, treatment of cells with perva nadate induces an increase in the phosphotyrosine content of kinase-in active 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-kD a phosphotyrosine containing protein; this interaction requires a func tional 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.