TFII-I is a ubiquitously expressed multifunctional transcription factor with broad biological roles in transcription and signal transduction in a variety of cell types. We and others have shown that TFII-I can interact physically and functionally with Bruton's tyrosine kinase (Btk), a hematopoietic non-receptor protein tyrosine kinase that is critical for B lymphocyte development. Although TFII-I-Btk interactions are impaired in B cells from X-linked immunodeficient mice, the precise molecular determinants governing TFII-I-Btk complex formation remain unknown. To this end, we have conducted a structural analysis of TFII-I-Btk interactions by using a panel of TFII-I mutants. These studies have revealed that a region within the N-terminal 90 amino acids of TFII-I, which includes a putative leucine zipper motif, is primarily responsible for its interaction with Btk. Mutations in the leucine zipper region itself were not sufficient to abrogate binding of TFII-I to Btk, suggesting that regions/residues outside the leucine zipper are responsible for such interactions. Because the first 90 amino acids of TFII-I are required for its dimerization, we propose that Btk tethers TFII-I to the cytoplasm by preventing its dimerization and its subsequent nuclear localization. We further examined the requirement of tyrosine phosphorylation for TFII-I-Btk complex formation. Our data showed that Src-dependent tyrosine phosphorylation sites in TFII-I are not targeted by Btk, suggesting that multiple kinases can independently target TFII-I via distinct signaling pathways. Our results provide a beginning step toward understanding the functional importance of the TFII-I-Btk pathway in B cell signaling and gene expression.Signaling through the surface antigen receptor in B cells leads to the activation of a variety of non-receptor protein tyrosine kinases (1). One of these key enzymes is Bruton's tyrosine kinase (Btk).1 Various mutations in Btk have been identified and are known to cause X-linked agammaglobulinemia in humans (2, 3) and X-linked immunodeficiency (Xid) in mice (4,5). Although the number of mature B cells is virtually absent in X-linked agammaglobulinemia, Xid is characterized by a less severe phenotype in which mature B cells capable of responding to antigenic stimulation are severely diminished (4). In addition, genetic depletion of Btk in mice also results in an Xid phenotype whereby pre-B to mature B cell differentiation is drastically hampered, further underscoring the importance of Btk in B cell function (6, 7). Despite these observations, the downstream targets of Btk are largely unknown. Results from our laboratory and the results of others (8 -10) have shown that Btk associates physically and functionally with the transcription factor TFII-I, thereby providing a mechanism for one of the pathways that connects Btk-mediated signaling to gene activation. Given that Btk is predominantly a cytoplasmic kinase and TFII-I is a nuclear transcription factor, it is important to decipher how they assemble. We present here a ...