Bruton's tyrosine kinase (Btk) 1 is a member of the Btk/Tec/ Itk subfamily of cytoplasmic tyrosine kinases (1-4). Btk contains a pleckstrin homology (PH) domain and adjacent Tec homology (TH) domain (5), in addition to the Src homology domain 3 (SH3), SH2, and SH1 (catalytic domain; Fig. 1A). Btk is crucial for B cell development and proliferation at the transition between the pre-B cell stage and later B cell stages. Mutations in the Btk gene cause a decrease in the number of mature B cells, resulting in human X-linked agammaglobulinemia (XLA) (1, 2) and murine X-linked immunodeficiency (Xid) (3, 4). Mutations in XLA patients, including missense and nonsense mutations, insertions, and deletions, have been found to be scattered all along the Btk gene (6). The mutations in the PH domain are important in determining the role of PH domain in signal transduction, because thus far Btk is the only protein in which mutations in the PH domain have been found to cause a disease. The PH domain is a divergent protein module of approximately 100 amino acids found in many proteins involved in signal transduction, and there is evidence that it is involved in protein-protein or protein-phospholipids interactions (for reviews, see Refs. 7 and 8). Although the PH domain of Btk binds the ␥ subunits of heterotrimeric G proteins (9, 10) and several protein kinase C (PKC) isoforms (11), the effect of the XLA and Xid mutations in the PH domain on this binding has not been fully elucidated.Very recently, we showed that the PH domain of Gap1 m , a member of a family of Ras GTPase-activating proteins, binds inositol 1,3,4,5-tetrakisphosphate (IP 4 ), inositol 1,3,4,5,6-pentakisphosphate (IP 5 ), and inositol 1,2,3,4,5,6-hexakisphosphate (IP 6 ) (12). Since the PH domain (or Btk homology domain) of Gap1 m is highly homologous to that of Btk (4, 13), Btk presumably binds these compounds. In this study, we show that Btk is an IP 4 -binding protein and that all of the XLA and Xid missense mutations in the PH domain result in dramatically reduced IP 4 binding activity. In addition, the constitutive activated form of Btk, which contains a point mutation (E41K) in the PH domain (14), binds IP 6 with higher affinity than normal Btk. On the basis of these findings, we discuss the role of IP 4 , IP 5 , and IP 6 binding to the PH domain of Btk in B cell development. 4 and inositol 1,4,5-trisphosphate (IP 3 ) were purchased from Boehringer Mannheim, and IP 5 and IP 6 were from Calbiochem. All other chemicals were commercial products of reagent grade. Solutions were prepared in deionized water.
MATERIALS AND METHODS
Chemicals-IPcDNA Cloning of Mouse Btk-cDNA encoding Btk (amino acids 1-659) from adult mouse spleen (BALB/c) was amplified by the reverse transcriptase polymerase chain reaction (PCR) (LA-PCR Kit; Takara Shuzo, Japan) for 40 cycles, each consisting of denaturation at 94°C for 1 min, annealing at 50°C for 2 min, and extension at 72°C for 3-4.5 min. The extension time was increased by 30 s every 10 cycles. The sense and antisense primer...
