Mutation of the Pleckstrin Homology Domain of Bruton's Tyrosine Kinase in Immunodeficiency Impaired Inositol 1,3,4,5-Tetrakisphosphate Binding Capacity

Abstract

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...

Constitutive membrane association potentiates activation of Bruton tyrosine kinase

“…As previously discussed, mounting biochemical evidence supports the hypothesis that PH domain containing proteins may be recruited to the plasma membrane via interaction with membrane proximal phospholipids or proteins (Hemmings, 1997;Harlan et al, 1994;Lemmon et al, 1995;Ferguson et al, 1995;Wang and Shaw, 1995;Salim et al, 1996;Fukuda et al, 1996;Wang et al, 1996;Touhara et al, 1994;Yao et al, 1994;Yenush et al, 1996). GagBtk proteins with PH domain mutations were used to determine if the Btk PH domain serves as essential signaling function in addition to membrane localization.…”

“…These diseases are characterized by defects in B cell development and function. Genetic and biochemical analyses indicate that Btk is a component of the signal transduction pathways utilized by the B cell antigen receptor (BCR), IL-5R, IL-6R, IL-10R, CD38, and FceRI (Wicker and Scher, 1986;Hinshelwood et al, 1995;Saouf et al, 1994;Aoki et al, 1994;de Weers et al, 1994;Koike et al, 1995;Sato et al, 1994;Hitoshi et al, 1993;Matsuda et al, 1995;Santos-Argumedo et al, 1995;Go et al, 1990;Kawakami et al, 1994). Activation of Btk by either receptor crosslinking or a point mutation E41K in the PH domain (Btk*) is dependent on Src family kinases Afar et al, 1996;Saouf et al, 1994;Mahajan et al, 1995) and is correlated with increased tyrosine phosphorylation Li et al, 1995;Mahajan et al, 1995;Hinshelwood et al, 1995;Aoki et al, 1994;de Weers et al, 1994;Saouf et al, 1994;Sato et al, 1994;Kawakami et al, 1994;Matsuda et al, 1995) and translocation to the plasma membrane (Li et al, 1995;Kawakami et al, 1994).…”

“…The Btk PH domain has been shown to bind in vitro to Gbg, protein kinase C, phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P 3 ) and the inositol phosphates IP 4 , IP 5 and IP 6 (Touhara et al, 1994;Tsukada et al, 1994;Yao et al, 1994;Salim et al, 1996;Fukuda et al, 1996). As these molecules are found at membrane proximal sites, these interactions may serve to target Btk to the membrane.…”

“…As these molecules are found at membrane proximal sites, these interactions may serve to target Btk to the membrane. Interestingly, mutations in the PH domain which result in XLA and xid abolish the binding of Btk to PI(3,4,5)P 3 , IP 4 , IP 5 and IP 6 (Salim et al, 1996;Fukuda et al, 1996). The PH domains of other molecules have also been implicated in membrane association.…”

“…Phosphatidylinositol-4,5-diphosphate (PIP 2 ) and many inositol phosphates bind in vitro to the PH domains of several proteins including PLCd, dynamin, b-spectrin, pleckstrin, RasGAP and others (Harlan et al, 1994;Lemmon et al, 1995;Salim et al, 1996;Ferguson et al, 1995;Wang and Shaw, 1995;Fukuda et al, 1996). The coupling of insulin receptor substrate (IRS) to the insulin receptor is mediated by the PH domain of IRS (Yenush et al, 1996).…”