Mutation of the pleckstrin homology domain of Bruton's tyrosine kinase in immunodeficiency impaired inositol 1,3,4,5-tetrakisphosphate binding capacity.

Bruton's tyrosine kinase (Btk), a cytoplasmic protein-tyrosine kinase, plays a pivotal role in B cell activation and development. Mutations in the pleckstrin homology (PH) domain of the Btk gene cause human X-linked agammaglobulinemia (XLA) and murine X-linked immunodeficiency (Xid). In this paper, we report that the PH domain of Btk functions as an inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate (IP6) binding domain (Kd of approximately 40 nM for IP4), and that all of the XLA (Phe replaced by Ser at position 25 (F25S), R28H, T33P, V64F, and V113D) and Xid mutations (R28C) found in the PH domain result in a dramatic reduction of IP4 binding activity. Furthermore, the rare alternative splicing variant, with 33 amino acids deleted in the PH domain, corresponding to exon 3 of the Btk gene, also impaired IP4 binding capacity. In contrast, a gain-of-function mutant called Btk*, which carries a E41K mutation in the PH domain, binds IP6 with two times higher affinity than the wild type. Our data suggest that B cell differentiation is closely correlated with the IP4 binding capacity of the PH domain of Btk.

Itk subfamily of cytoplasmic tyrosine kinases (1)(2)(3)(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.

Chemicals-IP
cDNA 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 primers were designed as follows (4): primer 1 (sense; amino acid residues 1-7), 5Ј-CGGATCCATGGCTGCAGT-GATACTGGA-3Ј, and primer 2 (antisense; amino acid residues 654 -659), 5Ј-CGGATCCTCAGGATTCTTCATCCATCA-3Ј. The PCR products were purified on a 0.7% agarose gel and extracted with a Geneclean kit II (Bio 101). After digestion with BamHI, the cDNA inserts were subcloned into the BamHI site of pGEX-2T (Pharmacia Biotech Inc.). Three clones were selected and verified by DNA sequencing using a BcaBEST dideoxy sequencing kit (Takara Shuzo). One of them has a deletion of amino acids 48 -80, which corresponds to exon 3 of the Btk gene (6).
Preparation of GST Fusion Proteins-cDNA encoding the PH domain of Btk was amplified by PCR with primers 3 and 4 (sense, amino acid residues 1-5, 5Ј-CGGATCCGAAGCTATGGCCGCAGTGAT-3Ј and antisense, amino acid residues 159 -165, 5Ј-CGAATTCGCAGCCCATAG-CATTTTTGG-3Ј, respectively). After digestion with BamHI and EcoRI, the cDNA inserts were subcloned into the BamHI-EcoRI site of pGEX-2T and verified by DNA sequencing. The PH domain of the mouse Btk was expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli JM109 and purified by glutathione-Sepharose 4B chromatography (Pharmacia) according to the manufacturer's recommendations. GST-Btk-PH coded for amino acids 1-165 of mouse Btk; GST-Btk-PH⌬E3, in which 33 amino acids corresponding to exon 3 of the Btk gene were deleted, amino acids 1-47 ϩ 81-165.

Measurement of [ 3 H]Ins 1,3,4,5-P 4 Binding to GST Fusion
Proteins-GST fusion proteins (1 g or 200 ng) were incubated with 9.6 nM [ 3 H]IP 4 (DuPont NEN) in 50 l of 50 mM HEPES-KOH, pH 7.2, for 10 min at 4°C. The sample was then mixed with 1 l of 50 mg/ml ␥-globulins and 51 l of a solution containing 30% PEG6000 and 50 mM HEPES-KOH, pH 7.2, and placed on ice for 5 min. The precipitate obtained by centrifugation at 10,000 ϫ g for 5 min was solubilized in 500 l of Solvable (Packard Instrument Co.), and radioactivity was measured in Aquasol 2 (Packard) with a liquid scintillation counter (16). Inhibition of specific [ 3 H]IP 4 binding to GST fusion proteins was also performed in the above reaction mixture containing various inositol phosphates.

IP 4 Binding
Properties of the PH Domain of Btk-To determine whether the PH domain of Btk serves as an IP 4 , IP 5 , and IP 6 binding site, the PH domain was expressed as a GST fusion protein (GST-Btk-PH), and IP 4 binding activity was tested. Consistent with the results of our previous study of the PH domain of Gap1 m (12), GST-Btk-PH bound IP 4 with a K d value of approximately 40 nM. The B max value was calculated at 0.27 pmol/pmol of protein by GraphPad-Prism (version 2.0), indicating substoichiometric binding of IP 4 to GST-Btk-PH. Such substoichiometric binding was also observed in the case of synaptotagmin II (15). This may result from instability of the IP 4sensitive form of GST-Btk-PH. Inositol phosphate binding specificity was also analyzed in competition experiments (Fig.  1B). Competitive potencies in decreasing order were IP 4 Ͼ IP 5 Ն IP 6 Ͼ Ͼ IP 3 . IP 5 and IP 6 were less effective than IP 4 , and IP 3 was almost completely inactive. Thus, the PH domain of Btk seems to function as an IP 4 , IP 5 , and IP 6 binding site.
An alternative splicing form of Btk, in which 33 amino acids within the PH domain had been deleted (amino acid residues 48 -80), was isolated in the course of cloning mouse Btk cDNA. This form of Btk (Btk⌬E3) was expressed at a much lower level in the adult mouse spleen (3-5% of normal Btk-PH; Fig. 3). The effect of deletion of subdomain IV ( Fig. 2A, underlined) on IP 4 binding capacity was also examined. GST-Btk-PH⌬E3 did not exhibit any significant IP 4 binding activity (data not shown).
Recently, Li et al. (14) isolated a constitutive activated form of Btk, designated Btk*, that contains a point mutation (E41K) in the PH domain (asterisk in Fig. 2A). Btk* induces fibroblast transformation by enhancing transphosphorylation of Tyr-551 by endogenous Src family tyrosine kinases and autophosphorylation of Tyr-223 in the SH3 domain (17,18). As shown in Fig. 4, GST-Btk-PH(E41K) bound IP 4 and IP 5 with the same affinities as those of GST-Btk-PH, but displayed about two times higher affinity for IP 6 (K i of approximately 60 nM) than wild type. Furthermore, the inositol phosphate binding specificity of GST-Btk-PH(E41K) was slightly different from that of the wild type. The competitive potencies were IP 4 Ն IP 6 Ͼ IP 5 Ͼ Ͼ IP 3 .

DISCUSSION
In our preceding paper we showed that the PH domain of Gap1 m binds IP 4 , IP 5 , and IP 6 (12). Although the PH domains are generally divergent, the PH domain of Gap1 m is much more homologous to that of Btk than other PH domains and designated Btk homology domain (4,13). In this study we have shown that Btk is an IP 4 -, IP 5 -, and IP 6 -binding protein, and that mutations in the PH domain observed in XLA and Xid, or deletion within the PH domain by alternative splicing, result in a dramatic reduction in IP 4 binding capacity. We have also shown that the gain-of-function mutant of Btk, Btk*, binds IP 6 with higher affinity than that of wild type Btk. These findings suggest that B cell differentiation is closely correlated with the IP 4 , IP 5 , and IP 6 binding capacity of the PH domain of Btk.
By analogy with the recent x-ray crystal structure of the high affinity complex between the PH domain from phospholipase C-␦1 and IP 3 (19), Phe-25 and Arg-28 may directly link to the 1-phosphate and 5-phosphate of IP 4 , respectively, via hydrogen bonds. Thus, mutation of these two residues decreases IP 4 binding capacity. In addition, substitution by Lys at position Glu-41 enhanced IP 6 but not IP 4 or IP 5 binding capacity, prob-ably as a result of interaction of Lys with the 2-phosphate of IP 6 . Other mutations (T33P, V64F, and V113D), or deletion of subdomain IV, may distort the IP 4 recognition site or the PH domain structure itself, because they displayed almost no significant IP 4 binding activity.
What is the function of IP 4 , IP 5 , and IP 6 binding to the PH domain of Btk? IP 5 is known to increase during hematopoietic cell differentiation and accumulates at higher concentrations than IP 3 and IP 4 (20). These observations raised the possibility that IP 5 is a physiological ligand and activates Btk function in vivo, although the activation mechanism remains unknown. One possibility is that IP 5 disrupts the interaction between the PH domain of Btk and certain PKC isoforms 2 and overcomes the negative regulation by PKC (11).
In this study, we showed that all of the XLA and Xid mutations in the PH domain impaired IP 4 binding capacity. Since Tec (21) and Itk (22), other members of Btk/Tec/Itk proteintyrosine kinase family expressed in hematopoietic cells, also have the similar PH domain and bind IP 4 , IP 5 , and IP 6 , 2 we propose that these inositol polyphosphates may be involved in hematopoietic cell differentiation by activating the Btk/Tec/Itk family.