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Characterization of the Pleckstrin Homology Domain of Btk as an Inositol Polyphosphate and Phosphoinositide Binding Domain
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
236, 333–339 (1997)
RC976947
Characterization of the Pleckstrin Homology Domain of Btk as an Inositol Polyphosphate and Phosphoinositide Binding Domain1 Toshio Kojima,*,† Mitsunori Fukuda,*,†,2 Yutaka Watanabe,‡ Fumiaki Hamazato,*,§ and Katsuhiko Mikoshiba*,†,Ø *Molecular Neurobiology Laboratory, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan; †Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan; ‡Faculty of Engineering, Ehime University, Matsuyama, 790-77, Japan; §Hitachi Advanced Research Laboratory, Hatoyama, Saitama, 350-03, Japan; and ØCalciosignal Net Project, Exploratory Research for Advanced Technology (ERATO), 2-9-3 Shimo-meguro, Meguro-ku, Tokyo 153, Japan
Received June 9, 1997
We previously reported that the pleckstrin homology (PH) domain of Bruton’s tyrosine kinase (Btk) binds Ins(1,3,4,5)P4 and that missense mutations in this domain which cause either human X-linked agammaglobulinemia (XLA) or murine X-linked immunodeficiency (Xid) also dramatically reduce the Ins(1,3,4,5)P4 binding activity. In this paper, we describe the inositol phosphate binding specificity of the Btk PH domain and different inositol polyphosphate binding properties among the PH domains of Tec family kinases. Our results suggest that certain inositol phosphates and/or phosphoinositides are physiological ligands of some Tec family kinases and that Tec family members are differently regulated by inositol molecules. q 1997 Academic Press
Bruton’s tyrosine kinase (Btk) is a member of the Tec family of cytoplasmic protein tyrosine kinases (11 This work was supported by grants from the Japanese Ministry of Education, Science, Sports, and Culture to K.M. and to Y.W. (06240105), the Science and Technology Agency of Japan to K.M., and the Japan Society for the Promotion of Science to M.F. 2 To whom correspondence should be addressed at Molecular Neurobiology Laboratory, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan. Fax: /81-298-36-9040. E-mail: fukuda@rtc. riken.go.jp. Abbreviations: Btk, Bruton’s tyrosine kinase; GST, glutathione Stransferase; InsP3 , inositol trisphosphate; InsP4 , inositol tetrakisphosphate; Ins(1,3,4,5,6)P5 , inositol 1,3,4,5,6-pentakisphosphate; InsP6 , inositol hexakisphosphate; PH, pleckstrin homology; PtdInsP2 , phosphatidylinositol bisphosphate; PtdIns(3,4,5)P3 , phosphatidylinositol 3,4,5-trisphosphate; Xid, X-linked immunodeficiency; XLA, X-linked agammaglobulinemia.
4). Since mutations in the Btk gene cause human Xlinked agammaglobulinemia (XLA) and murine Xlinked immunodeficiency (Xid), Btk appears to be crucial for B cell maturation. XLA patients have B cell differentiation defect which results in a deficiency of mature B cells and immunoglobulins, and thus these patients suffer from an increased susceptibility to infections (5). The Xid mice display a milder phenotype as compared with XLA patients, but the reason for this phenotypic discrepancy is not known (6). The most significant feature of the Tec family of tyrosine kinases is the presence of a pleckstrin homology (PH) domain in the amino-terminal region. The PH domain is composed of approximately 100 loosely conserved amino acids and is found in many proteins involved in signal transduction and cytoskeletal structures (7,8). Recently, the NMR structures of the PH domains have been determined for pleckstrin (9), bspectrin (10), and dynamin (11), and the X-ray structures for dynamin (12,13) and phospholipase C-d1 (14) have been calculated. Despite the low amino acid homology among PH domains of these molecules, all the PH domains have a similar structure: a fold that consists of a b-barrel formed by two b-sheets, with a Cterminal a-helix at one end of the b-barrel. Although the exact function of the PH domain is not known, the carboxy-terminal region of the PH domains has been shown to interact with the b/g subunits of heterotrimeric G proteins (15,16). The amino-terminal region has been shown to bind to PtdIns(4,5)P2 (17,18). These findings suggest a role for the PH domain anchoring the molecule to the membrane. Additionally, in some
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proteins, the PH domains have been reported to associate with several isoforms of protein kinase C (19,20). The Xid mutation and some of the XLA mutations are located in the PH domain of Btk, implying an important role for this domain in Btk function (3,4,21). We previously reported that the PH domain of Btk functions as an Ins(1,3,4,5)P4 binding domain and that the missense mutations of XLA and Xid found in the PH domain cause a dramatic reduction in Ins(1,3,4,5)P4 binding activity (22). In this study we examined the characteristics of the PH domains of Tec family kinases on the inositol phosphate and phosphoinositide binding in greater detail. We show that Btk specifically recognizes the Ins(1,3,4,5)P4 isomer with high affinity and that the PH domains of Tec family kinases are different with respect to inositol polyphosphate binding. MATERIALS AND METHODS Chemicals. D-myo-Ins(1,2,5,6)P4 and D-myo-Ins(3,4,5,6)P4 were purchased from Boehringer Mannheim. D-myo-Ins(1,3,4)P3 , D-myoIns(1,4,5)P3 , D-myo-Ins(1,5,6)P3 , D-myo-Ins(1,3,4,5)P4 , D-myoIns(1,3,4,6)P4 , D-myo-Ins(1,3,4,5,6)P5 , and D-myo-InsP6 were obtained from Calbiochem. PtdIns(3,4,5)P3 derivatives bearing benzoyl and heptanoyl groups were prepared according to the method reported (23). Dibenzoyl PtdIns(4,5)P2 was synthesized from 1,2-diO-cyclohexylidene-myo-inositol via regioselective phosphorylation of the 1,2-diol derivative (24). These phosphoinositide derivatives are soluble in water. All other chemicals were commercial products of reagent grade. Solutions were prepared in deionized water. cDNA cloning. Preparation of the cDNA encoding the PH domain of Btk and the site-directed mutagenesis of the PH domain were performed as described previously (22). cDNAs encoding the PH domains of Tec (amino acids 1-143; numbering according to GenBank accession number JU0215) (25) and Itk/Tsk (amino acids 1-143; GenBank accession number L05631) (26,27) from an adult mouse spleen (BALB/c) were amplified by the reverse transcriptase PCR (LA-PCR kit; Takara Shuzo, Japan) for 40 cycles. Each cycle consisted of denaturation at 94 7C for 1 min, annealing at 50 7C for 2 min, and extension at 72 7C for 3-4.5 min. The extension time was increased by 30 sec every 10 cycles. cDNA encoding the PH domain of Bmx (amino acids 1-143; GenBank accession number U88091) (28) from an adult mouse heart (ICR) was also amplified by the reverse transcriptase PCR for 30 cycles, each consisting of denaturation at 94 7C for 1 min, annealing at 48 7C for 2 min, and extension at 72 7C for 2 min. The sense and antisense primers were designed as follows: primer Tec1 (sense: amino acid residues 1-7) 5*-CGGATCCATGAATTTCAACACTATCCT-3*, primer Tec-2 (antisense: amino acid residues 138143) 5*-CGAATTCACATCCGGGTGCTAGTTTTT-3*, primer Itk-1 (sense: amino acid residues 1-7) 5*-CGGATCCATGAACAACTTCATCCTCCT-3*, primer Itk-2 (antisense: amino acid residues 138-143) 5*-CGAATTCACAGCCTACAGCAGGCTTCT-3*, primer Bmx-1 (sense: amino acid residues 1-3) 5*-CGGATCCATGGACGACAATATGGAGAG-3*, and primer Bmx-2 (antisense: amino acid residues 137143) 5*-CGAATTCGCATCCTGGGGCTGCTTTGC-3*. The PCR products were purified on a 0.7% agarose gel and extracted with a Geneclean kit II (Bio 101). After digestion with BamHI and EcoRI, the cDNA inserts were subcloned into the BamHI-EcoRI site of pGEX2T (Pharmacia Biotech Inc.). Clones were verified by DNA sequencing using a BcaBEST dideoxy sequencing kit (Takara Shuzo). Preparation of GST fusion protein. The PH domains of mouse Btk, Tec, Itk, and Bmx were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli JM109 and purified by glu-
tathione-Sepharose 4B column chromatography (Pharmacia) according to the manufacturer’s recommendations. The GST-Btk-PH protein contains amino acids 1-165 of mouse Btk (numbering according to GenBank accession number L08967). Similarly, GST-TecPH has amino acids 1-143 of mouse TecII, and GST-Itk-PH has amino acids 1-143 of mouse Itk. Since the Bmx-1 primer includes 5* flanking region of the putative initiation codon (28), GST-Bmx-PH contains additional four amino acids (Met-Asp-Asp-Asn) between GST and the mouse Bmx (amino acids 1-143). The mutants of the Btk-PH domain were XLA mutants: GST-Btk-PH (S14F) with Ser replaced by Phe at position 14, GST-Btk-PH (F25S), GST-Btk-PH (R28H), GST-Btk-PH (T33P), GST-Btk-PH (V64F), and GST-Btk-PH (V113D). Additionally a Xid mutant GST-Btk-PH (R28C) was constructed (22). The protein concentrations of the purified recombinant proteins were determined initially by using a Bio-Rad Protein Assay (Bio-Rad). Purified proteins were analyzed by 10% SDS-PAGE followed by Coomassie brilliant blue R-250 staining. The purity of each recombinant protein was estimated by scanning the Coomassie stained SDS-PAGE gel followed by densitometry with Bio Image (Millipore), and then the concentration of each GST-PH protein was determined. Measurement of [3H]Ins(1,3,4,5)P4 and [3H]InsP6 binding to GST fusion proteins. GST fusion proteins (0.5 or 1 mg) were incubated with 9.6 nM [3H]Ins(1,3,4,5)P4 (specific radioactivity 777 GBq/mmol) or 9.4 nM [3H]InsP6 (specific radioactivity 798.1 GBq/mmol) (DuPont NEN) in 50 ml of 50 mM HEPES-KOH, pH 7.2, for 10 min at 4 7C. The samples were then mixed with 1 ml of 50 mg/ml g-globulins and 49 ml of a solution containing 30% PEG6000 and 50 mM HEPESKOH, pH 7.2 and placed on ice for 10 min. The precipitate obtained via centrifugation at 10,000 1 g for 10 min was solubilized in 500 ml of Solvable (Packard Instrument Co.). The amount of radioactivity was measured in Aquasol 2 (Packard) with a liquid scintillation counter. Inhibition of specific [3H]Ins(1,3,4,5)P4 binding and specific [3H]InsP6 binding to GST fusion proteins was also performed in the above reaction mixture containing various inositol phosphates or phosphoinositides. Non-specific binding was defined as the binding observed in the absence of GST fusion proteins. Data processing. All statistical analyses and curve fitting were done using a GraphPad PRISM computer program.
RESULTS Inositol phosphate and phosphoinositide binding specificity of the Btk PH domain. In our previous study, we characterized the Btk PH domain as an inositol polyphosphate binding site (competitive potencies for Ins(1,3,4,5)P4 binding are Ins(1,3,4,5)P4 ú Ins(1,3,4,5,6)P5 § InsP6 ú Ins(1,4,5)P3). However, it remained undetermined whether the Btk PH domain also recognizes other InsP4 isomers and phosphoinositides. To answer this question, competition experiments for [3H]Ins(1,3,4,5)P4 binding were performed using several inositol phosphates and phosphoinositides as competitors (Figure 1). Competitive potencies for Ins(1,3,4,5)P4 binding to the PH domain of Btk in decreasing order were Ins(1,3,4,5)P4 (Kd Å 15 nM) ú Ins(1,3,4,5,6)P5 É InsP6 § PtdIns(3,4,5)P3 (Ki É 110 nM) ú Ins(3,4,5,6)P4 É Ins(1,3,4,6)P4 (Ki É 1 mM) ú Ins(1,5,6)P3 (Ki Å 6 mM) úIns(1,3,4)P3 § Ins(1,2,5,6)P4 § PtdIns(4,5)P2 § Ins(1,4,5)P3 (Ki § 10 mM) (Table 1). Both PtdIns(3,4,5)P3 derivatives (dibenzoyl and diheptanoyl PtdIns(3,4,5)P3) gave almost the same effect.
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FIG. 1. Competition curves of GST-Btk-PH for Ins(1,3,4,5)P4 with various inositol phosphates and phosphoinositides. The competition experiment was performed by using 0.5 mg of GST fusion proteins, as described in MATERIALS AND METHODS. Ins(1,3,4,5)P4 (closed squares), dibenzoyl PtdIns(3,4,5)P3 (open squares), Ins(3,4,5,6)P4 (closed triangles), Ins(1,2,5,6)P4 (closed reverse triangles), and dibenzoyl PtdIns(4,5)P2 (open triangles). Data are presented as means { S.D. of three measurements.
The Ki value for PtdIns(3,4,5)P3 presented here and in Table 1 is for dibenzoyl PtdIns(3,4,5)P3 . Ins(1,3,4,5)P4 and InsP6 binding properties of mutant PH domains of Btk. To characterize the effect of XLA and Xid mutations on the Ins(1,3,4,5)P4 and InsP6 binding properties of the PH domain of Btk, the mutant PH domains were expressed as GST fusion proteins. The GST fusion proteins were affinity-purified and analyzed by SDS-PAGE (Figure 2A). [3H]Ins(1,3,4,5)P4 and [3H]InsP6 binding assays showed that the Ins-
TABLE 1
Competition of [3H] Ins(1,3,4,5)P4 Binding to GST-Btk-PH with Various Inositol Phosphates and Phosphoinositides Inositol phosphate and phosphoinositide Ins(1,3,4)P3 Ins(1,4,5)P3 Ins(1,5,6)P3 Ins(1,2,5,6)P4 Ins(1,3,4,5)P4 Ins(1,3,4,6)P4 Ins(3,4,5,6)P4 Ins(1,3,4,5,6)P5 InsP6 PtdIns(4,5)P2 PtdIns(3,4,5)P3a
FIG. 2. Preparation of GST-Btk-PH bearing the XLA and Xid mutations and their Ins(1,3,4,5)P4 and InsP6 binding activity. (A) SDS-PAGE gel of purified wild type and mutant GST-Btk-PH was stained with Coomassie brilliant blue R-250 to visualize proteins. The band around 48 kDa corresponds to GST-Btk-PH. The bars and numbers on the left indicate the positions of the protein molecular mass standards (kDa). (B) Binding of Ins(1,3,4,5)P4 (open bars) and InsP6 (hatched bars) to GST-Btk-PH proteins. The binding assay was performed using either 0.5 mg (wild type) or 1 mg (mutants) of GST fusion proteins as described in MATERIALS AND METHODS. Note that in some mutations, especially R28C, InsP6 binding was not reduced to the same extent as Ins(1,3,4,5)P4 binding. Data are presented as means { S.D. of three measurements.
Ki (mM) 11 54 6.3 29 0.015 1.9 1.1 0.090 0.092 35 0.113
{ { { { { { { { { { {
2 8 0.5 1 0.005 0.1 0.1 0.028 0.061 2 0.005
a Dibenzoyl PtdIns(3,4,5)P3 . The binding assay was performed in 50 mM HEPES-KOH, pH 7.2, as described in MATERIALS AND METHODS. Determination of Ki values was carried out as shown in Fig. 1. Values are expressed as means { S.D. of two to three determinations.
(1,3,4,5)P4 binding activity of all mutants, including recently identified mutants S14F (21), was reduced, as reported previously (22), but InsP6 binding activity was less affected by several mutations (Figure 2B). It was particularly noteworthy that the R28C mutant maintained 77% of the wild type InsP6 binding activity. The inositol phosphate binding activity of the R28H mutant was weaker than that of the R28C mutant. To further evaluate the effect of mutation on Ins(1,3,4,5)P4 and InsP6 binding activity, competition experiments for [3H]Ins(1,3,4,5)P4 binding were performed using wild type GST-Btk-PH and the F25S and R28C mutants (Figure 3 and Table 2). With the F25S mutant, the Kd values for Ins(1,3,4,5)P4 (9 { 1 nM) and InsP6 (159 { 68 nM) were not significantly different from that for
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F25S and 0.16 { 0.10 pmol/pmol of protein for the wild type). In contrast, while the R28C mutant had a 10 times higher Kd value for Ins(1,3,4,5)P4 (184 { 17 nM), the Bmax (0.16 { 0.01 pmol/pmol of protein) was almost the same as with the wild type. The Kd value of the R28C mutant binding to InsP6 (Kd : 251 { 39 nM) was slightly higher than wild type. However, the Bmax value (0.77 { 0.07 pmol/pmol of protein) of the R28C mutant for InsP6 was about four times higher than that of the wild type (Bmax : 0.22 { 0.03 pmol/pmol of protein). According to the three dimensional structure of the PH domain of phospholipase C-d1 with Ins(1,4,5)P3 (14), PH domains are expected to have a single binding site for inositol phosphate. Our results, however, indicates substoichiometric binding of Ins(1,3,4,5)P4 (or InsP6) to GST-Btk-PHs. This may result from instability of the Ins(1,3,4,5)P4-sensitive form of GST-Btk-PH. XLA or Xid mutations may affect the proportion of Ins(1,3,4,5)P4-sensitive form. Since both Ins(1,3,4,5,6)P5 and InsP6 competed with [3H]Ins(1,3,4,5)P4 in binding to GST-Btk-PH with similar affinities (22), Ins(1,3,4,5,6)P5 and InsP6 were expected to have similar characteristics with respect to binding to the mutants. To confirm this hypothesis, experiments were performed where Ins(1,3,4,5,6)P5 and InsP6 competed with [3H]Ins(1,3,4,5)P4 in binding to either R28C or F25S mutants. As expected, Ins(1,3,4,5,6)P5 yielded a competition curve similar to that of InsP6 (data not shown).
FIG. 3. Characterization of Ins(1,3,4,5)P4 and InsP6 binding to wild type and mutant GST-Btk-PH. For Ins(1,3,4,5)P4 binding experiments (closed squares) 0.5 mg of wild type protein and 1 mg of either R28C or F25S recombinant protein were used. The conditions were changed for the InsP6 binding curves (open squares): with 0.5 mg or wild type protein, 3 mg of R28C, and 4 mg of F25S recombinant proteins. Each experiment shown is a representative of three independent experiments. Data are presented as means { S.D. of two measurements. The insets are Scatchard analyses of the depicted data. (A) Binding of Ins(1,3,4,5)P4 and InsP6 to wild type GST-Btk-PH. (B) Binding to the R28C mutant. (C) Binding to the F25S mutant.
the wild type (15 { 5 nM for Ins(1,3,4,5)P4 and 147 { 44 nM for Ins P6), but the Bmax value for Ins(1,3,4,5)P4 was much lower (0.02 { 0.01 pmol/pmol of protein for
Ins(1,3,4,5)P4 and InsP6 binding properties of the PH domains of Tec, Itk, and Bmx. To determine whether the PH domains of other Tec family members function as Ins(1,3,4,5)P4 binding domains, the PH domains of Tec, Itk, and Bmx were expressed as GST fusion proteins (Figure 4B). As shown in Figure 4C, the binding of Ins(1,3,4,5)P4 and InsP6 to the Tec PH domain was similar to binding to the Btk PH domain. In contrast, Ins(1,3,4,5)P4 bound poorly to the Bmx PH domain and Itk PH domain did not bind both Ins(1,3,4,5)P4 and InsP6 . Figure 5 shows the results of competition experiments for [3H]Ins(1,3,4,5)P4 binding to the Tec PH domain by Ins(1,4,5)P3 , Ins(1,3,4,5)P4 , Ins(1,3,4,5,6)P5 , and InsP6 . The Tec PH domain yielded competition curves for each of the inositol phosphate similar to those obtained for the PH domain of Btk (22). DISCUSSION In the present study, we demonstrated the binding properties of inositol phosphate and phosphoinositide to the PH domain of Btk. The Btk PH domain specifically recognizes the Ins(1,3,4,5)P4 isomer with high affinity (Kd Å 15 nM), while it also binds Ins(1,3,4,5,6)P5 , InsP6 , and PtdIns(3,4,5)P3 but with lower affinity (Ki Å 90-100 nM). Missense mutations
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Inositol Polyphosphate Binding Properties of Wild and Mutant PH Domains of Btk Wild
Inositol phosphate Ins 1,3,4,5-P4 InsP6
R28C
Kd (nM)
Bmax (pmol/pmol)
15 { 5 147 { 44
0.16 { 0.10 0.22 { 0.03
Kd (nM) 184 { 17 251 { 39
F25S Bmax (pmol/pmol) 0.16 { 0.01 0.77 { 0.07
Kd (nM) 9{1 159 { 68
Bmax (pmol/pmol) 0.02 { 0.01 0.24 { 0.03
Note. The affinities (Kd) and the Bmax values were obtained from the Scatchard analysis depicted in Fig. 3. Values are expressed as means { S.D. of three determinations.
in the Btk PH domain of XLA and Xid severely reduced the binding of Ins(1,3,4,5)P4 , but some mutations were less effective in reducing the binding of InsP6 . In particular, InsP6 binds more strongly to the mouse Xid mutant (R28C) than to the human XLA mutant (R28H), even though the mutation resides at the same position in both proteins. The Xid mice have been reported to demonstrate a milder phenotype as compared with XLA patients. Thus, a relationship may exist between inositol polyphosphates binding capacity and the severity of the disease. The general
absence of Btk protein, however, also results in a Xid phenotype in mice (6), indicating severity of the disease is not simply proper binding to the PH domain of Btk. In a previous paper we showed that the gain-of-function mutant (E41K) of Btk (29) had higher affinity for InsP6 than wild type (22). We hypothesize that Ins(1,3,4,5,6)P5 and InsP6 , which are major components of inositol phosphates in cytoplasm (30), may possibly compensate for the impaired Ins(1,3,4,5)P4 binding activity of Xid mutant, and activate the gain-of-function mutant of Btk.
FIG. 4. Comparison of the PH domain of the Tec protein tyrosine kinase family. (A) Amino acid sequence alignment of the PH domains of mouse Bmx, Itk, TecII, and Btk. Bold characters indicate residues which are identical in all four sequences. * indicates the conserved arginine residue, replaced by cisteine in Btk from the Xid mouse. # indicate the positions of missense mutations found in XLA patients. (B) Coomassie stained SDS-PAGE gel of purified GST fusion proteins (GST-Btk-PH, GST-Tec-PH, GST-Itk-PH, and GST-Bmx-PH). Bands around 48 or 45 kDa correspond to GST-PH. (C) Ins(1,3,4,5)P4 (open bars) and InsP6 (hatched bars) binding measurements of GST-BtkPH, GST-Tec-PH, GST-Itk-PH, and GST-Bmx-PH. For these experiments, 0.5 mg of either GST-Btk-PH or GST-Tec-PH or 1 mg of GST-ItkPH or GST-Bmx-PH purified protein was used. Data are presented as means { S.D. of three measurements.
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FIG. 5. Competition curves of GST-Tec-PH for Ins(1,3,4,5)P4 by several inositol phosphates. The binding of Ins(1,4,5)P3 is shown by closed squares, Ins(1,3,4,5)P4 by open circles, Ins(1,3,4,5,6)P5 by open squares, and InsP6 with closed circles. The protein amount used for the binding experiment was 0.5 mg of GST-Tec-PH. Data are presented as means { S.D. of three measurements.
Although all the amino acids altered in this study are conserved in the Itk PH domain (Figure 4A), Itk did not bind Ins(1,3,4,5)P4 and InsP6 , indicating that both are not physiological ligands of Itk. In addition, the Bmx PH domain poorly bound Ins(1,3,4,5)P4 , like in the case of Btk mutants, suggesting that Ins(1,3,4,5)P4 is not a physiological ligand of Bmx. However, at present, we cannot rule out the possibility that the intracellular concentration of IP4 is high enough to bind to Bmx PH domain in some cells (e.g. heart (28)). The similar binding profiles of Tec and Btk may indicate they have similar regulatory mechanisms. It has been suggested that Btk may function through reversible membrane association (29,31) and that the PH domain functions as membrane anchor by binding to phosphoinositides. Since the PH domain of Btk binds PtdIns(3,4,5)P3 with much higher affinity than PtdIns(4,5)P2 , Btk may bind PtdIns(3,4,5)P3 embedded in the membrane. Recently, a novel SH2-containing inositol polyphosphate 5-phosphatase named SHIP has been described (32-34). SHIP is expressed in a variety of hematopoietic cells and hydrolyzes Ins(1,3,4,5)P4 to Ins(1,3,4)P3 and PtdIns(3,4,5)P3 to PtdIns(3,4)P2 . Our studies have shown that the affinity of the Btk PH domain for Ins(1,3,4)P3 , the dephosphorylation product of Ins(1,3,4,5)P4 at the D-5 position, is about thousand fold lower than that for Ins(1,3,4,5)P4 (Table 2). Both the tyrosine phosphorylation of SHIP and its association with Shc or FcgRIIB have been suggested to be involved in negative signaling in B cells (35,36). These observations, combined with our studies, suggest that SHIP may have a role in regulating Btk. Since some mutations that lead to XLA are clustered
in the N-terminal half of the Btk PH domain, it has been suggested to be important (22). In order to understand the pathogenesis of XLA, the ligand(s) which bind to the Btk PH domain and how the binding is distorted by XLA mutations in vivo must be known. While elucidation of the physiological role of Ins(1,3,4,5)P4 is necessary for full understanding of the pathogenesis of XLA, we hypothesize that Ins(1,3,4,5)P4 is a ligand for Btk, and that disruption of the BtkIns(1,3,4,5)P4 interaction may lead to XLA. In summary, we have for the first time demonstrated that the PH domain of Btk binds at least two important signaling molecules, Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3 . We now propose that loss of these binding capacity of Btk results in immunodeficiency such as the human XLA and the mouse Xid. ACKNOWLEDGMENTS We thank Dr. Masato Hirata (Kyushu University) for advice. We are grateful to other members of the Mikoshiba Laboratory for the valuable discussions.
REFERENCES 1. Tsukada, S., Saffran, D. C., Rawlings, D. J., Parolini, O., Allen, R. C., Klisak, I., Sparkes, R. S., Kubagawa, H., Mohandas, T., Quan, S., Belmont, J. W., Cooper, M. D., Conley, M. E., and Witte, O. N. (1993) Cell 72, 279–290. 2. Vetrie, D., Vorechovsky, I., Sideras, P., Holland, J., Davies, A., Flinter, F., Hammarstro¨m, L., Kinnon, C., Levinsky, R., Bobrow, M., Smith, C. I. E., and Bentley, D. R. (1993) Nature 361, 226– 233. 3. Thomas, J. D., Sideras, P., Smith, C. I. E., Vorechovsky, I., Chapman, V., and Paul, W. E. (1993) Science 261, 355–358. 4. Rawlings, D. J., Saffran, D. C., Tsukada, S., Largaespada, D. A., Grimaldi, J. C., Cohen, L., Mohr, R. N., Bazan, J. F., Howard, M., Copeland, N. G., Jenkins, N. A., and Witte, O. N. (1993) Science 261, 358–361. 5. Conley, M. E. (1992) Annu. Rev. Immunol. 10, 215–238. 6. Khan, W. N., Alt, F. W., Gerstein, R. M., Malynn, B. A., Larsson, I., Rathbun, G., Davidson, L., Mu¨ller, S., Kantor, A. B., Herzenberg, L. A., Rosen, F. S., and Sideras, P. (1995) Immunity 3, 283– 299. 7. Musacchio, A., Gibson, T., Rice, P., Thompson, J., and Saraste, M. (1993) Trends. Biochem. Sci. 18, 343–348. 8. Shaw, G. (1996) BioEssays 18, 35–46. 9. Yoon, H. S., Hajduk, P. J., Petros, A. M., Olejniczak, E. T., Meadows, R. P., and Fesik, S. W. (1994) Nature 369, 672–675. 10. Macias, M. J., Musacchio, A., Ponstingl, H., Nilges, M., Saraste, M., and Oschkinat, H. (1994) Nature 369, 675–677. 11. Downing, A. K., Driscoll, P. C., Gout, I., Salim, K., Zvelebil, M. J., and Waterfield, M. D. (1994) Curr. Biol. 4, 884–891. 12. Timm, D., Salim, K., Gout, I., Guruprasad, L., Waterfield, M., and Blundell, T. (1994) Nature Struct. Biol. 1, 782–788. 13. Ferguson, K. M., Lemmon, M. A., Schlessinger, J., and Sigler, P. B. (1994) Cell 79, 199–209. 14. Ferguson, K. M., Lemmon, M. A., Schlessinger, J., and Sigler, P. B. (1995) Cell 83, 1037–1046.
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15. Touhara, K., Inglese, J., Pitcher, J. A., Shaw, G., and Lefkowitz, R. J. (1994) J. Biol. Chem. 269, 10217–10220. 16. Tsukada, S., Simon, M. I., Witte, O. N., and Katz, A. (1994) Proc. Natl. Acad. Sci. USA 91, 11256–11260. 17. Harlan, J. E., Hajduk, P. J., Yoon, H. S., and Fesik, S. W. (1994) Nature 371, 168–170. 18. Hyvo¨nen, M., Macias, M. J., Nilges, M., Oschkinat, H., Saraste, M., and Wilmanns, M. (1995) EMBO J. 14, 4676–4685. 19. Yao, L., Kawakami, Y., and Kawakami, T. (1994) Proc. Natl. Acad. Sci. USA 91, 9175–9179. 20. Konishi, H., Kuroda, S., and Kikkawa, U. (1994) Biochem. Biophys. Res. Commun. 205, 1770–1775. 21. Mattsson, P. T., Vihinen, M., and Smith, C. I. E. (1996) BioEssays 18, 825–834. 22. Fukuda, M., Kojima, T., Kabayama, H., and Mikoshiba, K. (1996) J. Biol. Chem. 271, 30303–30306. 23. Watanabe, Y., Hirofuji, H., and Ozaki, S. (1994) Tetrahedron Lett. 35, 123–124. 24. Watanabe, Y., Inada, E., Jinno, M., and Ozaki, S. (1993) Tetrahedron Lett. 34, 497–500. 25. Mano, H., Yamashita, Y., Yazaki, Y., and Hirai, H. (1995) Blood 85, 343–350. 26. Siliciano, J. D., Morrow, T. A., and Desiderio, S. V. (1992) Proc. Natl. Acad. Sci. USA 89, 11194–11198.
27. Heyeck, S. D., and Berg, L. J. (1993) Proc. Natl. Acad. Sci. USA 90, 669–673. 28. Tamagnone, L., Lahtinen, I., Mustonen, T., Virtaneva, K., Francis, F., Muscatelli, F., Alitalo, R., Smith, C. I. D., and Alitalo, K. (1994) Oncogene 9, 3683–3688. 29. Li, T., Tsukada, S., Satterthwaite, A., Havlik, M. H., Park, H., Takatsu, K., and Witte, O. N. (1995) Immunity 2, 451–460. 30. Mountford, J. C., Bunce, C. M., French, P. J., Michell. R. H., and Brown, G. (1994) Biochim. Biophys. Acta 1222, 101–108. 31. Kawakami, Y., Yao, L., Miura, T., Tsukada, S., Witte, O. N., and Kawakami, T. (1994) Mol. Cell. Biol. 14, 5108–5113. 32. Lioubin, M. N., Algate, P. A., Tsai, S., Carlberg, K., Aebersold, R., and Rohrschneider, L. R. (1996) Genes Dev. 10, 1084–1095. 33. Kavanaugh, W. M., Pot, D. A., Chin, S. M., Deuter-Reinhard, M., Jefferson, A. B., Norris, F. A., Masiarz, F. R., Cousens, L. S., Majerus, P. W., and Williams, L. T. (1996) Curr. Biol. 6, 438–445. 34. Damen, J. E., Liu, L., Rosten, P., Humphries, R. K., Jefferson, A. B., Majerus, P. W., and Krystal, G. (1996) Proc. Natl. Acad. Sci. USA 93, 1689–1693. 35. Ono, M., Bolland, S., Tempst, P., and Ravetch, J. V. (1996) Nature 383, 263–266. 36. Chacko, G. W., Tridandapani, S., Damen, J. E., Liu, L., Krystal, G., and Coggeshall, K. M. (1996) J. Immunol. 157, 2234–2238.
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Characterization of the Pleckstrin Homology Domain of Btk as an Inositol Polyphosphate and Phosphoinositide Binding Domain1 Toshio Kojima,*,† Mitsunori Fukuda,*,†,2 Yutaka Watanabe,‡ Fumiaki Hamazato,*,§ and Katsuhiko Mikoshiba*,†,Ø *Molecular Neurobiology Laboratory, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan; †Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan; ‡Faculty of Engineering, Ehime University, Matsuyama, 790-77, Japan; §Hitachi Advanced Research Laboratory, Hatoyama, Saitama, 350-03, Japan; and ØCalciosignal Net Project, Exploratory Research for Advanced Technology (ERATO), 2-9-3 Shimo-meguro, Meguro-ku, Tokyo 153, Japan
Received June 9, 1997
We previously reported that the pleckstrin homology (PH) domain of Bruton’s tyrosine kinase (Btk) binds Ins(1,3,4,5)P4 and that missense mutations in this domain which cause either human X-linked agammaglobulinemia (XLA) or murine X-linked immunodeficiency (Xid) also dramatically reduce the Ins(1,3,4,5)P4 binding activity. In this paper, we describe the inositol phosphate binding specificity of the Btk PH domain and different inositol polyphosphate binding properties among the PH domains of Tec family kinases. Our results suggest that certain inositol phosphates and/or phosphoinositides are physiological ligands of some Tec family kinases and that Tec family members are differently regulated by inositol molecules. q 1997 Academic Press
Bruton’s tyrosine kinase (Btk) is a member of the Tec family of cytoplasmic protein tyrosine kinases (11 This work was supported by grants from the Japanese Ministry of Education, Science, Sports, and Culture to K.M. and to Y.W. (06240105), the Science and Technology Agency of Japan to K.M., and the Japan Society for the Promotion of Science to M.F. 2 To whom correspondence should be addressed at Molecular Neurobiology Laboratory, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan. Fax: /81-298-36-9040. E-mail: fukuda@rtc. riken.go.jp. Abbreviations: Btk, Bruton’s tyrosine kinase; GST, glutathione Stransferase; InsP3 , inositol trisphosphate; InsP4 , inositol tetrakisphosphate; Ins(1,3,4,5,6)P5 , inositol 1,3,4,5,6-pentakisphosphate; InsP6 , inositol hexakisphosphate; PH, pleckstrin homology; PtdInsP2 , phosphatidylinositol bisphosphate; PtdIns(3,4,5)P3 , phosphatidylinositol 3,4,5-trisphosphate; Xid, X-linked immunodeficiency; XLA, X-linked agammaglobulinemia.
4). Since mutations in the Btk gene cause human Xlinked agammaglobulinemia (XLA) and murine Xlinked immunodeficiency (Xid), Btk appears to be crucial for B cell maturation. XLA patients have B cell differentiation defect which results in a deficiency of mature B cells and immunoglobulins, and thus these patients suffer from an increased susceptibility to infections (5). The Xid mice display a milder phenotype as compared with XLA patients, but the reason for this phenotypic discrepancy is not known (6). The most significant feature of the Tec family of tyrosine kinases is the presence of a pleckstrin homology (PH) domain in the amino-terminal region. The PH domain is composed of approximately 100 loosely conserved amino acids and is found in many proteins involved in signal transduction and cytoskeletal structures (7,8). Recently, the NMR structures of the PH domains have been determined for pleckstrin (9), bspectrin (10), and dynamin (11), and the X-ray structures for dynamin (12,13) and phospholipase C-d1 (14) have been calculated. Despite the low amino acid homology among PH domains of these molecules, all the PH domains have a similar structure: a fold that consists of a b-barrel formed by two b-sheets, with a Cterminal a-helix at one end of the b-barrel. Although the exact function of the PH domain is not known, the carboxy-terminal region of the PH domains has been shown to interact with the b/g subunits of heterotrimeric G proteins (15,16). The amino-terminal region has been shown to bind to PtdIns(4,5)P2 (17,18). These findings suggest a role for the PH domain anchoring the molecule to the membrane. Additionally, in some
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proteins, the PH domains have been reported to associate with several isoforms of protein kinase C (19,20). The Xid mutation and some of the XLA mutations are located in the PH domain of Btk, implying an important role for this domain in Btk function (3,4,21). We previously reported that the PH domain of Btk functions as an Ins(1,3,4,5)P4 binding domain and that the missense mutations of XLA and Xid found in the PH domain cause a dramatic reduction in Ins(1,3,4,5)P4 binding activity (22). In this study we examined the characteristics of the PH domains of Tec family kinases on the inositol phosphate and phosphoinositide binding in greater detail. We show that Btk specifically recognizes the Ins(1,3,4,5)P4 isomer with high affinity and that the PH domains of Tec family kinases are different with respect to inositol polyphosphate binding. MATERIALS AND METHODS Chemicals. D-myo-Ins(1,2,5,6)P4 and D-myo-Ins(3,4,5,6)P4 were purchased from Boehringer Mannheim. D-myo-Ins(1,3,4)P3 , D-myoIns(1,4,5)P3 , D-myo-Ins(1,5,6)P3 , D-myo-Ins(1,3,4,5)P4 , D-myoIns(1,3,4,6)P4 , D-myo-Ins(1,3,4,5,6)P5 , and D-myo-InsP6 were obtained from Calbiochem. PtdIns(3,4,5)P3 derivatives bearing benzoyl and heptanoyl groups were prepared according to the method reported (23). Dibenzoyl PtdIns(4,5)P2 was synthesized from 1,2-diO-cyclohexylidene-myo-inositol via regioselective phosphorylation of the 1,2-diol derivative (24). These phosphoinositide derivatives are soluble in water. All other chemicals were commercial products of reagent grade. Solutions were prepared in deionized water. cDNA cloning. Preparation of the cDNA encoding the PH domain of Btk and the site-directed mutagenesis of the PH domain were performed as described previously (22). cDNAs encoding the PH domains of Tec (amino acids 1-143; numbering according to GenBank accession number JU0215) (25) and Itk/Tsk (amino acids 1-143; GenBank accession number L05631) (26,27) from an adult mouse spleen (BALB/c) were amplified by the reverse transcriptase PCR (LA-PCR kit; Takara Shuzo, Japan) for 40 cycles. Each cycle consisted of denaturation at 94 7C for 1 min, annealing at 50 7C for 2 min, and extension at 72 7C for 3-4.5 min. The extension time was increased by 30 sec every 10 cycles. cDNA encoding the PH domain of Bmx (amino acids 1-143; GenBank accession number U88091) (28) from an adult mouse heart (ICR) was also amplified by the reverse transcriptase PCR for 30 cycles, each consisting of denaturation at 94 7C for 1 min, annealing at 48 7C for 2 min, and extension at 72 7C for 2 min. The sense and antisense primers were designed as follows: primer Tec1 (sense: amino acid residues 1-7) 5*-CGGATCCATGAATTTCAACACTATCCT-3*, primer Tec-2 (antisense: amino acid residues 138143) 5*-CGAATTCACATCCGGGTGCTAGTTTTT-3*, primer Itk-1 (sense: amino acid residues 1-7) 5*-CGGATCCATGAACAACTTCATCCTCCT-3*, primer Itk-2 (antisense: amino acid residues 138-143) 5*-CGAATTCACAGCCTACAGCAGGCTTCT-3*, primer Bmx-1 (sense: amino acid residues 1-3) 5*-CGGATCCATGGACGACAATATGGAGAG-3*, and primer Bmx-2 (antisense: amino acid residues 137143) 5*-CGAATTCGCATCCTGGGGCTGCTTTGC-3*. The PCR products were purified on a 0.7% agarose gel and extracted with a Geneclean kit II (Bio 101). After digestion with BamHI and EcoRI, the cDNA inserts were subcloned into the BamHI-EcoRI site of pGEX2T (Pharmacia Biotech Inc.). Clones were verified by DNA sequencing using a BcaBEST dideoxy sequencing kit (Takara Shuzo). Preparation of GST fusion protein. The PH domains of mouse Btk, Tec, Itk, and Bmx were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli JM109 and purified by glu-
tathione-Sepharose 4B column chromatography (Pharmacia) according to the manufacturer’s recommendations. The GST-Btk-PH protein contains amino acids 1-165 of mouse Btk (numbering according to GenBank accession number L08967). Similarly, GST-TecPH has amino acids 1-143 of mouse TecII, and GST-Itk-PH has amino acids 1-143 of mouse Itk. Since the Bmx-1 primer includes 5* flanking region of the putative initiation codon (28), GST-Bmx-PH contains additional four amino acids (Met-Asp-Asp-Asn) between GST and the mouse Bmx (amino acids 1-143). The mutants of the Btk-PH domain were XLA mutants: GST-Btk-PH (S14F) with Ser replaced by Phe at position 14, GST-Btk-PH (F25S), GST-Btk-PH (R28H), GST-Btk-PH (T33P), GST-Btk-PH (V64F), and GST-Btk-PH (V113D). Additionally a Xid mutant GST-Btk-PH (R28C) was constructed (22). The protein concentrations of the purified recombinant proteins were determined initially by using a Bio-Rad Protein Assay (Bio-Rad). Purified proteins were analyzed by 10% SDS-PAGE followed by Coomassie brilliant blue R-250 staining. The purity of each recombinant protein was estimated by scanning the Coomassie stained SDS-PAGE gel followed by densitometry with Bio Image (Millipore), and then the concentration of each GST-PH protein was determined. Measurement of [3H]Ins(1,3,4,5)P4 and [3H]InsP6 binding to GST fusion proteins. GST fusion proteins (0.5 or 1 mg) were incubated with 9.6 nM [3H]Ins(1,3,4,5)P4 (specific radioactivity 777 GBq/mmol) or 9.4 nM [3H]InsP6 (specific radioactivity 798.1 GBq/mmol) (DuPont NEN) in 50 ml of 50 mM HEPES-KOH, pH 7.2, for 10 min at 4 7C. The samples were then mixed with 1 ml of 50 mg/ml g-globulins and 49 ml of a solution containing 30% PEG6000 and 50 mM HEPESKOH, pH 7.2 and placed on ice for 10 min. The precipitate obtained via centrifugation at 10,000 1 g for 10 min was solubilized in 500 ml of Solvable (Packard Instrument Co.). The amount of radioactivity was measured in Aquasol 2 (Packard) with a liquid scintillation counter. Inhibition of specific [3H]Ins(1,3,4,5)P4 binding and specific [3H]InsP6 binding to GST fusion proteins was also performed in the above reaction mixture containing various inositol phosphates or phosphoinositides. Non-specific binding was defined as the binding observed in the absence of GST fusion proteins. Data processing. All statistical analyses and curve fitting were done using a GraphPad PRISM computer program.
RESULTS Inositol phosphate and phosphoinositide binding specificity of the Btk PH domain. In our previous study, we characterized the Btk PH domain as an inositol polyphosphate binding site (competitive potencies for Ins(1,3,4,5)P4 binding are Ins(1,3,4,5)P4 ú Ins(1,3,4,5,6)P5 § InsP6 ú Ins(1,4,5)P3). However, it remained undetermined whether the Btk PH domain also recognizes other InsP4 isomers and phosphoinositides. To answer this question, competition experiments for [3H]Ins(1,3,4,5)P4 binding were performed using several inositol phosphates and phosphoinositides as competitors (Figure 1). Competitive potencies for Ins(1,3,4,5)P4 binding to the PH domain of Btk in decreasing order were Ins(1,3,4,5)P4 (Kd Å 15 nM) ú Ins(1,3,4,5,6)P5 É InsP6 § PtdIns(3,4,5)P3 (Ki É 110 nM) ú Ins(3,4,5,6)P4 É Ins(1,3,4,6)P4 (Ki É 1 mM) ú Ins(1,5,6)P3 (Ki Å 6 mM) úIns(1,3,4)P3 § Ins(1,2,5,6)P4 § PtdIns(4,5)P2 § Ins(1,4,5)P3 (Ki § 10 mM) (Table 1). Both PtdIns(3,4,5)P3 derivatives (dibenzoyl and diheptanoyl PtdIns(3,4,5)P3) gave almost the same effect.
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FIG. 1. Competition curves of GST-Btk-PH for Ins(1,3,4,5)P4 with various inositol phosphates and phosphoinositides. The competition experiment was performed by using 0.5 mg of GST fusion proteins, as described in MATERIALS AND METHODS. Ins(1,3,4,5)P4 (closed squares), dibenzoyl PtdIns(3,4,5)P3 (open squares), Ins(3,4,5,6)P4 (closed triangles), Ins(1,2,5,6)P4 (closed reverse triangles), and dibenzoyl PtdIns(4,5)P2 (open triangles). Data are presented as means { S.D. of three measurements.
The Ki value for PtdIns(3,4,5)P3 presented here and in Table 1 is for dibenzoyl PtdIns(3,4,5)P3 . Ins(1,3,4,5)P4 and InsP6 binding properties of mutant PH domains of Btk. To characterize the effect of XLA and Xid mutations on the Ins(1,3,4,5)P4 and InsP6 binding properties of the PH domain of Btk, the mutant PH domains were expressed as GST fusion proteins. The GST fusion proteins were affinity-purified and analyzed by SDS-PAGE (Figure 2A). [3H]Ins(1,3,4,5)P4 and [3H]InsP6 binding assays showed that the Ins-
TABLE 1
Competition of [3H] Ins(1,3,4,5)P4 Binding to GST-Btk-PH with Various Inositol Phosphates and Phosphoinositides Inositol phosphate and phosphoinositide Ins(1,3,4)P3 Ins(1,4,5)P3 Ins(1,5,6)P3 Ins(1,2,5,6)P4 Ins(1,3,4,5)P4 Ins(1,3,4,6)P4 Ins(3,4,5,6)P4 Ins(1,3,4,5,6)P5 InsP6 PtdIns(4,5)P2 PtdIns(3,4,5)P3a
FIG. 2. Preparation of GST-Btk-PH bearing the XLA and Xid mutations and their Ins(1,3,4,5)P4 and InsP6 binding activity. (A) SDS-PAGE gel of purified wild type and mutant GST-Btk-PH was stained with Coomassie brilliant blue R-250 to visualize proteins. The band around 48 kDa corresponds to GST-Btk-PH. The bars and numbers on the left indicate the positions of the protein molecular mass standards (kDa). (B) Binding of Ins(1,3,4,5)P4 (open bars) and InsP6 (hatched bars) to GST-Btk-PH proteins. The binding assay was performed using either 0.5 mg (wild type) or 1 mg (mutants) of GST fusion proteins as described in MATERIALS AND METHODS. Note that in some mutations, especially R28C, InsP6 binding was not reduced to the same extent as Ins(1,3,4,5)P4 binding. Data are presented as means { S.D. of three measurements.
Ki (mM) 11 54 6.3 29 0.015 1.9 1.1 0.090 0.092 35 0.113
{ { { { { { { { { { {
2 8 0.5 1 0.005 0.1 0.1 0.028 0.061 2 0.005
a Dibenzoyl PtdIns(3,4,5)P3 . The binding assay was performed in 50 mM HEPES-KOH, pH 7.2, as described in MATERIALS AND METHODS. Determination of Ki values was carried out as shown in Fig. 1. Values are expressed as means { S.D. of two to three determinations.
(1,3,4,5)P4 binding activity of all mutants, including recently identified mutants S14F (21), was reduced, as reported previously (22), but InsP6 binding activity was less affected by several mutations (Figure 2B). It was particularly noteworthy that the R28C mutant maintained 77% of the wild type InsP6 binding activity. The inositol phosphate binding activity of the R28H mutant was weaker than that of the R28C mutant. To further evaluate the effect of mutation on Ins(1,3,4,5)P4 and InsP6 binding activity, competition experiments for [3H]Ins(1,3,4,5)P4 binding were performed using wild type GST-Btk-PH and the F25S and R28C mutants (Figure 3 and Table 2). With the F25S mutant, the Kd values for Ins(1,3,4,5)P4 (9 { 1 nM) and InsP6 (159 { 68 nM) were not significantly different from that for
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F25S and 0.16 { 0.10 pmol/pmol of protein for the wild type). In contrast, while the R28C mutant had a 10 times higher Kd value for Ins(1,3,4,5)P4 (184 { 17 nM), the Bmax (0.16 { 0.01 pmol/pmol of protein) was almost the same as with the wild type. The Kd value of the R28C mutant binding to InsP6 (Kd : 251 { 39 nM) was slightly higher than wild type. However, the Bmax value (0.77 { 0.07 pmol/pmol of protein) of the R28C mutant for InsP6 was about four times higher than that of the wild type (Bmax : 0.22 { 0.03 pmol/pmol of protein). According to the three dimensional structure of the PH domain of phospholipase C-d1 with Ins(1,4,5)P3 (14), PH domains are expected to have a single binding site for inositol phosphate. Our results, however, indicates substoichiometric binding of Ins(1,3,4,5)P4 (or InsP6) to GST-Btk-PHs. This may result from instability of the Ins(1,3,4,5)P4-sensitive form of GST-Btk-PH. XLA or Xid mutations may affect the proportion of Ins(1,3,4,5)P4-sensitive form. Since both Ins(1,3,4,5,6)P5 and InsP6 competed with [3H]Ins(1,3,4,5)P4 in binding to GST-Btk-PH with similar affinities (22), Ins(1,3,4,5,6)P5 and InsP6 were expected to have similar characteristics with respect to binding to the mutants. To confirm this hypothesis, experiments were performed where Ins(1,3,4,5,6)P5 and InsP6 competed with [3H]Ins(1,3,4,5)P4 in binding to either R28C or F25S mutants. As expected, Ins(1,3,4,5,6)P5 yielded a competition curve similar to that of InsP6 (data not shown).
FIG. 3. Characterization of Ins(1,3,4,5)P4 and InsP6 binding to wild type and mutant GST-Btk-PH. For Ins(1,3,4,5)P4 binding experiments (closed squares) 0.5 mg of wild type protein and 1 mg of either R28C or F25S recombinant protein were used. The conditions were changed for the InsP6 binding curves (open squares): with 0.5 mg or wild type protein, 3 mg of R28C, and 4 mg of F25S recombinant proteins. Each experiment shown is a representative of three independent experiments. Data are presented as means { S.D. of two measurements. The insets are Scatchard analyses of the depicted data. (A) Binding of Ins(1,3,4,5)P4 and InsP6 to wild type GST-Btk-PH. (B) Binding to the R28C mutant. (C) Binding to the F25S mutant.
the wild type (15 { 5 nM for Ins(1,3,4,5)P4 and 147 { 44 nM for Ins P6), but the Bmax value for Ins(1,3,4,5)P4 was much lower (0.02 { 0.01 pmol/pmol of protein for
Ins(1,3,4,5)P4 and InsP6 binding properties of the PH domains of Tec, Itk, and Bmx. To determine whether the PH domains of other Tec family members function as Ins(1,3,4,5)P4 binding domains, the PH domains of Tec, Itk, and Bmx were expressed as GST fusion proteins (Figure 4B). As shown in Figure 4C, the binding of Ins(1,3,4,5)P4 and InsP6 to the Tec PH domain was similar to binding to the Btk PH domain. In contrast, Ins(1,3,4,5)P4 bound poorly to the Bmx PH domain and Itk PH domain did not bind both Ins(1,3,4,5)P4 and InsP6 . Figure 5 shows the results of competition experiments for [3H]Ins(1,3,4,5)P4 binding to the Tec PH domain by Ins(1,4,5)P3 , Ins(1,3,4,5)P4 , Ins(1,3,4,5,6)P5 , and InsP6 . The Tec PH domain yielded competition curves for each of the inositol phosphate similar to those obtained for the PH domain of Btk (22). DISCUSSION In the present study, we demonstrated the binding properties of inositol phosphate and phosphoinositide to the PH domain of Btk. The Btk PH domain specifically recognizes the Ins(1,3,4,5)P4 isomer with high affinity (Kd Å 15 nM), while it also binds Ins(1,3,4,5,6)P5 , InsP6 , and PtdIns(3,4,5)P3 but with lower affinity (Ki Å 90-100 nM). Missense mutations
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Inositol Polyphosphate Binding Properties of Wild and Mutant PH Domains of Btk Wild
Inositol phosphate Ins 1,3,4,5-P4 InsP6
R28C
Kd (nM)
Bmax (pmol/pmol)
15 { 5 147 { 44
0.16 { 0.10 0.22 { 0.03
Kd (nM) 184 { 17 251 { 39
F25S Bmax (pmol/pmol) 0.16 { 0.01 0.77 { 0.07
Kd (nM) 9{1 159 { 68
Bmax (pmol/pmol) 0.02 { 0.01 0.24 { 0.03
Note. The affinities (Kd) and the Bmax values were obtained from the Scatchard analysis depicted in Fig. 3. Values are expressed as means { S.D. of three determinations.
in the Btk PH domain of XLA and Xid severely reduced the binding of Ins(1,3,4,5)P4 , but some mutations were less effective in reducing the binding of InsP6 . In particular, InsP6 binds more strongly to the mouse Xid mutant (R28C) than to the human XLA mutant (R28H), even though the mutation resides at the same position in both proteins. The Xid mice have been reported to demonstrate a milder phenotype as compared with XLA patients. Thus, a relationship may exist between inositol polyphosphates binding capacity and the severity of the disease. The general
absence of Btk protein, however, also results in a Xid phenotype in mice (6), indicating severity of the disease is not simply proper binding to the PH domain of Btk. In a previous paper we showed that the gain-of-function mutant (E41K) of Btk (29) had higher affinity for InsP6 than wild type (22). We hypothesize that Ins(1,3,4,5,6)P5 and InsP6 , which are major components of inositol phosphates in cytoplasm (30), may possibly compensate for the impaired Ins(1,3,4,5)P4 binding activity of Xid mutant, and activate the gain-of-function mutant of Btk.
FIG. 4. Comparison of the PH domain of the Tec protein tyrosine kinase family. (A) Amino acid sequence alignment of the PH domains of mouse Bmx, Itk, TecII, and Btk. Bold characters indicate residues which are identical in all four sequences. * indicates the conserved arginine residue, replaced by cisteine in Btk from the Xid mouse. # indicate the positions of missense mutations found in XLA patients. (B) Coomassie stained SDS-PAGE gel of purified GST fusion proteins (GST-Btk-PH, GST-Tec-PH, GST-Itk-PH, and GST-Bmx-PH). Bands around 48 or 45 kDa correspond to GST-PH. (C) Ins(1,3,4,5)P4 (open bars) and InsP6 (hatched bars) binding measurements of GST-BtkPH, GST-Tec-PH, GST-Itk-PH, and GST-Bmx-PH. For these experiments, 0.5 mg of either GST-Btk-PH or GST-Tec-PH or 1 mg of GST-ItkPH or GST-Bmx-PH purified protein was used. Data are presented as means { S.D. of three measurements.
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FIG. 5. Competition curves of GST-Tec-PH for Ins(1,3,4,5)P4 by several inositol phosphates. The binding of Ins(1,4,5)P3 is shown by closed squares, Ins(1,3,4,5)P4 by open circles, Ins(1,3,4,5,6)P5 by open squares, and InsP6 with closed circles. The protein amount used for the binding experiment was 0.5 mg of GST-Tec-PH. Data are presented as means { S.D. of three measurements.
Although all the amino acids altered in this study are conserved in the Itk PH domain (Figure 4A), Itk did not bind Ins(1,3,4,5)P4 and InsP6 , indicating that both are not physiological ligands of Itk. In addition, the Bmx PH domain poorly bound Ins(1,3,4,5)P4 , like in the case of Btk mutants, suggesting that Ins(1,3,4,5)P4 is not a physiological ligand of Bmx. However, at present, we cannot rule out the possibility that the intracellular concentration of IP4 is high enough to bind to Bmx PH domain in some cells (e.g. heart (28)). The similar binding profiles of Tec and Btk may indicate they have similar regulatory mechanisms. It has been suggested that Btk may function through reversible membrane association (29,31) and that the PH domain functions as membrane anchor by binding to phosphoinositides. Since the PH domain of Btk binds PtdIns(3,4,5)P3 with much higher affinity than PtdIns(4,5)P2 , Btk may bind PtdIns(3,4,5)P3 embedded in the membrane. Recently, a novel SH2-containing inositol polyphosphate 5-phosphatase named SHIP has been described (32-34). SHIP is expressed in a variety of hematopoietic cells and hydrolyzes Ins(1,3,4,5)P4 to Ins(1,3,4)P3 and PtdIns(3,4,5)P3 to PtdIns(3,4)P2 . Our studies have shown that the affinity of the Btk PH domain for Ins(1,3,4)P3 , the dephosphorylation product of Ins(1,3,4,5)P4 at the D-5 position, is about thousand fold lower than that for Ins(1,3,4,5)P4 (Table 2). Both the tyrosine phosphorylation of SHIP and its association with Shc or FcgRIIB have been suggested to be involved in negative signaling in B cells (35,36). These observations, combined with our studies, suggest that SHIP may have a role in regulating Btk. Since some mutations that lead to XLA are clustered
in the N-terminal half of the Btk PH domain, it has been suggested to be important (22). In order to understand the pathogenesis of XLA, the ligand(s) which bind to the Btk PH domain and how the binding is distorted by XLA mutations in vivo must be known. While elucidation of the physiological role of Ins(1,3,4,5)P4 is necessary for full understanding of the pathogenesis of XLA, we hypothesize that Ins(1,3,4,5)P4 is a ligand for Btk, and that disruption of the BtkIns(1,3,4,5)P4 interaction may lead to XLA. In summary, we have for the first time demonstrated that the PH domain of Btk binds at least two important signaling molecules, Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3 . We now propose that loss of these binding capacity of Btk results in immunodeficiency such as the human XLA and the mouse Xid. ACKNOWLEDGMENTS We thank Dr. Masato Hirata (Kyushu University) for advice. We are grateful to other members of the Mikoshiba Laboratory for the valuable discussions.
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