Binding of Bruton's Tyrosine Kinase to Fyn, Lyn, Or Hck Through A

View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Caltech Authors - Main Proc. Nati. Acad. Sci. USA Vol. 91, pp. 8152-8155, August 1994 Cell Biology Binding of Bruton's tyrosine kinase to Fyn, Lyn, or Hck through a Src homology 3 domain-mediated interaction GENHONG CHENG, ZHENG-SHENG YE, AND DAVID BALTIMORE The Rockefeller University, 1230 York Avenue, New York, NY 10021 Contributed by David Baltimore, April 25, 1994 ABSTRACT Bruton's tyrosine kinase (Btk) is a recently MATERIALS AND METHODS described B-cell-specific tyrosine kinase. Mutations in this gene Cloning. To isolate Btk cDNA clones, oligonucleotides lead to human X chromosome-linked agammaglobulinemia corresponding to either the amino terminus or carboxyl and murine X-linked inmunodeficiency. Although genetic terminus of the human Btk protein were made according to evidence strongly suggests that Btk plays a crucial role in the published sequences (5) and used to probe duplicate lifts B-lymphocyte differentiation and activation, its precise mech- of A phage plaques on filters. Ten independent doubly pos- anism of action remains unknown, primarily because the itive clones were isolated from -1 x 106 phage plaques. We proteins that it interacts with have not yet been identified. partially sequenced these 10 cDNA clones and found that Here, we show that Btk interacts with Src homology 3 domains they all contained the full-length Btk coding sequence. A of Fyn, Lyn, and Hck, protein-tyrosine kinases that get acti- DNA fragment containing the amino-terminal portion of a vated upon stimulation of B- and T-cell receptors. These mouse Btk cDNA clone was constructed in the EG202 interactions are mediated by two 10-aa motifs in Btk. An vector, which contains the LexA DNA binding domain as its analogous site with the same specificity is also present in Itk, the amino terminus (12). When this LexA-BtkN bait, along with T-cell-specific homologue of Btk. Our data extend the range of the reporter plasmid pSH18-34 containing the LexA binding interactions mediated by Src homology 3 domains and provide site driving the lacZ gene, was transformed into the yeast an indication of a link between Btk and established signaling strain EGY48 (MATa, trpl, ura3, his, Leu2.plexAop6-leu2), pathways in B lymphocytes. colonies required leucine to grow and did not turn blue on 5-bromo-4-chloro-3-indolyl -D-galactoside (X-Gal) medium. Thus, the bait did not contain intrinsic transactivation po- Tyrosine phosphorylation is one of the earliest events during tential and was suitable for library screening. Library plas- B-lymphocyte activation. When the B-cell receptors on the mids (pJG4-5) containing the B42 acidic activation domain surface of resting splenic B cells or certain B-cell lines are and cDNAs derived from HeLa cells were then transformed cross-linked by anti-immunoglobin antibody, within 10 min into the yeast clone carrying both the LexA-BtkN bait and more than a dozen proteins become phosphorylated on the reporter plasmid pSH18-34. A total of 4.5 x 106 yeast tyrosine (1). Among these are certain Src family tyrosine transformants were selected on Ura-, His-, Trp-, and Leu- kinases such as Fyn, Lyn, and Hck, which are associated X-Gal/galactose plates. Approximately 3000 colonies grew in with the B-cell receptor complex in resting B cells (2-4). How the absence of leucine, but only 90 turned blue. Because the clustering of the B-cell receptor complex might lead to library vector pJG4-5 used here contained a galactose- activation of protein-tyrosine kinases remains to be shown. inducible promotor, colonies of authentic interacting clones Recently, a kinase called Bruton's tyrosine kinase (Btk) has should turn blue when galactose but not glucose is used as a been discovered, which seems to play a central role in B-cell carbon source. Colony replicas of the 90 clones were plated signaling events. Mutations of this gene are responsible for onto both X-Gal/glucose and X-Gal/galactose plates, and 70 X-linked agammaglobulinemia, a severe human immunode- showed unambiguous galactose-dependent blue color. Plas- ficiency disease, and murine X-linked immunodeficiency mid DNAs were isolated from 12 of these clones, and library (5-8). It belongs to a subgroup of Src-related kinases includ- plasmids were selected through their tryptophan synthase ing Itk found in T cells and Tec II found in liver and myeloid gene. They were purified and reintroduced back into yeast cells (9, 10). Like all other members of the Src family, Btk strain EGY48 along with the reporter plasmid pSH18-34 plus contains a kinase a Src homology (SH) 2 either the pEG202-lexA-BtkN or pEG202 vector. Colonies tyrosine domain, were replicated onto either glucose/X-Gal or galactose/X- domain, and an SH3 domain. The long amino-terminal region Gal plates. Clones with specific Btk-interacting proteins in Btk is similar to those in Itk and Tec II but is not shared showed blue color on galactose/X-Gal plates only with by other Src kinases and contains an apparent pleckstrin pEG202-LexA-BtkN as bait. The insert from one clone was homology (PH) domain (11). To understand how Btk is sequenced and found to correspond to the amino-terminal involved in B-cell development and activation, we have region of the human FYN gene. searched for proteins with which Btk might interact. We The Source ofthe Various SH3 Domains. GST-Abl-SH3 and show that Btk has a specific binding site that interacts with GST-Src-SH3 have been described (13). The sequences cod- the SH3 domains of the Src-related Fyn, Lyn, and Hck ing for the SH3 domains of mouse Btk (amino acid residues kinases. These interactions occur through two proline- 218-276), bovine Blk (55-113), and chicken Fyn (80-144) containing 10-aa sites in the amino-terminal region of Btk, were amplified from the corresponding cDNAs, whereas just carboxyl-terminal to the PH domain. We suggest that Btk those of mouse Lyn (64-124) and mouse Hck (60-116) were is on the pathway of signaling by which fyn, lyn, and hck amplified by reverse transcriptase-PCR using polyadenylyl- control cellular events. ated mRNAs isolated from BALB/c mouse spleen. These various DNA fragments were ligated in-frame to the pGex2T The publication costs of this article were defrayed in part by page charge Abbreviations: Btk, Bruton's tyrosine kinase; SH, Src homology; payment. This article must therefore be hereby marked "advertisement" GST, glutathione S-transferase; IPTG, isopropyl ,-D-thiogalacto- in accordance with 18 U.S.C. §1734 solely to indicate this fact. side; PH, pleckstrin homology. 8152 Cell Biology: Cheng et al. Proc. Natl. Acad. Sci. USA 91 (1994) 8153 vector and were sequenced using a primer derived from the of the human FYN gene; it contained the SH3 domain and a vector. Glutathione S-transferase (GST)-SH3 fusion proteins portion of the SH2 domain. were purified from isopropyl 3-D-thiogalactoside (IPTG)- To test whether Fyn can interact directly with Btk, in vitro induced bacterial lysates and were subsequently biotinylated filter binding assays were performed. The cDNA fragment as described (13, 14). from the isolated clone was transferred into the vector Solution Binding Assays. One liter of IPTG-induced bacte- pGex-lZT, which can express in bacteria GST fusion pro- ria expressing either GST or GST-Hck-SH3 was lysed in 10 teins in the same frame as the LexA fusion proteins of yeast. ml of ice-cold lysis buffer (PBS/100 mM EDTA/1% Triton The GST fusion protein containing the amino-terminal por- X-100/1% aprotinin/1 mM phenylmethylsulfonyl fluoride) tion of human Fyn (GST-Fyn-N) was purified and biotiny- by sonication. The supernatants were incubated with 250 ,Ld lated as described (13, 14). Total IPTG-induced bacterial cell of glutathione-agarose for 30 min at 40C. These GST- or lysates expressing the GST fusion protein containing the GST-Hck-SH3-saturated agarose beads were then washed full-length Btk (GST-Btk-F) were fractionated by SDS/ four times with the lysis buffer and stored in an equal volume PAGE, transferred to nitrocellulose, and probed with biotin- of PBS. To produce and detect Btk, we used a mammalian ylated GST-Fyn-N fusion peptides. The GST-Fyn-N probe expression construct, pGD-BTK-FLU, made by ligating the specifically recognized the GST-Btk-F protein but not the full-length coding region of Btk cDNA in-frame to the 5' end GST alone (Fig. 1), showing that the amino-terminal portion of the influenza hemagglutinin tag (FLU) in the retrovirus of Fyn interacts directly with Btk. vector pGD-FLU. To create Btk(4A) mutations, PCR muta- When GST-Fyn-N was used to probe fragments ofBtk, the genesis was performed (15). Approximately 2 x 107 293 cells amino-terminal fragment, Btk-N, was recognized but Btk-H, either untransfected or transiently transfected with pGD- which lacked only 44 carboxyl-terminal amino acids of BTK-FLU or pGD-BTK(4A)-FLU were lysed in 1 ml of the Btk-N, did not bind (Fig. 1). A fragment of Itk similar to lysis buffer (1% Nonidet P-40/150 mM NaCl/20 mM Hepes, Btk-N was also tested and was bound by the GST-Fyn-N pH 7.5/10 mM NaF/0.4 mM EDTA/50 mM Na3VO4/1 mM probe as strongly as was the Btk fragment (9). These results phenylmethylsulfonyl fluoride/1 mM leupeptin/1% aproti- indicate that Btk can interact with Fyn through a relatively nin) on ice for 30 min.

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