- Email: [email protected]
Identification of BCAR-1 as a new substrate of Syk tyrosine kinase through a determination of amino acid sequence preferences surrounding the substrate tyrosine residue
Immunology Letters 135 (2011) 151–157
Contents lists available at ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/IMLET
Identification of BCAR-1 as a new substrate of Syk tyrosine kinase through a determination of amino acid sequence preferences surrounding the substrate tyrosine residue Ji-Yeon Kim 1 , Kyungmin Huh 1 , Rara Jung, Tae Jin Kim ∗ Division of Immunology, Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Jangan-gu, Chunchun-dong 300, Suwon 440-746, Gyeonggi-do, Republic of Korea
a r t i c l e
i n f o
Article history: Received 30 August 2010 Received in revised form 6 October 2010 Accepted 23 October 2010 Available online 31 October 2010 Keywords: Syk Tyrosine kinase Signal transduction BCAR-1 In vitro kinase assay
a b s t r a c t Syk, a non-receptor tyrosine kinase, is an essential signaling molecule in B cells and other hematopoietic cells. Recently, its unexpected diverse functions were recognized in the regulation of cellular adhesion, innate immune recognition, vascular development, and carcinogenesis. Despite its pleiotropic role, only a few substrate proteins have been identified. To find new substrate proteins for Syk, we performed a systemic in vitro kinase assay using GST fusion peptides to determine the substrate specificity surrounding the tyrosine residue to be phosphorylated. Substitution of amino acid residues surrounding tyrosine 178 of BLNK, a principal Syk substrate in B cell receptor-mediated signaling, revealed that acidic residues at sites −5 to −1 were necessary for phosphorylation by Syk. Valine at site +1 was also influential in phosphorylation and a substitution of Pro on site +3 to a basic amino acid residue, Lys, resulted in attenuated phosphorylation. On the basis of these results, a general consensus phosphorylation motif for Syk was determined and several new candidate target proteins were identified in protein database searches. Of the candidate proteins, BCAR-1 (breast cancer anti-estrogen resistance 1) was confirmed to be phosphorylated by Syk in an in vitro kinase assay using a full-length protein of BCAR-1. Furthermore, BCAR-1 was tyrosine phosphorylated upon the overexpression of Syk in HEK-293T cells. These results suggest that more Syk substrates can be found using an in vitro kinase approach and show for the first time that BCAR-1 is a physiological substrate of Syk. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Syk family tyrosine kinases, Syk (spleen tyrosine kinase) and ZAP-70, are critical in the signal transduction of antigen receptors in adaptive immunity [1]. Because the checkpoint signaling through antigen receptors is essential for lymphocyte development, Sykor ZAP-70-deficient mice show an absence or functional defects in B cells or T cells, respectively [2–4]. Whereas the expression of ZAP-70 is limited to T lineage cells, Syk is expressed more widely than ZAP-70 and it is found not only in B cells but also in myeloid cells, developing T cells, endothelial cells, and epithelial cells [5–7]. Besides its critical role in the B cell receptor (BCR)-mediated signaling, increasingly more functions of Syk have been identified, such as an involvement in integrin signaling [8–10], the invasion of cancer cells into surrounding stroma [11], and an involvement in the chemotaxis and phagocytosis of myeloid cells [12–15]. Therefore,
∗ Corresponding author. Tel.: +82 31 299 6161; fax: +82 31 299 6179. E-mail address: [email protected] (T.J. Kim). 1 These authors contributed equally. 0165-2478/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2010.10.016
it is highly likely that many unknown substrates of Syk may explain the pleiotropic functions of Syk [1]. The most representative substrate of Syk is BLNK (also known as SLP-65 or BASH), which is a key adapter protein that plays an integral role in linking the BCR-mediated activation of tyrosine kinases to other pathways [16–19]. BLNK has multiple tyrosine residues that can mediate interactions between distinct signaling effectors such as PLC␥, BTK, and Vav upon its phosphorylation. It plays a critical signaling function in B cell activation and in the regulation of downstream signaling pathways [20–22]. Upon BCR’s recognition of specific antigens, Syk is activated by its recruitment to ITAMs (immune receptor tyrosine-based activation motifs) within the Ig-␣ and Ig- chains and autophosphorylation and then phosphorylates BLNK [23–25]. The substrate specificity of a kinase is primarily determined by the structural feature of the kinase active site [26]. Within the substrate, the amino acids situated immediately N-terminal or Cterminal to the phosphorylation site often contribute greatly to the kinase recognition of substrate. Here, in order to find new substrates of Syk, we investigated the substrate specificity of Syk using GST-linked peptides that were produced based on the sequence of
152
J.-Y. Kim et al. / Immunology Letters 135 (2011) 151–157
a known Syk substrate, BLNK. New putative substrates were identified by determining a general phosphorylation consensus sequence and searching for proteins with the consensus sequence from protein database. Many of these substrates were tested using an in vitro kinase assay. Of those identified, BCAR-1 was confirmed to be a new physiological Syk substrate. 2. Materials and methods 2.1. Plasmid construction Human Syk cDNA was PCR-amplified from the cDNA pool of Jurkat cells and subcloned into pDEST–SG5–FLAG (Invitrogen, Carlsbad, CA) for mammalian expression. A short alternatively spliced form of Syk, referred to as SykB or SykS, was used for in vitro kinase assay with GST-peptides [1]. The predominant long form of Syk (SykL) was also generated and used for the investigation of its ability to phosphorylate BCAR-1 in HEK-293T cells. The kinase-dead forms of Syk, Syk-K402R for SykL and Syk-K379R for SykB, were generated using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). Human BCAR-1 cDNA was PCR-amplified and subcloned into the BamHI–SmaI site of pENTR1A and was transferred into pDEST–CG3–MT for the expression of Nterminal Myc-tagged protein using LR recombinase (Invitrogen). For GST fusion peptide expression vectors, complementary oligonucleotides encoding the desired peptides (11 amino acids) were cloned into the BamHI–SmaI site of pGEX-4T-1 (GE Biosciences, Piscataway, NJ). The truncated form of BCAR-1 (aa 570–910) was subcloned into the BamHI–SmaI site of GEX-4T-1. All constructs were confirmed by restriction enzyme digestion and DNA sequencing. 2.2. Production of GST-tagged peptides and proteins The GST-peptides were overexpressed in Escherichia coli DH5␣. The culture was incubated at 37 ◦ C until OD595 reached 0.4–0.5, after which 0.4 mM IPTG was added and the culture was incubated for an additional 3 h. The cells were harvested and lysated in lysis buffer (PBS containing 1% Triton X-100, 2 mM PMSF, and 2 mM Na3 VO4 ). The crude lysates were incubated with Glutathione–Sepharose 4B Fast Flow (GE Biosciences) at 4 ◦ C for 3 h with rolling. The beads were washed three times with PBS, and the binding protein was eluted with elution buffer (20 mM glutathione in 100 mM Tris–HCl, pH 8.0) at room temperature for 10 min. GST-peptide expressions were checked by SDS-PAGE. 2.3. In vitro kinase assay Cell extracts were prepared from 293T cells that had been transfected with pDEST–FLAG–Syk (wild-type) or pDEST–FLAG–Syk (kinase-dead) by lysing cells in NETN buffer (50 mM Tris–HCl, pH 7.5; 150 mM NaCl; 1 mM EDTA; 0.5% Nonidet P-40; 1 mM sodium fluoride; 2 mM Na3 VO4 ; and protease inhibitors) for 1 h at 4 ◦ C. The supernatants were immunoprecipitated with anti-FLAG antibody (M2; Sigma–Aldrich, Oakville, Ontario, Canada) and protein A sepharose beads (GE Biosciences). The beads were washed three times with NETN buffer and twice with kinase buffer (50 mM Tris–HCl, pH 7.5; 50 mM NaCl; and 5 mM MgCl2 ). The kinase reaction was prepared by resuspending the beads in 40 l kinase buffer containing 5 Ci [␥-32P]ATP (GE Biosciences), 10 M ATP, and 2 g GST-tagged protein as a substrate. A kinase reaction was performed at 30 ◦ C for 30 min and was terminated by adding 10 l of 5× SDSPAGE loading buffer, after which the proteins were transferred to nitrocellulose membrane. Radiolabeled proteins were visualized by BAS (Fujifilm, Tokyo, Japan).
2.4. Immunoblotting After exposure to BAS, the membranes were washed briefly with TBS-T and immersed in TBS-T with 3% bovine serum albumin (BSA) for 2 h to block non-specific background. The membranes were incubated with anti-Syk antibody (N-14; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 2 h and then washed with TBS-T six times for 10 min each time. The membranes were then incubated with horseradish peroxidase-labeled goat anti-rabbit antibody (Dako, Carpentaria, CA) for 1 h and washed with TBS-T. An enhanced chemiluminescence solution (GE Biosciences) was used for visualization.
2.5. Cell culture and transfection HEK-293T cells were grown as monolayers in Dulbecco’s modified Eagle medium (Gibco/BRL, Gaithersburg, MD) containing 10% (v/v) fetal calf serum (HyClone, Logan, UT) and penicillin–streptomycin (Gibco/BRL) at 37 ◦ C in a 5% CO2 atmosphere. HEK-293T cells were transiently transfected with a combination of cDNA expression constructs including Bcar-1, wild-type Syk, and/or kinase-dead Syk using the calcium phosphate method. After 2 days, myc-Bcar-1 from the cell lysates was immunoprecipitated with anti-Myc antibody (9E10; Roche, Basel, Switzerland) and subsequently immunoblotted with an anti-Myc antibody, anti-FLAG antibody, anti-Syk antibody, or antiphosphotyrosine (4G10; Upstate Biotechnology, Lake Placid, NY).
3. Results 3.1. Cloning of substrate GST-peptides with a partial sequence of BLNK and confirmation of in vitro kinase assay The sequence of amino acids surrounding the target phosphorylation site is important for the substrate recognition by a kinase [27]. In order to determine the substrate specificity of Syk, we produced GST proteins fused to 11 amino acid peptide residues from BLNK sequences. Inserted peptide amino acid sequences were designed so that the tyrosine residue is in the center of 11 amino acid residues. BLNK has five tyrosine residues phosphorylated by Syk, which are all located in the N-terminal acidic motif (Fig. 1A). We selected a peptide sequence surrounding tyrosine 178 (Y178), because Y178 was the most strongly phosphorylated tyrosine in chicken BLNK under BCR stimulation, and the mutation of Y178 to phenylalanine significantly attenuated the association of BLNK with PLC␥2 and intracellular calcium influx [18]. Therefore, the GST-tagged peptide comprising amino acids 173–183 (Y178) of BLNK was prepared by inserting the corresponding sequence oligonucleotides into pGEX-4T-1. Then, we confirmed the specific phosphorylation of GST–BLNK 173–183 by FLAG-tagged Syk (SykB) that was generated as described in Section 2. SykB is a short alternative form of Syk that has a deletion of 23 amino acid residues in the intervening region between SH2 and kinase domains compared to the predominant form of Syk, SykL [1]. An in vitro kinase assay with GST–BLNK 173–183 showed strong phosphorylation by Syk, which was confirmed to be targeted on tyrosine, because the replacement of Y178 with phenylalanine (GST–BLNK 173–183/Y178F) resulted in a total loss of phosphorylation (Fig. 2). This in vitro kinase system required kinase activity, because the kinase-dead mutant Syk (SykB K379R) failed to phosphorylate GST–BLNK 173–183 with no autophosphorylation. This finding confirmed the validity of our in vitro kinase system.
J.-Y. Kim et al. / Immunology Letters 135 (2011) 151–157
153
Fig. 1. (A) Schematic diagram of human BLNK. (B) Illustration of substitution mutants based on the sequence of BLNK 173–183 (Y178).
3.2. Identification of the sequence specificity of Syk To determine the amino acid sequence preference for the Syk phosphorylation of BLNK Y178, we produced variant GST-tagged peptides by substituting each amino acid residue of BLNK 173–183 for alanine, except Y178 and A176, and performed the in vitro kinase assay using Syk (Fig. 1B). The in vitro kinase assay using the substituted mutants showed that the amino acid residues on the amino side of Y178 were critical for tyrosine phosphorylation because the intensity of in vitro phosphorylation was significantly attenuated in GST-peptides with an alanine substitution at sites −5 to −1 (Fig. 3A). Tyrosine phosphorylation was more strongly affected in the GSTpeptides with substitution at sites closer to Y178. Of the amino acid residues on the carboxy side of Y178, only substitution of V179 at site +1 (V179A) resulted in a decrease in phosphorylation comparable with the phosphorylation level of D174A replacement mutant
Fig. 2. Phosphorylation of GST–BLNK peptides by Syk kinase in vitro. Wild-type or kinase-dead Syk proteins were immunoprecipitated with anti-FLAG antibody and were used in an in vitro kinase assay with 5 Ci [␥-32P]ATP, 10 M cold ATP, and 2 g GST–BLNK 173–183 Y178 (wild type), or Y178F mutant peptides in the presence of 10 mM MgCl2 . Similar amounts of FLAG-tagged Syk in the different lanes were shown by immunoblotting with anti-FLAG antibody (top panel). The amount of radiolabeled Syk (middle panel) and GST–BLNK (lower panel) in the reaction was assessed.
GST-peptide, whereas the substitution at sites +2 to +5 did not influence phosphorylation significantly. Therefore, Syk was likely to interact preferentially with amino acid residues on the amino side of Y178 and with valine residues on the +1 carboxy position for the phosphorylation of Y178. Because all residues at sites −5 to −1 comprised acidic amino acids, except alanine at site −2, the results suggest that acidic residues are required on the amino side of phosphotyrosine for phosphorylation by Syk. To further examine the preferred residues based on chemical properties, each amino acid residue was substituted to a basic residue—lysine. There is no basic residue in the BLNK 173–183 peptide sequence. In general, an in vitro kinase assay using GSTpeptides with substitution to lysine showed results similar to those using GST-peptides with substitution to alanine (Fig. 3B). Substitutions to lysine at sites +1 to +3 resulted in weaker phosphorylation, which suggests that substrate phosphorylation by Syk is negatively affected by the presence of basic amino acids on the carboxy side of phosphotyrosine in the substrate, especially at site +1 of phosphotyrosine. Next, we generated more substitution mutants in which each hydrophobic residue in BLNK 173–183 was switched to the acidic amino acid aspartate or each acidic amino acid was switched to the hydrophobic residue leucine. The results with this set of mutants were nearly identical to those with the alanine mutation (Fig. 3C). Collectively, these results demonstrated that acidic residues at sites −5 to −1 are crucial for the phosphorylation of BLNK Y178 by Syk. Valine at site +1 also contributes to phosphorylation by Syk. An amino acid residue at site +2 could be substituted with other groups of amino acids, except for basic residues.
3.3. Screening of short GST-tagged peptides from candidate proteins On the basis of sequence specificity, the following query was made to search for potential new substrates using PATTINPROT21 (http://npsa-pbil.ibcp.fr/cgi-bin/npsa automat.pl?page=
154
J.-Y. Kim et al. / Immunology Letters 135 (2011) 151–157
Fig. 3. Phosphorylation of GST–BLNK 173–183 with substitutions by Syk. Each amino acid residue in BLNK was replaced with (A) alanine, (B) lysine, or (C) leucine or aspartic acid. Phosphorylation of the modified peptides was evaluated using an in vitro kinase assay. Wild-type BLNK 173–183 and the substitution mutant of Y178 for phenylalanine (Y178F) were used as positive and negative controls, respectively.
npsa pattinprot.html) against the UNIPROT-SWISSPROT protein database: [ED]-[ED]-[ED]-X-[ED]-Y-X-X-{KR} [28,29]. The brackets [ ] denote inclusion of the specified residues, whereas the brackets { } exclude the specified residues. Of the 7727 human proteins suggested by PATTINPROT, 36 sequences from 34 proteins were selected based on the potential functional relevance with Syk (Table 1).
To confirm whether the selected proteins are genuine substrates for Syk, GST-tagged 11-amino acid peptides from selected candidates were produced for the in vitro kinase assay using the same method used for GST–BLNK 173–183. Screening of the peptides using an in vitro kinase assay revealed that 22 peptides were indeed phosphorylated by Syk (Fig. 4 and Table 1). Positive candidates include proteins involved in actin cytoskeleton regula-
Table 1 Peptide sequences from candidate substrates for in vitro kinase assay with the Syk and GST-peptides tested in Fig. 4. Accession number
Gene name
Amino acid sequences
Q16537
PPP2R5E
NELVDYITISR
P78314 Q9BYF1 Q13023 P31749 Q9NTI5 Q14155 O95260 Q13315 Q13535 Q8NDB2 Q8NDB2 P56945 P56945 Q9P287 P41182 Q96G01 P33151 Q14511 P41180 P24863 P41597 Q9Y5K6 P06127 Q76N32 P25106 Q8N6F7 P14317 Q8IWV1 P01106 Q13469 P04637 P62491 Q96IW2 O75563 P51692
SH3BP2 ACE2 AKAP6 AKT1 APRIN ARHGEF7 ATE1 ATM ATR BANK1 BANK1 BCAR1 BCAR1 BCCIP BCL6 BICD1 CDH5 NEDD9 CASR CCNC CCR2 CD2AP CD5 CEP68 CXCR7 GCET2 HCLS1 LAX1 MYC NFATC2 p53 RB11A SHD SKAP2 STAT5B
DSDEDYEKVPL PLYEEYVVLKN DFDSEYQELWD MNEFEYLKLLG AHDPDYVKVQD SEDSDYDSIWT YYDPDYSFLSL LPGEEYPLPME ATPEEYNTVVQ QEPEDYISVIQ IDDSEYDMILA TEQDEYDTPRH MEDYDYVHLQG LSDNDYDGIKK PVPGEYSRPTL ELEAEYDSLKQ DSDVDYDFLND MDDYDYVHLQG AADDDYGRPGI LSEEEYWKLQI FFDYDYGAPCH IVEYDYDAVHD HVDNEYSQPPM ESDDEYLALPA VSETEYSALEQ GTETEYSLLHM EPENDYEDVEE EDSSDYENVLT NYDLDYDSVQP LFDYEYLNPNE PLDGEYFTLQI TRDDEYDYLFK EADTEYLDPFD EDGEEYDDPFA SHLEDYSGLSV
Tyrosine no. 99 448 183 968 152 1005 776 374 959 26 146 588 235 666 71 242 59 757 631 218 57 28 10 453 482 354 128 378 373 16 38 327 8 106 75 548
Protein name Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit epsilon isoform SH3 domain-binding protein 2 Angiotensin-converting enzyme 2 precursor A-kinase anchor protein 6 RAC-alpha serine/threonine-protein kinase Androgen-induced proliferation inhibitor Rho guanine nucleotide exchange factor 7 Arginyl-tRNA–protein transferase 1 Serine-protein kinase ATM Serine/threonine-protein kinase ATR B-cell scaffold protein with ankyrin repeats B-cell scaffold protein with ankyrin repeats Breast cancer anti-estrogen resistance protein 1 Breast cancer anti-estrogen resistance protein 1 BRCA2 and CDKN1A-interacting protein B-cell lymphoma 6 protein Protein bicaudal D homolog 1 Cadherin-5 precursor Enhancer of filamentation 1 Extracellular calcium-sensing receptor precursor Cyclin C C–C chemokine receptor type 2 CD2-associated protein T-cell surface glycoprotein CD5 precursor CEP68 centrosomal protein 68 kDa Chemokine (C–X–C motif) receptor 7 Germinal center B-cell-expressed transcript Hematopoietic lineage cell-specific protein Lymphocyte transmembrane adapter 1 Myc proto-oncogene protein Nuclear factor of activated T-cells, cytoplasmic 2 Tumor suppressor protein p53 Ras-related protein Rab-11A SH2 domain-containing adapter protein D Src kinase-associated phosphoprotein 2 Signal transducer and activator of transcription 5B
J.-Y. Kim et al. / Immunology Letters 135 (2011) 151–157
155
3.4. In vitro and in vivo phosphorylation of BCAR-1 by Syk
Fig. 4. Phosphorylation of candidate target proteins for Syk. GST peptides containing the putative target sequences were used as substrates for the in vitro kinase assay. The amino acid sequences of the GST peptides are shown in Table 1. Phosphorylation of candidate peptides was evaluated using an in vitro kinase assay. Original GST–BLNK 173–183 and GST–BLNK 173–183 Y178F were used as positive and negative controls, respectively.
tion (ARHGEF7, BCAR-1, NEDD9, and HCLS1), lymphocyte signaling (SH3BP2, BANK1, CD5, GCET2, LAX1, and STAT5B), cell cycle regulation (ATR and CCNC), or others (AKAP, ATE1, CDH5, CEP68, CXCR7, and SKAP2). Of these proteins, SH3BP2, GCET2, HCLS1, and LAX1 are previously reported substrates of Syk, which supports the validity of our study [30–33]. Strong phosphorylation of the GST-peptide substrates from the reported proteins was confirmed in an in vitro kinase assay (Fig. 4).
We next attempted to confirm substrate phosphorylation in the context of a full-sized protein and within a cell. Here we selected BCAR-1 for an additional study because BCAR-1 is an adapter protein involved in cell migration and is also implicated in breast cancer progression related to clinical resistance to tamoxifen similarly to Syk [34,35]. BCAR-1 is also known as a Crk-associated substrate (CAS or p130Cas ). It seemed to us that BCAR-1 could be an interesting Syk substrate and might explain the migratory behavior of lymphocytes and cancer cells. BCAR-1 has 19 phosphotyrosine residues, many of which are known to be phosphorylated by c-Src or PTK2 (Fig. 5A) [36]. First, a partial protein—GST-tagged BCAR-1 571–710—was prepared from a BCAR-1 cDNA clone obtained from KRIBB. Although PATTINPROT suggested two potential phosphotyrosines, Y235 and Y666, which were both phosphorylated in short peptides, only the partial protein containing Y666 (BCAR-1 571–710) was produced for the assay. GST–BCAR-1 571–710 was phosphorylated by Syk in vitro, whereas A kinase-associated protein 6 (AKAP6) 965–1102, another partial protein we generated, was not phosphorylated by Syk (Fig. 5B, data of AKAP6 not shown). Y666 of BCAR-1 appeared to be a target of Syk phosphorylation, but there was another tyrosine residue, Y664, near to Y666. To further delineate the specific tyrosine residues phosphorylated by Syk, we generated two GST-peptide fusion proteins from BCAR-1—GST–BCAR-1 661–671 and GST–BCAR-1 405–415. The tyrosine residue Y410 is within the interior substrate domain of BCAR-1 and is included in 1 of 15 YxxP motifs, which was reported to be phosphorylated by Src family tyrosine kinases [37]. The in vitro kinase assay revealed the specific phosphorylation of GST–BCAR-1 661–671, but no phosphorylation of GST–BCAR-1 405–415, which indicates that Syk preferentially phosphorylates tyrosine residues only in the context of our prediction (Fig. 6). To ask whether Y666 or Y664 is really phosphorylated by Syk, we performed the in vitro kinase assay using mutants of GST–BCAR-1 661–671 with replacement mutations of Y664F, Y666F, or Y664F/Y666F. We found that both Y664 and Y666 were phosphorylated by Syk because mutations of both Y664 and Y666, but not of either Y664 or Y666, abolished the Syk phosphorylation. We further investigated whether a full-length protein of BCAR-1 is phosphorylated within cells by a predominant form of Syk (SykL).
Fig. 5. Phosphorylation of GST-BCAR-1 (aa 571–910) by Syk. (A) Domain structure of BCAR-1 showing phosphotyrosine 666 in the Src binding domain. (B) Wild-type Syk, but not kinase-dead Syk, phosphorylated GST–hBCAR-1 (Src binding domain).
156
J.-Y. Kim et al. / Immunology Letters 135 (2011) 151–157
Fig. 6. Phosphorylation of GST-BCAR-1 405–415 and 661–671 by Syk. Wild-type BLNK 173–183 and BLNK 173–183 Y178F were used as positive and negative controls, respectively.
We transfected the BCAR-1 construct with the predominant form of wild-type or kinase-dead Syk (SykL) constructs into 293T cells and checked the state of tyrosine phosphorylation of BCAR-1. Myctagged BCAR-1 was immunoprecipitated with anti-myc antibody and blotted with anti-phosphotyrosine antibody. Upon cotransfection with Syk, BCAR-1 was phosphorylated, but BCAR-1 was not tyrosine phosphorylated upon cotransfection with kinase-dead Syk (Fig. 7). Collectively, these results suggest that BCAR-1 Y664 and Y666 residues are new target residues of Syk phosphorylation, which was confirmed by in vitro and in vivo assays. 4. Discussion It is generally thought that BCR engagement leads to phosphorylation of ITAMs by Src family kinases (SFKs) and, in turn, Syk is activated by its recruitment to doubly phosphorylated ITAMs and its phosphorylation by SFKs or Syk themselves [38]. Syk is rapidly phosphorylated and activated upon BCR engagement, but its continued activity is required for B cell development and differentiation [39,40]. Activated Syk can move from plasma membrane to other cellular compartments and phosphorylate critical substrates such as BLNK for B cell proliferation and differentiation. Recently, Syk was demonstrated to be also expressed by various types of non-hematopoietic cells including breast epithelial cells and to have a negative regulatory role in carcinogenesis [41,11]. However, any relevant effectors, which are Syk substrates, have not been identified to date. Therefore, it is thought that there are a lot more substrates of Syk to explain the diverse functions of Syk in many types of cells.
We designed this study with the expectation that more substrates could be found by determining the substrate specificity surrounding the phosphotyrosine residue and by searching for proteins with similar sequences in the protein database. Here we found that the amino acid residues on the amino side of phosphotyrosine were important for the substrate phosphorylation by Syk. We suggest that this substrate preference may hold for proteins with BLNK-like sequences. The sequence around Y72 of BLNK also fit with our consensus sequence, and other tyrosine residues—Y84, Y96, and Y189—had acidic residues in their amino sides [19]. However, some Syk substrates do not contain BLNK-like sequences, such as the recently reported substrate, STAT3 [42]. This suggests that Syk may also phosphorylate tyrosines in a dissimilar context. Although this study cannot find Syk substrates with STAT3-like sequences, it has value because it elucidated more Syk substrates with BLNK-like sequences. It was interesting to us that the kinase domain of Syk preferentially recognized the amino acid residues located amino-terminal to the substrate tyrosine residue, whereas the amino acid residues carboxy-terminal to the phosphotyrosine were reported to determine the binding specificities of the SH2 domain. The SH2 domains were previously grouped according to their binding sequence specificities, and some common binding motifs, such as YEEI or YMXM, were found [43]. Therefore, we propose that an extended motif composed of the Syk substrate motif and certain specific SH2-binding motifs may define a signaling module connecting Syk activation to specific signaling pathway. For BLNK, all five tyrosine residues were in the YxxP motif, which is also known as a binding site for the SH2 domain. It was shown that each YxxP motif of BLNK bound distinct signaling molecules such as Vav, Nck, BTK, and PLC␥ [19]. Therefore, the five tyrosine residues of BLNK make signaling connections from Syk activation to downstream pathways of Vav, Nck, BTK, and PLC␥. In contrast, BCAR-1 appears to have a different signaling connection. BCAR-1, also identified as p130Cas , is composed of an N-terminal SH3 domain, a central substrate domain with 15 YxxP motifs, and a C-terminal Src-binding region [37]. Here the Syk substrate tyrosines were not present in the central substrate domain, but in the C-terminal Src-binding domain. Our in vitro kinase assay with GST-peptide confirmed that Syk could clearly phosphorylate the C-terminal tyrosines Y664 and Y666, but not Y410 in the central substrate domain. Since the C-terminal YDYVHL sequence was shown as a binding site for Src family tyrosine kinases, we think that Syk activation-driven phosphorylation of Y664 and Y666 within the YDYVHL sequence lead to the binding of SH2 domain of Src family tyrosine kinases. Thus, this extended motif in BCAR-1 is thought to connect Syk activation to Src family tyrosine kinase binding, which may lead to phosphorylation of multiple tyrosines in the central substrate domain. In summary, we successfully identified some new Syk substrate candidate proteins using a systemic in vitro kinase assay using GST fusion peptides with BLNK-like sequences. We further confirmed that BCAR-1 is a new Syk substrate by showing its tyrosine phosphorylation upon co-transfection with Syk. With this approach, it is expected that more significant Syk substrates can be identified, which would help to elucidate the multi-faceted functions of Syk. Conflict of interest The authors have no financial conflicts of interest.
Fig. 7. Syk-dependent phosphorylation of BCAR-1. 293T cells were co-transfected with Myc-BCAR-1 along with empty vector, FLAG–Syk (wild-type SykL), or FLAG–Syk (kinase-dead K402R SykL). FLAG–Syk in each cell lysate was assessed by immunoblotting with anti-Syk antibody or anti-FLAG antibody. Immunoprecipitated Myc-BCAR-1 was blotted with anti-phosphotyrosine antibody (4G10).
Acknowledgements This work was supported by the Korean Research Foundation Grant funded by the Korean Government (MEST) (KRF-313-2007-2-
J.-Y. Kim et al. / Immunology Letters 135 (2011) 151–157
E00205) and a grant (01-PJ3-PG6-01GN12-0001) from the 21 Good Health R and D Project, Ministry of Health, Welfare, and Family Affairs, Republic of Korea. References [1] Mocsai A, Ruland J, Tybulewicz VL. The SYK tyrosine kinase: a crucial player in diverse biological functions. Nat Rev Immunol 2010;10:387–402. [2] Turner M, Mee PJ, Costello PS, Williams O, Price AA, Duddy LP, et al. Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk. Nature 1995;378:298–302. [3] Cheng AM, Rowley B, Pao W, Hayday A, Bolen JB, Pawson T. Syk tyrosine kinase required for mouse viability and B-cell development. Nature 1995;378:303–6. [4] Arpaia E, Shahar M, Dadi H, Cohen A, Roifman CM. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase. Cell 1994;76:947–58. [5] Zou W, Reeve JL, Liu Y, Teitelbaum SL, Ross FP. DAP12 couples c-Fms activation to the osteoclast cytoskeleton by recruitment of Syk. Mol Cell 2008;31:422–31. [6] Chu DH, van Oers NS, Malissen M, Harris J, Elder M, Weiss A. Pre-T cell receptor signals are responsible for the down-regulation of Syk protein tyrosine kinase expression. J Immunol 1999;163:2610–20. [7] Chan AC, van Oers NS, Tran A, Turka L, Law CL, Ryan JC, et al. Differential expression of ZAP-70 and Syk protein tyrosine kinases, and the role of this family of protein tyrosine kinases in TCR signaling. J Immunol 1994;152:4758–66. [8] Zarbock A, Lowell CA, Ley K. Spleen tyrosine kinase Syk is necessary for E-selectin-induced alpha(L)beta(2) integrin-mediated rolling on intercellular adhesion molecule-1. Immunity 2007;26:773–83. [9] Mocsai A, Zhou M, Meng F, Tybulewicz VL, Lowell CA. Syk is required for integrin signaling in neutrophils. Immunity 2002;16:547–58. [10] Hirahashi J, Mekala D, Van Ziffle J, Xiao L, Saffaripour S, Wagner DD, et al. Mac-1 signaling via Src-family and Syk kinases results in elastase-dependent thrombohemorrhagic vasculopathy. Immunity 2006;25:271–83. [11] Luangdilok S, Box C, Patterson L, Court W, Harrington K, Pitkin L, et al. Syk tyrosine kinase is linked to cell motility and progression in squamous cell carcinomas of the head and neck. Cancer Res 2007;67:7907–16. [12] Tohyama Y, Yamamura H. Protein tyrosine kinase, syk: a key player in phagocytic cells. J Biochem 2009;145:267–73. [13] Berton G, Mocsai A, Lowell CA. Src and Syk kinases: key regulators of phagocytic cell activation. Trends Immunol 2005;26:208–14. [14] Gevrey JC, Isaac BM, Cox D. Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine). J Immunol 2005;175:3737–45. [15] Turner M, Schweighoffer E, Colucci F, Di Santo JP, Tybulewicz VL. Tyrosine kinase SYK: essential functions for immunoreceptor signalling. Immunol Today 2000;21:148–54. [16] Janssen E, Zhang W. Adaptor proteins in lymphocyte activation. Curr Opin Immunol 2003;15:269–76. [17] Ishiai M, Kurosaki M, Pappu R, Okawa K, Ronko I, Fu C, et al. BLNK required for coupling Syk to PLC gamma 2 and Rac1-JNK in B cells. Immunity 1999;10:117–25. [18] Chiu CW, Dalton M, Ishiai M, Kurosaki T, Chan AC. BLNK: molecular scaffolding through ‘cis’-mediated organization of signaling proteins. EMBO J 2002;21:6461–72. [19] Wu JN, Koretzky GA. The SLP-76 family of adapter proteins. Semin Immunol 2004;16:379–93. [20] Kurosaki T, Tsukada S. BLNK: connecting Syk and Btk to calcium signals. Immunity 2000;12:1–5. [21] Pappu R, Cheng AM, Li B, Gong Q, Chiu C, Griffin N, et al. Requirement for B cell linker protein (BLNK) in B cell development. Science 1999;286:1949–54.
157
[22] Koretzky GA, Abtahian F, Silverman MA. SLP76 and SLP65: complex regulation of signalling in lymphocytes and beyond. Nat Rev Immunol 2006;6:67–78. [23] Latour S, Veillette A. Proximal protein tyrosine kinases in immunoreceptor signaling. Curr Opin Immunol 2001;13:299–306. [24] Tsang E, Giannetti AM, Shaw D, Dinh M, Tse JK, Gandhi S, et al. Molecular mechanism of the Syk activation switch. J Biol Chem 2008;283:32650–9. [25] Keshvara LM, Isaacson CC, Yankee TM, Sarac R, Harrison ML, Geahlen RL. Sykand Lyn-dependent phosphorylation of Syk on multiple tyrosines following B cell activation includes a site that negatively regulates signaling. J Immunol 1998;161:5276–83. [26] Ubersax JA, Ferrell JEJ. Mechanisms of specificity in protein phosphorylation. Nat Rev Mol Cell Biol 2007;8:530–41. [27] Seo GJ, Kim SE, Lee YM, Lee JW, Lee JR, Hahn MJ, et al. Determination of substrate specificity and putative substrates of Chk2 kinase. Biochem Biophys Res Commun 2003;304:339–43. [28] Combet C, Blanchet C, Geourjon C, Deleage G. NPS@: network protein sequence analysis. Trends Biochem Sci 2000;25:147–50. [29] Bairoch A, Apweiler R. The SWISS-PROT protein sequence data bank and its supplement TrEMBL. Nucleic Acids Res 1997;25:31–6. [30] Foucault I, Le Bras S, Charvet C, Moon C, Altman A, Deckert M. The adaptor protein 3BP2 associates with VAV guanine nucleotide exchange factors to regulate NFAT activation by the B-cell antigen receptor. Blood 2005;105:1106–13. [31] Pan Z, Shen Y, Ge B, Du C, McKeithan T, Chan WC. Studies of a germinal centre Bcell expressed gene, GCET2, suggest its role as a membrane associated adapter protein. Br J Haematol 2007;137:578–90. [32] Hao JJ, Carey GB, Zhan X. Syk-mediated tyrosine phosphorylation is required for the association of hematopoietic lineage cell-specific protein 1 with lipid rafts and B cell antigen receptor signalosome complex. J Biol Chem 2004;279:33413–20. [33] Zhu M, Janssen E, Leung K, Zhang W. Molecular cloning of a novel gene encoding a membrane-associated adaptor protein (LAX) in lymphocyte signaling. J Biol Chem 2002;277:46151–8. [34] Cary LA, Han DC, Polte TR, Hanks SK, Guan JL. Identification of p130Cas as a mediator of focal adhesion kinase-promoted cell migration. J Cell Biol 1998;140:211–21. [35] van der Flier S, Brinkman A, Look MP, Kok EM, Meijer-van Gelder ME, Klijn JG, et al. Bcar1/p130Cas protein and primary breast cancer: prognosis and response to tamoxifen treatment. J Natl Cancer Inst 2000;92:120–7. [36] Goldberg GS, Alexander DB, Pellicena P, Zhang ZY, Tsuda H, Miller WT. Src phosphorylates Cas on tyrosine 253 to promote migration of transformed cells. J Biol Chem 2003;278:46533–40. [37] Bouton AH, Riggins RB, Bruce-Staskal PJ. Functions of the adapter protein Cas: signal convergence and the determination of cellular responses. Oncogene 2001;20:6448–58. [38] Saouaf SJ, Mahajan S, Rowley RB, Kut SA, Fargnoli J, Burkhardt AL, et al. Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement. Proc Natl Acad Sci USA 1994;91:9524–8. [39] Oh H, Ozkirimli E, Shah K, Harrison ML, Geahlen RL. Generation of an analogsensitive Syk tyrosine kinase for the study of signaling dynamics from the B cell antigen receptor. J Biol Chem 2007;282:33760–8. [40] Cornall RJ, Cheng AM, Pawson T, Goodnow CC. Role of Syk in B-cell development and antigen–receptor signaling. Proc Natl Acad Sci USA 2000;97:1713–8. [41] Coopman PJ, Mueller SC. The Syk tyrosine kinase: a new negative regulator in tumor growth and progression. Cancer Lett 2006;241:159–73. [42] Uckun FM, Qazi S, Ma H, Tuel-Ahlgren L, Ozer Z. STAT3 is a substrate of SYK tyrosine kinase in B-lineage leukemia/lymphoma cells exposed to oxidative stress. Proc Natl Acad Sci USA 2010;107:2902–7. [43] Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, et al. SH2 domains recognize specific phosphopeptide sequences. Cell 1993;72:767–78.
Contents lists available at ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/IMLET
Identification of BCAR-1 as a new substrate of Syk tyrosine kinase through a determination of amino acid sequence preferences surrounding the substrate tyrosine residue Ji-Yeon Kim 1 , Kyungmin Huh 1 , Rara Jung, Tae Jin Kim ∗ Division of Immunology, Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Jangan-gu, Chunchun-dong 300, Suwon 440-746, Gyeonggi-do, Republic of Korea
a r t i c l e
i n f o
Article history: Received 30 August 2010 Received in revised form 6 October 2010 Accepted 23 October 2010 Available online 31 October 2010 Keywords: Syk Tyrosine kinase Signal transduction BCAR-1 In vitro kinase assay
a b s t r a c t Syk, a non-receptor tyrosine kinase, is an essential signaling molecule in B cells and other hematopoietic cells. Recently, its unexpected diverse functions were recognized in the regulation of cellular adhesion, innate immune recognition, vascular development, and carcinogenesis. Despite its pleiotropic role, only a few substrate proteins have been identified. To find new substrate proteins for Syk, we performed a systemic in vitro kinase assay using GST fusion peptides to determine the substrate specificity surrounding the tyrosine residue to be phosphorylated. Substitution of amino acid residues surrounding tyrosine 178 of BLNK, a principal Syk substrate in B cell receptor-mediated signaling, revealed that acidic residues at sites −5 to −1 were necessary for phosphorylation by Syk. Valine at site +1 was also influential in phosphorylation and a substitution of Pro on site +3 to a basic amino acid residue, Lys, resulted in attenuated phosphorylation. On the basis of these results, a general consensus phosphorylation motif for Syk was determined and several new candidate target proteins were identified in protein database searches. Of the candidate proteins, BCAR-1 (breast cancer anti-estrogen resistance 1) was confirmed to be phosphorylated by Syk in an in vitro kinase assay using a full-length protein of BCAR-1. Furthermore, BCAR-1 was tyrosine phosphorylated upon the overexpression of Syk in HEK-293T cells. These results suggest that more Syk substrates can be found using an in vitro kinase approach and show for the first time that BCAR-1 is a physiological substrate of Syk. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Syk family tyrosine kinases, Syk (spleen tyrosine kinase) and ZAP-70, are critical in the signal transduction of antigen receptors in adaptive immunity [1]. Because the checkpoint signaling through antigen receptors is essential for lymphocyte development, Sykor ZAP-70-deficient mice show an absence or functional defects in B cells or T cells, respectively [2–4]. Whereas the expression of ZAP-70 is limited to T lineage cells, Syk is expressed more widely than ZAP-70 and it is found not only in B cells but also in myeloid cells, developing T cells, endothelial cells, and epithelial cells [5–7]. Besides its critical role in the B cell receptor (BCR)-mediated signaling, increasingly more functions of Syk have been identified, such as an involvement in integrin signaling [8–10], the invasion of cancer cells into surrounding stroma [11], and an involvement in the chemotaxis and phagocytosis of myeloid cells [12–15]. Therefore,
∗ Corresponding author. Tel.: +82 31 299 6161; fax: +82 31 299 6179. E-mail address: [email protected] (T.J. Kim). 1 These authors contributed equally. 0165-2478/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2010.10.016
it is highly likely that many unknown substrates of Syk may explain the pleiotropic functions of Syk [1]. The most representative substrate of Syk is BLNK (also known as SLP-65 or BASH), which is a key adapter protein that plays an integral role in linking the BCR-mediated activation of tyrosine kinases to other pathways [16–19]. BLNK has multiple tyrosine residues that can mediate interactions between distinct signaling effectors such as PLC␥, BTK, and Vav upon its phosphorylation. It plays a critical signaling function in B cell activation and in the regulation of downstream signaling pathways [20–22]. Upon BCR’s recognition of specific antigens, Syk is activated by its recruitment to ITAMs (immune receptor tyrosine-based activation motifs) within the Ig-␣ and Ig- chains and autophosphorylation and then phosphorylates BLNK [23–25]. The substrate specificity of a kinase is primarily determined by the structural feature of the kinase active site [26]. Within the substrate, the amino acids situated immediately N-terminal or Cterminal to the phosphorylation site often contribute greatly to the kinase recognition of substrate. Here, in order to find new substrates of Syk, we investigated the substrate specificity of Syk using GST-linked peptides that were produced based on the sequence of
152
J.-Y. Kim et al. / Immunology Letters 135 (2011) 151–157
a known Syk substrate, BLNK. New putative substrates were identified by determining a general phosphorylation consensus sequence and searching for proteins with the consensus sequence from protein database. Many of these substrates were tested using an in vitro kinase assay. Of those identified, BCAR-1 was confirmed to be a new physiological Syk substrate. 2. Materials and methods 2.1. Plasmid construction Human Syk cDNA was PCR-amplified from the cDNA pool of Jurkat cells and subcloned into pDEST–SG5–FLAG (Invitrogen, Carlsbad, CA) for mammalian expression. A short alternatively spliced form of Syk, referred to as SykB or SykS, was used for in vitro kinase assay with GST-peptides [1]. The predominant long form of Syk (SykL) was also generated and used for the investigation of its ability to phosphorylate BCAR-1 in HEK-293T cells. The kinase-dead forms of Syk, Syk-K402R for SykL and Syk-K379R for SykB, were generated using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). Human BCAR-1 cDNA was PCR-amplified and subcloned into the BamHI–SmaI site of pENTR1A and was transferred into pDEST–CG3–MT for the expression of Nterminal Myc-tagged protein using LR recombinase (Invitrogen). For GST fusion peptide expression vectors, complementary oligonucleotides encoding the desired peptides (11 amino acids) were cloned into the BamHI–SmaI site of pGEX-4T-1 (GE Biosciences, Piscataway, NJ). The truncated form of BCAR-1 (aa 570–910) was subcloned into the BamHI–SmaI site of GEX-4T-1. All constructs were confirmed by restriction enzyme digestion and DNA sequencing. 2.2. Production of GST-tagged peptides and proteins The GST-peptides were overexpressed in Escherichia coli DH5␣. The culture was incubated at 37 ◦ C until OD595 reached 0.4–0.5, after which 0.4 mM IPTG was added and the culture was incubated for an additional 3 h. The cells were harvested and lysated in lysis buffer (PBS containing 1% Triton X-100, 2 mM PMSF, and 2 mM Na3 VO4 ). The crude lysates were incubated with Glutathione–Sepharose 4B Fast Flow (GE Biosciences) at 4 ◦ C for 3 h with rolling. The beads were washed three times with PBS, and the binding protein was eluted with elution buffer (20 mM glutathione in 100 mM Tris–HCl, pH 8.0) at room temperature for 10 min. GST-peptide expressions were checked by SDS-PAGE. 2.3. In vitro kinase assay Cell extracts were prepared from 293T cells that had been transfected with pDEST–FLAG–Syk (wild-type) or pDEST–FLAG–Syk (kinase-dead) by lysing cells in NETN buffer (50 mM Tris–HCl, pH 7.5; 150 mM NaCl; 1 mM EDTA; 0.5% Nonidet P-40; 1 mM sodium fluoride; 2 mM Na3 VO4 ; and protease inhibitors) for 1 h at 4 ◦ C. The supernatants were immunoprecipitated with anti-FLAG antibody (M2; Sigma–Aldrich, Oakville, Ontario, Canada) and protein A sepharose beads (GE Biosciences). The beads were washed three times with NETN buffer and twice with kinase buffer (50 mM Tris–HCl, pH 7.5; 50 mM NaCl; and 5 mM MgCl2 ). The kinase reaction was prepared by resuspending the beads in 40 l kinase buffer containing 5 Ci [␥-32P]ATP (GE Biosciences), 10 M ATP, and 2 g GST-tagged protein as a substrate. A kinase reaction was performed at 30 ◦ C for 30 min and was terminated by adding 10 l of 5× SDSPAGE loading buffer, after which the proteins were transferred to nitrocellulose membrane. Radiolabeled proteins were visualized by BAS (Fujifilm, Tokyo, Japan).
2.4. Immunoblotting After exposure to BAS, the membranes were washed briefly with TBS-T and immersed in TBS-T with 3% bovine serum albumin (BSA) for 2 h to block non-specific background. The membranes were incubated with anti-Syk antibody (N-14; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 2 h and then washed with TBS-T six times for 10 min each time. The membranes were then incubated with horseradish peroxidase-labeled goat anti-rabbit antibody (Dako, Carpentaria, CA) for 1 h and washed with TBS-T. An enhanced chemiluminescence solution (GE Biosciences) was used for visualization.
2.5. Cell culture and transfection HEK-293T cells were grown as monolayers in Dulbecco’s modified Eagle medium (Gibco/BRL, Gaithersburg, MD) containing 10% (v/v) fetal calf serum (HyClone, Logan, UT) and penicillin–streptomycin (Gibco/BRL) at 37 ◦ C in a 5% CO2 atmosphere. HEK-293T cells were transiently transfected with a combination of cDNA expression constructs including Bcar-1, wild-type Syk, and/or kinase-dead Syk using the calcium phosphate method. After 2 days, myc-Bcar-1 from the cell lysates was immunoprecipitated with anti-Myc antibody (9E10; Roche, Basel, Switzerland) and subsequently immunoblotted with an anti-Myc antibody, anti-FLAG antibody, anti-Syk antibody, or antiphosphotyrosine (4G10; Upstate Biotechnology, Lake Placid, NY).
3. Results 3.1. Cloning of substrate GST-peptides with a partial sequence of BLNK and confirmation of in vitro kinase assay The sequence of amino acids surrounding the target phosphorylation site is important for the substrate recognition by a kinase [27]. In order to determine the substrate specificity of Syk, we produced GST proteins fused to 11 amino acid peptide residues from BLNK sequences. Inserted peptide amino acid sequences were designed so that the tyrosine residue is in the center of 11 amino acid residues. BLNK has five tyrosine residues phosphorylated by Syk, which are all located in the N-terminal acidic motif (Fig. 1A). We selected a peptide sequence surrounding tyrosine 178 (Y178), because Y178 was the most strongly phosphorylated tyrosine in chicken BLNK under BCR stimulation, and the mutation of Y178 to phenylalanine significantly attenuated the association of BLNK with PLC␥2 and intracellular calcium influx [18]. Therefore, the GST-tagged peptide comprising amino acids 173–183 (Y178) of BLNK was prepared by inserting the corresponding sequence oligonucleotides into pGEX-4T-1. Then, we confirmed the specific phosphorylation of GST–BLNK 173–183 by FLAG-tagged Syk (SykB) that was generated as described in Section 2. SykB is a short alternative form of Syk that has a deletion of 23 amino acid residues in the intervening region between SH2 and kinase domains compared to the predominant form of Syk, SykL [1]. An in vitro kinase assay with GST–BLNK 173–183 showed strong phosphorylation by Syk, which was confirmed to be targeted on tyrosine, because the replacement of Y178 with phenylalanine (GST–BLNK 173–183/Y178F) resulted in a total loss of phosphorylation (Fig. 2). This in vitro kinase system required kinase activity, because the kinase-dead mutant Syk (SykB K379R) failed to phosphorylate GST–BLNK 173–183 with no autophosphorylation. This finding confirmed the validity of our in vitro kinase system.
J.-Y. Kim et al. / Immunology Letters 135 (2011) 151–157
153
Fig. 1. (A) Schematic diagram of human BLNK. (B) Illustration of substitution mutants based on the sequence of BLNK 173–183 (Y178).
3.2. Identification of the sequence specificity of Syk To determine the amino acid sequence preference for the Syk phosphorylation of BLNK Y178, we produced variant GST-tagged peptides by substituting each amino acid residue of BLNK 173–183 for alanine, except Y178 and A176, and performed the in vitro kinase assay using Syk (Fig. 1B). The in vitro kinase assay using the substituted mutants showed that the amino acid residues on the amino side of Y178 were critical for tyrosine phosphorylation because the intensity of in vitro phosphorylation was significantly attenuated in GST-peptides with an alanine substitution at sites −5 to −1 (Fig. 3A). Tyrosine phosphorylation was more strongly affected in the GSTpeptides with substitution at sites closer to Y178. Of the amino acid residues on the carboxy side of Y178, only substitution of V179 at site +1 (V179A) resulted in a decrease in phosphorylation comparable with the phosphorylation level of D174A replacement mutant
Fig. 2. Phosphorylation of GST–BLNK peptides by Syk kinase in vitro. Wild-type or kinase-dead Syk proteins were immunoprecipitated with anti-FLAG antibody and were used in an in vitro kinase assay with 5 Ci [␥-32P]ATP, 10 M cold ATP, and 2 g GST–BLNK 173–183 Y178 (wild type), or Y178F mutant peptides in the presence of 10 mM MgCl2 . Similar amounts of FLAG-tagged Syk in the different lanes were shown by immunoblotting with anti-FLAG antibody (top panel). The amount of radiolabeled Syk (middle panel) and GST–BLNK (lower panel) in the reaction was assessed.
GST-peptide, whereas the substitution at sites +2 to +5 did not influence phosphorylation significantly. Therefore, Syk was likely to interact preferentially with amino acid residues on the amino side of Y178 and with valine residues on the +1 carboxy position for the phosphorylation of Y178. Because all residues at sites −5 to −1 comprised acidic amino acids, except alanine at site −2, the results suggest that acidic residues are required on the amino side of phosphotyrosine for phosphorylation by Syk. To further examine the preferred residues based on chemical properties, each amino acid residue was substituted to a basic residue—lysine. There is no basic residue in the BLNK 173–183 peptide sequence. In general, an in vitro kinase assay using GSTpeptides with substitution to lysine showed results similar to those using GST-peptides with substitution to alanine (Fig. 3B). Substitutions to lysine at sites +1 to +3 resulted in weaker phosphorylation, which suggests that substrate phosphorylation by Syk is negatively affected by the presence of basic amino acids on the carboxy side of phosphotyrosine in the substrate, especially at site +1 of phosphotyrosine. Next, we generated more substitution mutants in which each hydrophobic residue in BLNK 173–183 was switched to the acidic amino acid aspartate or each acidic amino acid was switched to the hydrophobic residue leucine. The results with this set of mutants were nearly identical to those with the alanine mutation (Fig. 3C). Collectively, these results demonstrated that acidic residues at sites −5 to −1 are crucial for the phosphorylation of BLNK Y178 by Syk. Valine at site +1 also contributes to phosphorylation by Syk. An amino acid residue at site +2 could be substituted with other groups of amino acids, except for basic residues.
3.3. Screening of short GST-tagged peptides from candidate proteins On the basis of sequence specificity, the following query was made to search for potential new substrates using PATTINPROT21 (http://npsa-pbil.ibcp.fr/cgi-bin/npsa automat.pl?page=
154
J.-Y. Kim et al. / Immunology Letters 135 (2011) 151–157
Fig. 3. Phosphorylation of GST–BLNK 173–183 with substitutions by Syk. Each amino acid residue in BLNK was replaced with (A) alanine, (B) lysine, or (C) leucine or aspartic acid. Phosphorylation of the modified peptides was evaluated using an in vitro kinase assay. Wild-type BLNK 173–183 and the substitution mutant of Y178 for phenylalanine (Y178F) were used as positive and negative controls, respectively.
npsa pattinprot.html) against the UNIPROT-SWISSPROT protein database: [ED]-[ED]-[ED]-X-[ED]-Y-X-X-{KR} [28,29]. The brackets [ ] denote inclusion of the specified residues, whereas the brackets { } exclude the specified residues. Of the 7727 human proteins suggested by PATTINPROT, 36 sequences from 34 proteins were selected based on the potential functional relevance with Syk (Table 1).
To confirm whether the selected proteins are genuine substrates for Syk, GST-tagged 11-amino acid peptides from selected candidates were produced for the in vitro kinase assay using the same method used for GST–BLNK 173–183. Screening of the peptides using an in vitro kinase assay revealed that 22 peptides were indeed phosphorylated by Syk (Fig. 4 and Table 1). Positive candidates include proteins involved in actin cytoskeleton regula-
Table 1 Peptide sequences from candidate substrates for in vitro kinase assay with the Syk and GST-peptides tested in Fig. 4. Accession number
Gene name
Amino acid sequences
Q16537
PPP2R5E
NELVDYITISR
P78314 Q9BYF1 Q13023 P31749 Q9NTI5 Q14155 O95260 Q13315 Q13535 Q8NDB2 Q8NDB2 P56945 P56945 Q9P287 P41182 Q96G01 P33151 Q14511 P41180 P24863 P41597 Q9Y5K6 P06127 Q76N32 P25106 Q8N6F7 P14317 Q8IWV1 P01106 Q13469 P04637 P62491 Q96IW2 O75563 P51692
SH3BP2 ACE2 AKAP6 AKT1 APRIN ARHGEF7 ATE1 ATM ATR BANK1 BANK1 BCAR1 BCAR1 BCCIP BCL6 BICD1 CDH5 NEDD9 CASR CCNC CCR2 CD2AP CD5 CEP68 CXCR7 GCET2 HCLS1 LAX1 MYC NFATC2 p53 RB11A SHD SKAP2 STAT5B
DSDEDYEKVPL PLYEEYVVLKN DFDSEYQELWD MNEFEYLKLLG AHDPDYVKVQD SEDSDYDSIWT YYDPDYSFLSL LPGEEYPLPME ATPEEYNTVVQ QEPEDYISVIQ IDDSEYDMILA TEQDEYDTPRH MEDYDYVHLQG LSDNDYDGIKK PVPGEYSRPTL ELEAEYDSLKQ DSDVDYDFLND MDDYDYVHLQG AADDDYGRPGI LSEEEYWKLQI FFDYDYGAPCH IVEYDYDAVHD HVDNEYSQPPM ESDDEYLALPA VSETEYSALEQ GTETEYSLLHM EPENDYEDVEE EDSSDYENVLT NYDLDYDSVQP LFDYEYLNPNE PLDGEYFTLQI TRDDEYDYLFK EADTEYLDPFD EDGEEYDDPFA SHLEDYSGLSV
Tyrosine no. 99 448 183 968 152 1005 776 374 959 26 146 588 235 666 71 242 59 757 631 218 57 28 10 453 482 354 128 378 373 16 38 327 8 106 75 548
Protein name Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit epsilon isoform SH3 domain-binding protein 2 Angiotensin-converting enzyme 2 precursor A-kinase anchor protein 6 RAC-alpha serine/threonine-protein kinase Androgen-induced proliferation inhibitor Rho guanine nucleotide exchange factor 7 Arginyl-tRNA–protein transferase 1 Serine-protein kinase ATM Serine/threonine-protein kinase ATR B-cell scaffold protein with ankyrin repeats B-cell scaffold protein with ankyrin repeats Breast cancer anti-estrogen resistance protein 1 Breast cancer anti-estrogen resistance protein 1 BRCA2 and CDKN1A-interacting protein B-cell lymphoma 6 protein Protein bicaudal D homolog 1 Cadherin-5 precursor Enhancer of filamentation 1 Extracellular calcium-sensing receptor precursor Cyclin C C–C chemokine receptor type 2 CD2-associated protein T-cell surface glycoprotein CD5 precursor CEP68 centrosomal protein 68 kDa Chemokine (C–X–C motif) receptor 7 Germinal center B-cell-expressed transcript Hematopoietic lineage cell-specific protein Lymphocyte transmembrane adapter 1 Myc proto-oncogene protein Nuclear factor of activated T-cells, cytoplasmic 2 Tumor suppressor protein p53 Ras-related protein Rab-11A SH2 domain-containing adapter protein D Src kinase-associated phosphoprotein 2 Signal transducer and activator of transcription 5B
J.-Y. Kim et al. / Immunology Letters 135 (2011) 151–157
155
3.4. In vitro and in vivo phosphorylation of BCAR-1 by Syk
Fig. 4. Phosphorylation of candidate target proteins for Syk. GST peptides containing the putative target sequences were used as substrates for the in vitro kinase assay. The amino acid sequences of the GST peptides are shown in Table 1. Phosphorylation of candidate peptides was evaluated using an in vitro kinase assay. Original GST–BLNK 173–183 and GST–BLNK 173–183 Y178F were used as positive and negative controls, respectively.
tion (ARHGEF7, BCAR-1, NEDD9, and HCLS1), lymphocyte signaling (SH3BP2, BANK1, CD5, GCET2, LAX1, and STAT5B), cell cycle regulation (ATR and CCNC), or others (AKAP, ATE1, CDH5, CEP68, CXCR7, and SKAP2). Of these proteins, SH3BP2, GCET2, HCLS1, and LAX1 are previously reported substrates of Syk, which supports the validity of our study [30–33]. Strong phosphorylation of the GST-peptide substrates from the reported proteins was confirmed in an in vitro kinase assay (Fig. 4).
We next attempted to confirm substrate phosphorylation in the context of a full-sized protein and within a cell. Here we selected BCAR-1 for an additional study because BCAR-1 is an adapter protein involved in cell migration and is also implicated in breast cancer progression related to clinical resistance to tamoxifen similarly to Syk [34,35]. BCAR-1 is also known as a Crk-associated substrate (CAS or p130Cas ). It seemed to us that BCAR-1 could be an interesting Syk substrate and might explain the migratory behavior of lymphocytes and cancer cells. BCAR-1 has 19 phosphotyrosine residues, many of which are known to be phosphorylated by c-Src or PTK2 (Fig. 5A) [36]. First, a partial protein—GST-tagged BCAR-1 571–710—was prepared from a BCAR-1 cDNA clone obtained from KRIBB. Although PATTINPROT suggested two potential phosphotyrosines, Y235 and Y666, which were both phosphorylated in short peptides, only the partial protein containing Y666 (BCAR-1 571–710) was produced for the assay. GST–BCAR-1 571–710 was phosphorylated by Syk in vitro, whereas A kinase-associated protein 6 (AKAP6) 965–1102, another partial protein we generated, was not phosphorylated by Syk (Fig. 5B, data of AKAP6 not shown). Y666 of BCAR-1 appeared to be a target of Syk phosphorylation, but there was another tyrosine residue, Y664, near to Y666. To further delineate the specific tyrosine residues phosphorylated by Syk, we generated two GST-peptide fusion proteins from BCAR-1—GST–BCAR-1 661–671 and GST–BCAR-1 405–415. The tyrosine residue Y410 is within the interior substrate domain of BCAR-1 and is included in 1 of 15 YxxP motifs, which was reported to be phosphorylated by Src family tyrosine kinases [37]. The in vitro kinase assay revealed the specific phosphorylation of GST–BCAR-1 661–671, but no phosphorylation of GST–BCAR-1 405–415, which indicates that Syk preferentially phosphorylates tyrosine residues only in the context of our prediction (Fig. 6). To ask whether Y666 or Y664 is really phosphorylated by Syk, we performed the in vitro kinase assay using mutants of GST–BCAR-1 661–671 with replacement mutations of Y664F, Y666F, or Y664F/Y666F. We found that both Y664 and Y666 were phosphorylated by Syk because mutations of both Y664 and Y666, but not of either Y664 or Y666, abolished the Syk phosphorylation. We further investigated whether a full-length protein of BCAR-1 is phosphorylated within cells by a predominant form of Syk (SykL).
Fig. 5. Phosphorylation of GST-BCAR-1 (aa 571–910) by Syk. (A) Domain structure of BCAR-1 showing phosphotyrosine 666 in the Src binding domain. (B) Wild-type Syk, but not kinase-dead Syk, phosphorylated GST–hBCAR-1 (Src binding domain).
156
J.-Y. Kim et al. / Immunology Letters 135 (2011) 151–157
Fig. 6. Phosphorylation of GST-BCAR-1 405–415 and 661–671 by Syk. Wild-type BLNK 173–183 and BLNK 173–183 Y178F were used as positive and negative controls, respectively.
We transfected the BCAR-1 construct with the predominant form of wild-type or kinase-dead Syk (SykL) constructs into 293T cells and checked the state of tyrosine phosphorylation of BCAR-1. Myctagged BCAR-1 was immunoprecipitated with anti-myc antibody and blotted with anti-phosphotyrosine antibody. Upon cotransfection with Syk, BCAR-1 was phosphorylated, but BCAR-1 was not tyrosine phosphorylated upon cotransfection with kinase-dead Syk (Fig. 7). Collectively, these results suggest that BCAR-1 Y664 and Y666 residues are new target residues of Syk phosphorylation, which was confirmed by in vitro and in vivo assays. 4. Discussion It is generally thought that BCR engagement leads to phosphorylation of ITAMs by Src family kinases (SFKs) and, in turn, Syk is activated by its recruitment to doubly phosphorylated ITAMs and its phosphorylation by SFKs or Syk themselves [38]. Syk is rapidly phosphorylated and activated upon BCR engagement, but its continued activity is required for B cell development and differentiation [39,40]. Activated Syk can move from plasma membrane to other cellular compartments and phosphorylate critical substrates such as BLNK for B cell proliferation and differentiation. Recently, Syk was demonstrated to be also expressed by various types of non-hematopoietic cells including breast epithelial cells and to have a negative regulatory role in carcinogenesis [41,11]. However, any relevant effectors, which are Syk substrates, have not been identified to date. Therefore, it is thought that there are a lot more substrates of Syk to explain the diverse functions of Syk in many types of cells.
We designed this study with the expectation that more substrates could be found by determining the substrate specificity surrounding the phosphotyrosine residue and by searching for proteins with similar sequences in the protein database. Here we found that the amino acid residues on the amino side of phosphotyrosine were important for the substrate phosphorylation by Syk. We suggest that this substrate preference may hold for proteins with BLNK-like sequences. The sequence around Y72 of BLNK also fit with our consensus sequence, and other tyrosine residues—Y84, Y96, and Y189—had acidic residues in their amino sides [19]. However, some Syk substrates do not contain BLNK-like sequences, such as the recently reported substrate, STAT3 [42]. This suggests that Syk may also phosphorylate tyrosines in a dissimilar context. Although this study cannot find Syk substrates with STAT3-like sequences, it has value because it elucidated more Syk substrates with BLNK-like sequences. It was interesting to us that the kinase domain of Syk preferentially recognized the amino acid residues located amino-terminal to the substrate tyrosine residue, whereas the amino acid residues carboxy-terminal to the phosphotyrosine were reported to determine the binding specificities of the SH2 domain. The SH2 domains were previously grouped according to their binding sequence specificities, and some common binding motifs, such as YEEI or YMXM, were found [43]. Therefore, we propose that an extended motif composed of the Syk substrate motif and certain specific SH2-binding motifs may define a signaling module connecting Syk activation to specific signaling pathway. For BLNK, all five tyrosine residues were in the YxxP motif, which is also known as a binding site for the SH2 domain. It was shown that each YxxP motif of BLNK bound distinct signaling molecules such as Vav, Nck, BTK, and PLC␥ [19]. Therefore, the five tyrosine residues of BLNK make signaling connections from Syk activation to downstream pathways of Vav, Nck, BTK, and PLC␥. In contrast, BCAR-1 appears to have a different signaling connection. BCAR-1, also identified as p130Cas , is composed of an N-terminal SH3 domain, a central substrate domain with 15 YxxP motifs, and a C-terminal Src-binding region [37]. Here the Syk substrate tyrosines were not present in the central substrate domain, but in the C-terminal Src-binding domain. Our in vitro kinase assay with GST-peptide confirmed that Syk could clearly phosphorylate the C-terminal tyrosines Y664 and Y666, but not Y410 in the central substrate domain. Since the C-terminal YDYVHL sequence was shown as a binding site for Src family tyrosine kinases, we think that Syk activation-driven phosphorylation of Y664 and Y666 within the YDYVHL sequence lead to the binding of SH2 domain of Src family tyrosine kinases. Thus, this extended motif in BCAR-1 is thought to connect Syk activation to Src family tyrosine kinase binding, which may lead to phosphorylation of multiple tyrosines in the central substrate domain. In summary, we successfully identified some new Syk substrate candidate proteins using a systemic in vitro kinase assay using GST fusion peptides with BLNK-like sequences. We further confirmed that BCAR-1 is a new Syk substrate by showing its tyrosine phosphorylation upon co-transfection with Syk. With this approach, it is expected that more significant Syk substrates can be identified, which would help to elucidate the multi-faceted functions of Syk. Conflict of interest The authors have no financial conflicts of interest.
Fig. 7. Syk-dependent phosphorylation of BCAR-1. 293T cells were co-transfected with Myc-BCAR-1 along with empty vector, FLAG–Syk (wild-type SykL), or FLAG–Syk (kinase-dead K402R SykL). FLAG–Syk in each cell lysate was assessed by immunoblotting with anti-Syk antibody or anti-FLAG antibody. Immunoprecipitated Myc-BCAR-1 was blotted with anti-phosphotyrosine antibody (4G10).
Acknowledgements This work was supported by the Korean Research Foundation Grant funded by the Korean Government (MEST) (KRF-313-2007-2-
J.-Y. Kim et al. / Immunology Letters 135 (2011) 151–157
E00205) and a grant (01-PJ3-PG6-01GN12-0001) from the 21 Good Health R and D Project, Ministry of Health, Welfare, and Family Affairs, Republic of Korea. References [1] Mocsai A, Ruland J, Tybulewicz VL. The SYK tyrosine kinase: a crucial player in diverse biological functions. Nat Rev Immunol 2010;10:387–402. [2] Turner M, Mee PJ, Costello PS, Williams O, Price AA, Duddy LP, et al. Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk. Nature 1995;378:298–302. [3] Cheng AM, Rowley B, Pao W, Hayday A, Bolen JB, Pawson T. Syk tyrosine kinase required for mouse viability and B-cell development. Nature 1995;378:303–6. [4] Arpaia E, Shahar M, Dadi H, Cohen A, Roifman CM. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase. Cell 1994;76:947–58. [5] Zou W, Reeve JL, Liu Y, Teitelbaum SL, Ross FP. DAP12 couples c-Fms activation to the osteoclast cytoskeleton by recruitment of Syk. Mol Cell 2008;31:422–31. [6] Chu DH, van Oers NS, Malissen M, Harris J, Elder M, Weiss A. Pre-T cell receptor signals are responsible for the down-regulation of Syk protein tyrosine kinase expression. J Immunol 1999;163:2610–20. [7] Chan AC, van Oers NS, Tran A, Turka L, Law CL, Ryan JC, et al. Differential expression of ZAP-70 and Syk protein tyrosine kinases, and the role of this family of protein tyrosine kinases in TCR signaling. J Immunol 1994;152:4758–66. [8] Zarbock A, Lowell CA, Ley K. Spleen tyrosine kinase Syk is necessary for E-selectin-induced alpha(L)beta(2) integrin-mediated rolling on intercellular adhesion molecule-1. Immunity 2007;26:773–83. [9] Mocsai A, Zhou M, Meng F, Tybulewicz VL, Lowell CA. Syk is required for integrin signaling in neutrophils. Immunity 2002;16:547–58. [10] Hirahashi J, Mekala D, Van Ziffle J, Xiao L, Saffaripour S, Wagner DD, et al. Mac-1 signaling via Src-family and Syk kinases results in elastase-dependent thrombohemorrhagic vasculopathy. Immunity 2006;25:271–83. [11] Luangdilok S, Box C, Patterson L, Court W, Harrington K, Pitkin L, et al. Syk tyrosine kinase is linked to cell motility and progression in squamous cell carcinomas of the head and neck. Cancer Res 2007;67:7907–16. [12] Tohyama Y, Yamamura H. Protein tyrosine kinase, syk: a key player in phagocytic cells. J Biochem 2009;145:267–73. [13] Berton G, Mocsai A, Lowell CA. Src and Syk kinases: key regulators of phagocytic cell activation. Trends Immunol 2005;26:208–14. [14] Gevrey JC, Isaac BM, Cox D. Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine). J Immunol 2005;175:3737–45. [15] Turner M, Schweighoffer E, Colucci F, Di Santo JP, Tybulewicz VL. Tyrosine kinase SYK: essential functions for immunoreceptor signalling. Immunol Today 2000;21:148–54. [16] Janssen E, Zhang W. Adaptor proteins in lymphocyte activation. Curr Opin Immunol 2003;15:269–76. [17] Ishiai M, Kurosaki M, Pappu R, Okawa K, Ronko I, Fu C, et al. BLNK required for coupling Syk to PLC gamma 2 and Rac1-JNK in B cells. Immunity 1999;10:117–25. [18] Chiu CW, Dalton M, Ishiai M, Kurosaki T, Chan AC. BLNK: molecular scaffolding through ‘cis’-mediated organization of signaling proteins. EMBO J 2002;21:6461–72. [19] Wu JN, Koretzky GA. The SLP-76 family of adapter proteins. Semin Immunol 2004;16:379–93. [20] Kurosaki T, Tsukada S. BLNK: connecting Syk and Btk to calcium signals. Immunity 2000;12:1–5. [21] Pappu R, Cheng AM, Li B, Gong Q, Chiu C, Griffin N, et al. Requirement for B cell linker protein (BLNK) in B cell development. Science 1999;286:1949–54.
157
[22] Koretzky GA, Abtahian F, Silverman MA. SLP76 and SLP65: complex regulation of signalling in lymphocytes and beyond. Nat Rev Immunol 2006;6:67–78. [23] Latour S, Veillette A. Proximal protein tyrosine kinases in immunoreceptor signaling. Curr Opin Immunol 2001;13:299–306. [24] Tsang E, Giannetti AM, Shaw D, Dinh M, Tse JK, Gandhi S, et al. Molecular mechanism of the Syk activation switch. J Biol Chem 2008;283:32650–9. [25] Keshvara LM, Isaacson CC, Yankee TM, Sarac R, Harrison ML, Geahlen RL. Sykand Lyn-dependent phosphorylation of Syk on multiple tyrosines following B cell activation includes a site that negatively regulates signaling. J Immunol 1998;161:5276–83. [26] Ubersax JA, Ferrell JEJ. Mechanisms of specificity in protein phosphorylation. Nat Rev Mol Cell Biol 2007;8:530–41. [27] Seo GJ, Kim SE, Lee YM, Lee JW, Lee JR, Hahn MJ, et al. Determination of substrate specificity and putative substrates of Chk2 kinase. Biochem Biophys Res Commun 2003;304:339–43. [28] Combet C, Blanchet C, Geourjon C, Deleage G. NPS@: network protein sequence analysis. Trends Biochem Sci 2000;25:147–50. [29] Bairoch A, Apweiler R. The SWISS-PROT protein sequence data bank and its supplement TrEMBL. Nucleic Acids Res 1997;25:31–6. [30] Foucault I, Le Bras S, Charvet C, Moon C, Altman A, Deckert M. The adaptor protein 3BP2 associates with VAV guanine nucleotide exchange factors to regulate NFAT activation by the B-cell antigen receptor. Blood 2005;105:1106–13. [31] Pan Z, Shen Y, Ge B, Du C, McKeithan T, Chan WC. Studies of a germinal centre Bcell expressed gene, GCET2, suggest its role as a membrane associated adapter protein. Br J Haematol 2007;137:578–90. [32] Hao JJ, Carey GB, Zhan X. Syk-mediated tyrosine phosphorylation is required for the association of hematopoietic lineage cell-specific protein 1 with lipid rafts and B cell antigen receptor signalosome complex. J Biol Chem 2004;279:33413–20. [33] Zhu M, Janssen E, Leung K, Zhang W. Molecular cloning of a novel gene encoding a membrane-associated adaptor protein (LAX) in lymphocyte signaling. J Biol Chem 2002;277:46151–8. [34] Cary LA, Han DC, Polte TR, Hanks SK, Guan JL. Identification of p130Cas as a mediator of focal adhesion kinase-promoted cell migration. J Cell Biol 1998;140:211–21. [35] van der Flier S, Brinkman A, Look MP, Kok EM, Meijer-van Gelder ME, Klijn JG, et al. Bcar1/p130Cas protein and primary breast cancer: prognosis and response to tamoxifen treatment. J Natl Cancer Inst 2000;92:120–7. [36] Goldberg GS, Alexander DB, Pellicena P, Zhang ZY, Tsuda H, Miller WT. Src phosphorylates Cas on tyrosine 253 to promote migration of transformed cells. J Biol Chem 2003;278:46533–40. [37] Bouton AH, Riggins RB, Bruce-Staskal PJ. Functions of the adapter protein Cas: signal convergence and the determination of cellular responses. Oncogene 2001;20:6448–58. [38] Saouaf SJ, Mahajan S, Rowley RB, Kut SA, Fargnoli J, Burkhardt AL, et al. Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement. Proc Natl Acad Sci USA 1994;91:9524–8. [39] Oh H, Ozkirimli E, Shah K, Harrison ML, Geahlen RL. Generation of an analogsensitive Syk tyrosine kinase for the study of signaling dynamics from the B cell antigen receptor. J Biol Chem 2007;282:33760–8. [40] Cornall RJ, Cheng AM, Pawson T, Goodnow CC. Role of Syk in B-cell development and antigen–receptor signaling. Proc Natl Acad Sci USA 2000;97:1713–8. [41] Coopman PJ, Mueller SC. The Syk tyrosine kinase: a new negative regulator in tumor growth and progression. Cancer Lett 2006;241:159–73. [42] Uckun FM, Qazi S, Ma H, Tuel-Ahlgren L, Ozer Z. STAT3 is a substrate of SYK tyrosine kinase in B-lineage leukemia/lymphoma cells exposed to oxidative stress. Proc Natl Acad Sci USA 2010;107:2902–7. [43] Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, et al. SH2 domains recognize specific phosphopeptide sequences. Cell 1993;72:767–78.