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Molecular Biology, Pathobiology and Genetics |
1 Department of Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Houston, Texas; 2 Department of Gastrointestinal Surgery, 1st Affiliated Hospital of Sun Yat-sen University, Guangzhou, China; and 3 Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland
Requests for reprints: Jiale Dai, Department of Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Box 89, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-8995; Fax: 713-792-4324; E-mail: jldai{at}mdanderson.org.
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Abstract |
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Introduction |
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Spleen tyrosine kinase (SYK), a candidate tumor suppressor gene, is expressed in normal mammary epithelial cells, but is lost in >30% of breast tumors as a result of DNA hypermethylation in the SYK promoter region (2, 3). This epigenetic inactivation of SYK suggests an important role as a suppressor of breast cancer progression. Indeed, among breast cancer patients, the loss of Syk expression has been correlated with shorter survival (4, 5); overexpression of the full-length Syk [also known as Syk(L)] leads to decreased proliferation, invasiveness, and mobility of breast cancer cells (6–8). These findings agree with earlier observations of a progressive decrease of SYK mRNA from normal mammary glands, to ductal carcinoma in situ, and to invasive breast carcinoma (3). Biochemically, expression of Syk(L) has been shown to lower epidermal growth factor–initiated signaling and phosphatidylinositol-3 kinase activity in mammary epithelial cells (8, 9). Whether these effects are directly related to the suppression response remains to be determined.
The role of SYK as a tumor suppressor gene is unusual in that most tyrosine kinases instead have oncogenic activity, suggesting that some of the Syk biological functions is separate from its catalytic activity. In examining Syk expression in breast tumors and cell lines, we previously found a SYK variant in breast cancer cells that is created by alternative RNA splicing (6). This tumor-specific exon skipping generates a Syk isoform, Syk(S), with an in-frame deletion of 23 residues within interdomain B (IDB) of Syk. Syk(S) differs from Syk(L) in biological activity despite their striking structural similarity. Syk(L) suppresses breast cancer invasion, whereas Syk(S) lacks this activity. Coincident with their differing phenotypic responses, the two Syk isoforms have different subcellular distributions. Syk(L) is present in both the cytoplasm and nucleus, whereas Syk(S) is localized exclusively in the cytoplasm (6, 10). Five basic amino acids within the deleted 23 residues act as an autonomous nuclear localization signal required for Syk(L) nuclear translocation. The correlation of anti-invasive action and nuclear presence of Syk(L) suggests that nuclear translocation may be a rate-limiting step in the Syk(L) biological makeup. Consistent with this notion, cells expressing nuclear translocation–deficient Syk(L) mutants do not exhibit anti-invasion phenotypes (6). These findings indicate that the nuclear activity of Syk(L) is indispensable for its tumor suppression function.
For a given protein, one potential action in the nucleus is to regulate gene transcription. In the present study, we examined the possible effect of Syk(L) on gene transcription and looked for its downstream targets.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thymidine incorporation, chemoinvasion, and reporter assays. Cell proliferation rates were measured by [3H]thymidine incorporation as described previously (11). Chemoinvasion assays were done using Boyden chambers coated with Matrigel as described previously (6).
For Gal4 reporter experiments, cells cultured in six-well dishes received 0.5 µg of pFA-CMV constructs [Syk(L), SH2D, IDB(L), or KD], 0.5 µg of RF-Luc (Stratagene), and 0.2 µg of pCMVß. pRF-Luc that contains five copies of Gal4-response element was used as a Gal4 transactivation readout. Forty-eight hours after transfection, cells were collected and assayed for luciferase and ß-galactosidase activity.
For the promoter response tests, Flag-Syk(L) stable and Neo control cells each received 1.5 µg of pGL3-Basic (Promega, Madison, WI) constructs fused with FRA1 promoter regions.
Glutathione S-transferase pull-down assays. GST-Syk(L) was purified from pGEX4T2-SYK(L)–transformed Escherichia coli using glutathione-Sepharose beads (GE Healthcare) and quantified by SDS-PAGE and Coomassie blue staining. Radiolabeled histone deacetylases (HDAC) were synthesized by in vitro TNT reaction (Promega) from the cDNAs encoding HDACs 1 to 7 in the presence of [35S]methionine (Perkin-Elmer, Boston, MA). Pull-down assays were done by incubating the TNT products with the GST beads in the presence of 20 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1% NP40, 1 mmol/L EDTA, and 1 mmol/L DTT. After extensive washes, the bound proteins were eluted by boiling in 1x SDS sample buffer, separated by SDS-PAGE, and exposed for autoradiography. The HDAC cDNAs were gifts from Drs. Ronald Evans (Salk Institute, San Diego, CA), Tony Kouzarides (Wellcome Cancer Research UK Gurdon Institute, Cambridge, United Kingdom), and Jiemin Wong (Baylor College of Medicine, Houston, TX).
Adenovirus. Recombinant adenovirus expression Syk(L) or Syk(S) were prepared as described previously (11). MDA-MB-231 cells were infected for 48 hours with adeno-Syk(L), adeno-Syk(S), or adeno-LacZ at a multiplicity of infection of 20.
Northern blotting. Total RNA was extracted from cells using TRIzol (Molecular Research Center, Cincinnati, OH). Twenty micrograms of total RNA were fractionated on a 1% formaldehyde-agarose gel, transferred onto membranes, and detected by hybridization with probe that was prepared by random-primer labeling with [
-32P]dCTP (Perkin-Elmer) using PCR products as templates. Gene-specific PCR products were amplified from I.M.A.G.E. clones (Invitrogen). To control for loading variability, the membrane was stripped and rehybridized with a human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe.
Microarray analyses. DNase-digested total RNA was converted to cDNA and labeled with Cy3 or Cy5 dye. Labeled cDNAs (100 µg) were hybridized with an in-house CG11 pathway array as described previously (12). The gene list, which is available at http://www3.mdanderson.org/~genomics/, includes 1,126 genes with defined tumor-related activities. All test genes on chips were spotted in duplicate. The slides were washed and scanned with a GeneTAC UC 4 laser scanner (Genomic Solutions, Ann Arbor, MI), and the signal intensities were quantified with ArrayVision (Imaging Research, St. Catherines, Ontario, Canada) and defined as spot density minus background density divided by the SD of the background density. Hybridization procedures were carried out in the Genomics Core Facility and the array data were analyzed by the Biostatistics and Data Management Bioinformatics Core Facility at M. D. Anderson Cancer Center.
Immunoprecipitation and immunoblotting. For SYK(L) and HDAC1 cotransfection experiments, HEK293 cells were cotransfected with pCMX-hHDAC1-HA (from Dr. Ronald Evans) and pFlag-SYK(L) [or pFlag-SH2D, pFlag-IDB(L), and pFlag-KD]. Cells were then lysed in 1x radioimmunoprecipitation assay (RIPA) buffer and the soluble components were subjected to immunoprecipitation using agarose-conjugated anti-Flag antibody (M2 affinity gel; Sigma). The immunocomplex was separated by SDS-PAGE and detected by antihemagglutinin (anti-HA) antibody (Y11; Santa Cruz Biotechnology).
To detect Syk interaction with HDAC1 at the endogenous level, cultured MDA-MB-468 cells were lysed with 1x RIPA buffer. The cleared lysates were immunoprecipitated with anti-HDAC1 antibody (Cell Signaling, Beverly, MA) using an equivalent amount of normal rabbit IgG as a control. The immunocomplex was collected by addition of protein A/G plus agarose and detected with anti-Syk antibody (4D10).
To measure binding of transfected Syk to endogenous HDACs, HEK293 cells were transfected with pFlag-SYK(L) or control pFlag-bovine alkaline phosphatase (BAP; Sigma) plasmids. Cells were lysed in 50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton-X 100, and 1x protease inhibitor cocktail (Sigma). The Flag-tagged proteins were immunoprecipitated by M2 affinity gel and the immunocomplex was analyzed with antibodies against HDACs 1, 3, 4, 5, 6, and 7 (Cell Signaling). Similar experiments were done to measure the binding between transfected Syk(L) and endogenous Sp1. Sp1 in the immunocomplex was analyzed by an anti-Sp1 antibody (PEP2; Santa Cruz Biotechnology).
Additional antibodies used were against Gal4 DNA-binding domain (RK5C1) and Syk (N19; Santa Cruz Biotechnology). Immunodetection was done using horseradish peroxidase–conjugated secondary antibody and Supersignal reagents (Pierce, Rockford, IL).
Measurement of histone deacetylase. HEK293 cells transfected with pFlag-SYK(L) or pCMV-FLAG were lysed and subjected to M2 immunoprecipitation. The beads were washed extensively with the lysis buffer, once with HDAC reaction buffer, and then assayed for deacetylase activity using a fluorescence-based HDAC testing kit (BioMol Research Laboratories, Plymouth Meeting, PA). The beads were incubated with 200 µmol/L acetylated substrate in 50 µL assay buffer in the presence or absence of 5 µmol/L trichostatin A. The reaction was incubated at 25°C for 90 minutes to allow deacetylation of the substrate. The developer for the deacetylated substrate was then added to produce a fluorophore detectable on a fluorometric reader (excitation at 360 nm and emission at 450 nm). The trichostatin A–containing reaction was used to assess the non-HDAC background signal. The difference in signal intensity between the trichostatin A–negative and trichostatin A–positive reactions reflects specific HDAC activity.
In vitro kinase assay. Wild-type or mutant (K402R) pFlag-SYK(L) expression vectors were transfected into COS7 cells. The M2-immunoprecipitated product was incubated for 30 minutes at room temperature with 2 µg of purified recombinant cdb3 (from Dr. Philip Low, Purdue University, West Lafayette, IN) in a 20 µL in vitro kinase reaction in the presence of 20 mmol/L Tris (pH 7.5), 10 mmol/L MnCl2, 10 mmol/L MgCl2, 1 µmol/L ATP, and 5 µCi [
-32P]ATP (Perkin-Elmer). The reaction supernatant was run on SDS-PAGE and subjected to autoradiography. A Syk inhibitor, piceatannol (100 nmol/L; Sigma), was also used as a control for reaction specificity.
Chromatin immunoprecipitation. A commercial chromatin immunoprecipitation (ChIP) assay kit (Active Motif, Carlsbad, CA) was used to analyze the in vivo binding of Syk(L) to the FRA1 promoter region using MDA-MB-231/Flag-Syk(L) stable cells. Fragmented chromatin was immunoprecipitated by overnight incubation at 4°C with protein G beads and 3 µg of either anti-Syk (4D10), anti-Flag (M2), anti-TFIIB antibodies, or mouse IgG control. Recovered DNA was analyzed by semiquantitative PCR. The primers for FRA1 were as follows: set 1, 5'-GGCAGGAGAATCCCTTTAGC-3' (forward) and 5'-TCTTTCCACTGGCCTTGTTT-3' (reverse) and set 2, 5'-GGAAAGACCTCACTCCACGA-3' (forward) and 5'-CTGACGTAGCTGCCCATACA-3' (reverse). The control primers 5'-ATGGTTGCCACTGGGGATCT-3' (forward) and 5'-TGCCAAAGCCTAGGGGAAGA-3' (reverse) were used to normalize the DNA output of ChIP.
Electrophoretic mobility shift assays. Approximately 4 µg of K562 nuclear extract (Active Motif) was incubated for 30 minutes at 4°C in binding buffer [10% glycerol, 5 mmol/L MgCl2, 35 mmol/L KCl, 80 mmol/L NaCl, 1 mmol/L DTT, 25 mmol/L Tris-HCl (pH 7.5), 1% NP40, and 50 µg/mL poly(deoxyinosinic-deoxycytidylic acid)] in a 20 µL reaction. Radiolabeled probe (20,000 cpm) was subsequently added to each reaction and incubated at room temperature for an additional 20 minutes. DNA-protein complexes were resolved on 4% native Tris-borate-EDTA gels that were exposed for autoradiography. For competition gel shift assays, 10- or 100-fold excess unlabeled fragment was included in reaction before the probe was added. For supershift, 1 µg of antibodies or normal IgG was incubated with nuclear extracts for 30 minutes at 4°C before probe addition. The 100 bp FRA1 probe was released from the pGL3 vector by digestion with Acc65I and HindIII and labeled by end fill in. For experiments using the recombinant proteins, each reaction included 25 ng of recombinant Sp1 (Active Motif) or 150 ng of GST or GST-Syk(L).
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Results |
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We used this reporter model to further determine which Syk domain(s) is responsible for the repression activity. Syk(L) has three major domains: two tandem SH2D (residues 1-259) at the amino terminus, the KD (residues 395-635) at the carboxyl terminus, and IDB(L) (residues 259-395) in between. These domains, which cover the entire Syk sequence, were analyzed in MDA-MB-231 and HEK293 cells. Both SH2D and the KD had a repressor activity similar to that of Syk(L) (Fig. 1D) despite the comparable expression level of all Gal4 fusion proteins (Fig. 1E). In contrast, IDB(L), a domain shown to be responsible for Syk(L) nuclear localization, did not show repressor function (Fig. 1D). Thus, Syk(L) contains two distinct domains, SH2D and KD, that mediate the intrinsic repressor function of Syk(L).
Tyrosine kinase activity has been shown to be important for a variety of Syk signaling events (13), although evidence from certain experimental settings indicates that this kinase activity is not crucial for some of the downstream effects of Syk (14). To determine whether kinase activity is required for the transcription repression function of Syk(L), we tested the repressor activity of a kinase-inactive Syk mutant (K402R) in the Gal4 model. The Lys402 residue in the KD is a crucial ATP-binding site. As expected, replacing that residue with an arginine rendered the tyrosine kinase inert, as determined by in vitro kinase assay of cdb3, a classic Syk substrate (Fig. 1F). Transfection experiments in MDA-MB-231 and HEK293 cells showed that the KI mutant suppressed the Gal4 reporter at a level similar to that of the corresponding wild-type Syk (Fig. 1G), suggesting that the catalytic kinase activity is dispensable for the intrinsic transcription repression function of Syk(L).
Interaction of Syk(L) with histone deacetylases. Many transcription repressors or corepressors exert their functions by modulating chromatin configuration, which is largely determined by histone acetylation status (15). We tested the hypothesis that Syk(L) and HDACs are physically associated. We transfected HEK293 cells with pFlag-Syk(L) to determine whether the Flag-tagged Syk(L) forms complexes with the endogenous HDACs. Immunoprecipitation of cell lysates with anti-Flag antibody and immunoblotting of the precipitates with antibodies to HDACs 1 and 3 to 7 revealed an association between Syk(L) and HDACs 1, 3, 6, and 7 (Fig. 2A). HDACs 4 and 5, however, were not detectable (data not shown). We further examined whether Syk(L) bound HDACs at the endogenous expression level. A breast cancer cell line, MDA-MB-468, which expresses high levels of HDAC1 and Syk, was used for this experiment. Coimmunoprecipitation experiments identified a significant interaction between the two proteins (Fig. 2B), demonstrating that Syk(L) associates with HDAC1 in vivo at the endogenous level.
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Transcription repressors function by forming complexes with HDAC by either direct binding or recruitment of corepressors (16). We used a GST pull-down assay to analyze whether Syk(L) directly interacts with HDAC. A GST-Syk(L) fusion protein attached to glutathione beads (Fig. 2D) was incubated with radiolabeled HDACs. Whereas HDAC5 did not seem to interact with GST-Syk(L), HDACs 1 to 4, 6, and 7 showed significant binding to GST-Syk(L) (Fig. 2E). The binding was specific as the interaction did not occur with the GST control or an unrelated luciferase protein. These experiments using purified recombinant protein indicated that Syk(L) is likely to interact with HDACs directly.
The physical interaction of Syk(L) with HDACs was also reflected by the association of Syk(L) with active HDAC. HEK293 cells were transfected with pFlag-SYK(L) or the control vector, and the Flag-immunoprecipitated products were assayed for HDAC activity. Syk(L) was associated with high levels of HDAC activity (trichostatin A sensitive), which was marginal in the mock- or vector-transfected cells (Fig. 2F). This Syk(L)-associated HDAC activity is likely to be instrumental in the transcription repression property of Syk(L).
Downstream genes regulated by Syk(L). Because nuclear translocation is crucial to the tumor suppressor function of Syk(L), we surmised that genes transcriptionally repressed by Syk(L), but not by Syk(S), are candidates responsible for Syk(L)-inducible invasion and growth suppression.
DNA microarrays were used to uncover changes in global gene expression profiles following Syk expression in the Syk-negative breast cancer cell line, MDA-MB-231. The epigenetic abrogation of Syk function in these cells predicts that restoration of Syk(L) would suppress their aggressiveness (2). In agreement with our observations in MDA-MB-435S stable lines, we found that expression of Syk(L) brought by adenoviral delivery (Fig. 3A) led to lowered invasion (Fig. 3B). In addition, we found that Syk(L) expression led to modest but consistent inhibition of cell growth (Fig. 3C). Syk(S), however, had neither such activity, likely due to its lack of nuclear translocation property (6). To confirm that these gene expression changes were not artifacts caused by adenoviral infection other than Syk, we used adeno-LacZ as a control to compare the relative transcript levels in adeno-Syk- and adeno-LacZ–infected cells. cDNA was synthesized and labeled from infected cells. Competitive hybridization was done on an in-house CG11 oligo-DNA array on which 1126 essential tumor-associated genes were spotted in duplicate, including those related to adhesion (12 genes), angiogenesis (129 genes), apoptosis (37 genes), cell cycle (250 genes), DNA repair (104 genes), immunomodulation (13 genes), invasion (17 genes), metastasis (230 genes), proliferation (34 genes), tumor suppression (198 genes), and other cellular functions (102 genes), together with positive (48 spots), negative (48 spots), and blank (24 spots) controls. We reasoned that genes activated by Syk(L), but not by Syk(S), might be responsible for Syk(L)-inducible invasion suppression. We found two genes, CCND1 (also known as cyclin D1) and FOSL1 (also known as FRA1), that were down-regulated by the expression of Syk(L) but were not affected by Syk(S). These observations were verified by Northern blotting (Fig. 3D).
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900 bp FRA1 promoter reporter construct (nucleotides –861 to +32) to Syk(L). This FRA1 regulatory region is critical for FRA1 induction by mitogens (17) and was shown to be highly active in MDA-MB-231 cells (40-fold higher activity than the promoter-less reporter) but significantly repressed in the presence of Syk(L) (Fig. 4B). Progressive 5' deletions in this 900 bp region allowed us to narrow the Syk(L)-responsive region to a fragment containing nucleotides –283 to –68. Testing of this fragment by nested deletion revealed a 100 bp fragment (nucleotides –283 to –184) that responded fully to Syk(L) (Fig. 4B). Electrophoretic mobility shift assay (EMSA) was then done to verify the physical association of Syk(L) with the 100 bp response region using the commercial nuclear extract from K562 cells that express high level of endogenous Syk(L) (Fig. 4C). The K562 nuclear factors resulted in multiple shifted bands (lane 2). To determine whether Syk is a component in the DNA-protein complex, we carried out an EMSA supershift experiment using two anti-Syk antibodies (4D10 and N19). Addition of either antibody led to the disappearance of a major band (arrow) accompanied by a complex supershift (arrowhead; lanes 3 and 4), whereas the respective mouse or rabbit IgG control had no such effect (lanes 5 and 6). Sequence analysis revealed a putative GC box (5'-GGGGCGGGCC-3'; nucleotides –238 to –229) within the 100 bp FRA1 fragment that resembled the consensus Sp1-binding sequence 5'-GGGGCGGGGC-3' (18). We found that the Syk-containing DNA complex was supershifted by an anti-Sp1 antibody (*, lane 7), indicating that Sp1 and Syk coexist in the same DNA complex.
To determine whether Syk(L) and Sp1 bind directly to DNA, we did EMSA using purified recombinant proteins. The recombinant GST-Syk(L) alone did not form complexes with the 100 bp FRA1 probe (Fig. 4D, lane 5), suggesting that Syk(L) does not bind to DNA directly. This was consistent with our earlier cyclic amplification and selection of targets (a.k.a. CASTing) studies in which we did not identify a consensus DNA sequence for Syk(L) (data not shown). As expected, we found that recombinant Sp1 protein created a shifted band (arrow) that was supershifted (arrowhead) by anti-Sp1 antibody (Fig. 4D, lanes 2 and 3). Interestingly, addition of GST-Syk(L), but not GST alone, led to further complex retardation (*, compare lanes 2 and 6), indicative of the formation of a Syk(L)-Sp1-DNA complex. The interaction between Syk(L) and Sp1 was further verified by coimmunoprecipitation analyses (Fig. 4E). All these results collectively indicate that Syk(L) influences FRA1 gene transcription by indirect binding to DNA through the Sp1 transcription factor.
To elucidate the biological significance of the Sp1 site in Syk(L)-mediated transcription repression, we generated a version of the 100 bp fragment in which the Sp1 site was disrupted by nucleotide substitution (from 5'-GGGGCGGGCC-3' to 5'-GGTTGGGGCC-3'). As expected, the mutation led to a markedly lower affinity for Sp1 (lanes 2 and 4) and an inability to compete with the wild-type DNA (lanes 7, 8 and lanes 9, 10), as assessed by EMSA (Fig. 4F). Consistently, the mutation rendered the 100 bp FRA1 promoter region resistant to repression by Syk(L) (Fig. 4B, bottom column), substantiating the importance of Sp1 in mediation of Syk(L)-regulated gene transcription.
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Discussion |
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Based on the current experimental evidence, we propose a model in which Syk counterbalances the transcription of Sp1-activated genes. By binding to Sp1, nuclear Syk(L) recruits HDACs to affect chromatin remodeling of Sp1-activated genes (Fig. 5). This model resembles the pathway for retinoblastoma (Rb)-mediated transcription repression of E2F-induced gene transcription (34, 35). Sp1-activated gene transcription has been to shown to be a key contributor to tumor growth, survival, invasion, and angiogenesis (36). Aberrant activation or overexpression of Sp1 is frequently observed in tumors (37, 38). We hypothesize that the loss of Syk(L) during mammary tumorigenesis promotes disease progression by derepression of Sp1-mediated oncoprotein expression. Sp1 is a ubiquitous transcription factor that transactivates a large number of genes. It is not clear how Syk(L) affects only limited variety of transcripts. This specificity may be attributed to DNA sequences adjacent to the Sp1-binding element in a promoter context. Alternatively, other Sp1-interacting nuclear factors may also compete with Syk(L) in a gene-specific fashion. These possibilities need further investigation.
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In summary, we have identified a distinct Syk pathway to specify gene transcription repression that is highly relevant to the suppression of breast cancer. Presumably, the shuttling of Syk(L) between the cytoplasm and the nucleus is highly regulated by upstream signals; the understanding of stimuli that trigger Syk(L) nuclear translocation is thus crucial for the development of therapeutic strategies. Moreover, it would be useful to evaluate the means to overcome the rate-limiting nature of this nuclear translocation process. The identification of chemical compounds or small peptides that would facilitate the Syk(L) nuclear translocation may serve as a way to rectify tumor suppression.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Drs. Ronald Evans, Tony Kouzarides, Philip Low, and Jiemin Wong for the reagents; Dr. Kun-Sang Chang for critical reading of the manuscript; and Drs. Wei Zhang and Keith Baggerly for help with the microarray analyses.
Received 6/24/05. Revised 8/18/05. Accepted 9/ 7/05.
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B-mediated secretion of urokinase type plasminogen activator by inhibiting the phosphatidylinositol 3'-kinase activity in breast cancer cells. J Biol Chem 2003;278:6209–21.This article has been cited by other articles:
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  R. Harfouche, D. M. Hentschel, S. Piecewicz, S. Basu, C. Print, D. Eavarone, T. Kiziltepe, R. Sasisekharan, and S. Sengupta Glycome and Transcriptome Regulation of Vasculogenesis Circulation, November 10, 2009; 120(19): 1883 - 1892. [Abstract] [Full Text] [PDF]
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 M. Z. Chen, X. Zhu, H.-Q. Sun, Y. S. Mao, Y. Wei, M. Yamamoto, and H. L. Yin Oxidative Stress Decreases Phosphatidylinositol 4,5-Bisphosphate Levels by Deactivating Phosphatidylinositol- 4-phosphate 5-Kinase {beta} in a Syk-dependent Manner J. Biol. Chem., August 28, 2009; 284(35): 23743 - 23753. [Abstract] [Full Text] [PDF]
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 X. Zhang, U. Shrikhande, B. M. Alicie, Q. Zhou, and R. L. Geahlen Role of the Protein Tyrosine Kinase Syk in Regulating Cell-Cell Adhesion and Motility in Breast Cancer Cells Mol. Cancer Res., May 1, 2009; 7(5): 634 - 644. [Abstract] [Full Text] [PDF]
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 O. Bailet, N. Fenouille, P. Abbe, G. Robert, S. Rocchi, N. Gonthier, C. Denoyelle, M. Ticchioni, J.-P. Ortonne, R. Ballotti, et al. Spleen Tyrosine Kinase Functions as a Tumor Suppressor in Melanoma Cells by Inducing Senescence-like Growth Arrest Cancer Res., April 1, 2009; 69(7): 2748 - 2756. [Abstract] [Full Text] [PDF]
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A. Abudula, A. Grabbe, M. Brechmann, C. Polaschegg, N. Herrmann, I. Goldbeck, K. Dittmann, and J. Wienands SLP-65 Signal Transduction Requires Src Homology 2 domain-mediated Membrane Anchoring and a Kinase-independent Adaptor Function of Syk J. Biol. Chem., September 28, 2007; 282(39): 29059 - 29066. [Abstract] [Full Text] [PDF]
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 M. Gururajan, T. Dasu, S. Shahidain, C. D. Jennings, D. A. Robertson, V. M. Rangnekar, and S. Bondada Spleen Tyrosine Kinase (Syk), a Novel Target of Curcumin, Is Required for B Lymphoma Growth J. Immunol., January 1, 2007; 178(1): 111 - 121. [Abstract] [Full Text] [PDF]
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 F. Zhou, J. Hu, H. Ma, M. L. Harrison, and R. L. Geahlen Nucleocytoplasmic trafficking of the syk protein tyrosine kinase. Mol. Cell. Biol., May 1, 2006; 26(9): 3478 - 3491. [Abstract] [Full Text] [PDF]
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