Three Potential Tumor Markers Promote Metastasis and Recurrence of Colorectal Cancer by Regulating the Inflammatory Response: ADAM8, LYN, and S100A9
Jiawei LiuJing LiHaolin DuLiming XuZhenbang YangMengjiao YuanKaiyue ZhangJialei LiWenjun XingShoujie WangTingting HuJinjin WangJin WangQian Gong
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Metastasis and recurrence are major causes of colorectal cancer (CRC) death, but their molecular mechanisms are unclear. In this study, genes associated with CRC metastasis and recurrence were identified by weighted gene coexpression network analysis, selecting the top 25% most variant genes in the dataset GSE33113. By average linkage hierarchical clustering, a total of 21 modules were generated. One key module was identified as the most relevant to the prognosis of CRC. Gene Ontology analysis indicated that genes associated with tumor metastasis and recurrence in this module were significantly enriched in inflammatory biological functions. Functional analysis was performed on the key module, and candidate hub genes (ADAM8, LYN, and S100A9) were screened out by expression and survival analysis. In summary, the three core genes identified in this study could greatly improve our understanding of CRC metastasis and recurrence. The results also provide a theoretical basis for the use of three core genes (ADAM8, LYN, and S100A9) as a combined marker for early diagnosis, which could benefit CRC patients.Keywords:
S100A9
LYN
Abstract The Src family kinase Lyn plays both stimulatory and inhibitory roles in hemopoietic cells. In this report we provide evidence that Lyn is involved in dendritic cell (DC) generation and maturation. Loss of Lyn promoted DC expansion in vitro from bone marrow precursors due to enhanced generation and accelerated differentiation of Lyn-deficient DC progenitors. Differentiated Lyn-deficient DCs also had a higher survival rate. Similarly, the CD11c-positive cell number was increased in aged Lyn-deficient mice in vivo. In contrast to their enhanced generation, lyn−/− DCs failed to mature appropriately in response to innate stimuli, resulting in DCs with lower levels of MHC class II and costimulatory molecules. In addition, IL-12 production and Ag-specific T cell activation were reduced in lyn−/− DCs after maturation, resulting in impaired Th1 responses. This is the first study to characterize Lyn-deficient DCs. Our results suggest that Lyn kinase plays uniquely negative and positive regulatory roles in DC generation and maturation, respectively.
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Tyrosine-protein kinase CSK
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SRC-family kinases (SFKs) have been implicated in Alzheimer's disease (AD), but their mode of action was scarcely understood. Here, we show that LYN plays an essential role in amyloid β (Aβ)-triggered neurotoxicity and tau hyperphosphorylation by phosphorylating Fcγ receptor IIb2 (FcγRIIb2). We found that enzyme activity of LYN was increased in the brain of AD patients and was promoted in neuronal cells exposed to Aβ 1–42 (Aβ1–42). Knockdown of LYN expression inhibited Aβ1–42-induced neuronal cell death. Of note, LYN interacted with FcγRIIb2 upon exposure to Aβ1–42 and phosphorylated FcγRIIb2 at Tyr273 within immunoreceptor tyrosine-based inhibitory motif in neuronal cells. With the use of the structure-based drug design, we isolated KICG2576, an ATP-competitive inhibitor of LYN. Determination of cocrystal structure illustrated that KICG2576 bound to the cleft in the LYN kinase domain and inhibited LYN with a half-maximal inhibitory concentration value of 0.15 µM. KICG2576 inhibited Aβ- or FcγRIIb2-induced cell death, and this effect was better than pyrazolopyrimidine 1, a widely used inhibitor of SFK. Upon exposure to Aβ, KICG2576 blocked the phosphorylation of FcγRIIb2 and translocation of phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 2, a binding protein to the phosphorylated FcγRIIb2, to the plasma membrane, resulting in the inhibition of tau hyperphosphorylation, the downstream event of Aβ1–42-FcγRIIb2 binding. Furthermore, intracerebroventricular injection of KICG2576 into mice ameliorated Aβ-induced memory impairment. These results suggest that LYN plays a crucial role in Aβ1–42-mediated neurotoxicity and tau pathology, providing a therapeutic potential of LYN in AD.—Gwon, Y., Kim, S.-H., Kim, H. T., Kam, T.-I., Park, J., Lim, B., Cha, H., Chang, H.-J., Hong, Y. R., Jung, Y.-K. Amelioration of amyloid β-FcγRIIb neurotoxicity and tau pathologies by targeting LYN. FASEB J. 33, 4300–4313 (2019). www.fasebj.org
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Src family kinase
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The Lyn protein-tyrosine kinase is activated in the cellular response to DNA-damaging agents. Here we demonstrate that Lyn associates constitutively with the SHPTP1 protein-tyrosine phosphatase. The SH3 domain of Lyn interacts directly with SHPTP1. The results show that Lyn phosphorylates SHPTP1 at the C-terminal Tyr-564 site. Lyn-mediated phosphorylation of SHPTP1 stimulates SHPTP1 tyrosine phosphatase activity. We also demonstrate that treatment of cells with 1-β-d-arabinofuranosylcytosine and other genotoxic agents induces Lyn-dependent phosphorylation and activation of SHPTP1. The significance of the Lyn-SHPTP1 interaction is supported by the demonstration that activation of Lyn contributes in part to the apoptotic response to ara-C treatment and that SHPTP1 attenuates this response. These findings support a functional interaction between Lyn and SHPTP1 in the response to DNA damage. The Lyn protein-tyrosine kinase is activated in the cellular response to DNA-damaging agents. Here we demonstrate that Lyn associates constitutively with the SHPTP1 protein-tyrosine phosphatase. The SH3 domain of Lyn interacts directly with SHPTP1. The results show that Lyn phosphorylates SHPTP1 at the C-terminal Tyr-564 site. Lyn-mediated phosphorylation of SHPTP1 stimulates SHPTP1 tyrosine phosphatase activity. We also demonstrate that treatment of cells with 1-β-d-arabinofuranosylcytosine and other genotoxic agents induces Lyn-dependent phosphorylation and activation of SHPTP1. The significance of the Lyn-SHPTP1 interaction is supported by the demonstration that activation of Lyn contributes in part to the apoptotic response to ara-C treatment and that SHPTP1 attenuates this response. These findings support a functional interaction between Lyn and SHPTP1 in the response to DNA damage. DNA-dependent protein kinase DNA-PK catalytic subunit ataxia telangiectasia mutated 1-β-d -arabinofuranosylcytosine glutathioneS-transferase phosphate-buffered saline polyacrylamide gel electrophoresis The response of mammalian cells to DNA damage includes cell cycle arrest, activation of DNA repair, and induction of apoptosis. The signaling events responsible for regulation of the genotoxic stress response, however, are largely unknown. Certain insights have been derived from the finding that DNA-damaging agents activate a nuclear complex that consists in part of the c-Abl and Lyn protein-tyrosine kinases. c-Abl associates with the DNA-dependent protein kinase (DNA-PK)1 and is activated in the response to DNA damage by a DNA-PK-dependent mechanism (1Kharbanda S. Ren R. Pandey P. Shafman T.D. Feller S.M. Weichselbaum R.R. Kufe D.W. Nature. 1995; 376: 785-788Crossref PubMed Scopus (463) Google Scholar, 2Kharbanda S. Pandey P. Ren R. Feller S. Mayer B. Zon L. Kufe D. J. Biol. Chem. 1995; 270: 30278-30281Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 3Kharbanda S. Pandey P. Jin S. Inoue S. Bharti A. Yuan Z.-M. Weichselbaum R. Weaver D. Kufe D. Nature. 1997; 386: 732-735Crossref PubMed Scopus (239) Google Scholar). The ataxia telangiectasia mutated (ATM) gene product also associates with c-Abl and contributes to c-Abl activation (4Shafman T. Khanna K.K. Kedar P. Spring K. Kozlov S. Yen T. Hobson K. Gatei M. Zhang N. Watters D. Egerton M. Shiloh Y. Kharbanda S. Kufe D. Lavin M.F. Nature. 1997; 387: 520-523Crossref PubMed Scopus (419) Google Scholar, 5Baskaran R. Wood L.D. Whitaker L.L. Xu Y. Barlow C. Canman C.E. Morgan S.E. Baltimore D. Wynshaw-Boris A. Kastan M.B. Wang J.Y.J. Nature. 1997; 387: 516-519Crossref PubMed Scopus (487) Google Scholar). The demonstration that c-Abl binds to p53, induces the transactivation function of p53, and activates p21 expression has supported involvement of c-Abl in regulation of the p53-dependent G1 arrest response (6Yuan Z.M. Huang Y. Whang Y. Sawyers C. Weichselbaum R. Kharbanda S. Kufe D. Nature. 1996; 382: 272-274Crossref PubMed Scopus (212) Google Scholar, 7Yuan Z.-M. Huang Y. Fan M.M. Sawyers C. Kharbanda S. Kufe D. J. Biol. Chem. 1996; 271: 26457-26460Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 8Goga A. Liu X. Hambuch T.M. Senechal K. Major E. Berk A.J. Witte O.N. Sawyers C.L. Oncogene. 1995; 11: 791-799PubMed Google Scholar). Other studies have demonstrated that c-Abl interacts with the p73 homolog of p53 in the apoptotic response to DNA damage (9Yuan Z.M. Shioya H. Ishiko T. Sun X. Huang Y. Lu H. Kharbanda S. Weichselbaum R. Kufe D. Nature. 1999; 399: 814-817Crossref PubMed Scopus (543) Google Scholar). The interaction of c-Abl and the Rad51 protein has also provided support for involvement of c-Abl in recombinational repair of DNA strand breaks (10Yuan Z.M. Huang Y. Ishiko T. Nakada S. Utsugisawa T. Kharbanda S. Sung P. Shinohara A. Weichselbaum R. Kufe D. J. Biol. Chem. 1998; 273: 3799-3802Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). In addition, a proapoptotic function for c-Abl in the response to DNA damage has been attributed to negative regulation of phosphatidylinositol 3-kinase and induction of the stress-activated protein kinase pathway (1Kharbanda S. Ren R. Pandey P. Shafman T.D. Feller S.M. Weichselbaum R.R. Kufe D.W. Nature. 1995; 376: 785-788Crossref PubMed Scopus (463) Google Scholar, 2Kharbanda S. Pandey P. Ren R. Feller S. Mayer B. Zon L. Kufe D. J. Biol. Chem. 1995; 270: 30278-30281Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 11Yuan Z.M. Utsugisawa T. Huang Y. Ishiko T. Nakada S. Kharbanda S. Weichselbaum R. Kufe D. J. Biol. Chem. 1997; 272: 23485-23488Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 12Yuan Z.-M. Huang Y. Ishiko T. Kharbanda S. Weichselbaum R. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1437-1440Crossref PubMed Scopus (178) Google Scholar). These findings have supported a role for c-Abl in the growth arrest, DNA repair, and apoptotic responses to genotoxic stress. The Lyn tyrosine kinase is a member of the Src family that contains SH2 and SH3 domains and an N-terminal sequence, which when myristoylated, serves as a membrane localization signal (13Clark S.G. Stern M.J. Horowitz H.R. Nature. 1992; 356: 340-344Crossref PubMed Scopus (511) Google Scholar). Cell fractionation and confocal microscopy studies have also demonstrated expression of Lyn in the nucleus (14Kharbanda S. Saleem A. Yuan Z.-M. Kraeft S. Weichselbaum R. Chen L.B. Kufe D. Cancer Res. 1996; 56: 3617-3621PubMed Google Scholar). Nuclear Lyn is activated by DNA damage associated with exposure to 1-β-d-arabinofuranosylcytosine (ara-C), ionizing radiation, and certain alkylating agents (15Kharbanda S. Yuan Z.M. Weichselbaum R. Kufe D. J. Biol. Chem. 1994; 269: 20739-20743Abstract Full Text PDF PubMed Google Scholar, 16Kharbanda S. Yuan Z.-M. Taneja N. Weichselbaum R. Kufe D. Oncogene. 1994; 9: 3005-3011PubMed Google Scholar, 17Yuan Z.-M. Kharbanda S. Kufe D. Biochemistry. 1995; 34: 1058-1063Crossref PubMed Scopus (23) Google Scholar). Lyn, like c-Abl, interacts with the DNA-PK complex (18Kumar S. Pandey P. Bharti A. Jin S. Weichselbaum R. Weaver D. Kufe D. Kharbanda S. J. Biol. Chem. 1998; 273: 25654-25658Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The interaction between Lyn and DNA-PKcs may play a role in releasing DNA-PKcs from Ku·DNA complexes after repair to permit relocation at new sites of DNA damage (18Kumar S. Pandey P. Bharti A. Jin S. Weichselbaum R. Weaver D. Kufe D. Kharbanda S. J. Biol. Chem. 1998; 273: 25654-25658Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The activation of nuclear Lyn by DNA damage is also associated with binding of Lyn to Cdc2 (14Kharbanda S. Saleem A. Yuan Z.-M. Kraeft S. Weichselbaum R. Chen L.B. Kufe D. Cancer Res. 1996; 56: 3617-3621PubMed Google Scholar, 15Kharbanda S. Yuan Z.M. Weichselbaum R. Kufe D. J. Biol. Chem. 1994; 269: 20739-20743Abstract Full Text PDF PubMed Google Scholar, 16Kharbanda S. Yuan Z.-M. Taneja N. Weichselbaum R. Kufe D. Oncogene. 1994; 9: 3005-3011PubMed Google Scholar, 17Yuan Z.-M. Kharbanda S. Kufe D. Biochemistry. 1995; 34: 1058-1063Crossref PubMed Scopus (23) Google Scholar). Lyn phosphorylates Cdc2 on Tyr-15 and inhibits Cdc2 activity (14Kharbanda S. Saleem A. Yuan Z.-M. Kraeft S. Weichselbaum R. Chen L.B. Kufe D. Cancer Res. 1996; 56: 3617-3621PubMed Google Scholar, 15Kharbanda S. Yuan Z.M. Weichselbaum R. Kufe D. J. Biol. Chem. 1994; 269: 20739-20743Abstract Full Text PDF PubMed Google Scholar, 16Kharbanda S. Yuan Z.-M. Taneja N. Weichselbaum R. Kufe D. Oncogene. 1994; 9: 3005-3011PubMed Google Scholar, 17Yuan Z.-M. Kharbanda S. Kufe D. Biochemistry. 1995; 34: 1058-1063Crossref PubMed Scopus (23) Google Scholar). As activation of Cdc2 in a complex with cyclin B is required for the transition of cells from G2 to M phase (19Nurse P. Nature. 1990; 344: 503-507Crossref PubMed Scopus (2473) Google Scholar), Lyn may function as an effector of G2/M regulation in response to DNA damage. Other studies have demonstrated that the arrest of cells at G1/S phase by ara-C treatment is associated with binding of activated Lyn to Cdk2 (20Yuan Z.M. Huang Y. Kraeft S.-K. Chen L.B. Kharbanda S. Kufe D. Oncogene. 1996; 13: 939-946PubMed Google Scholar). These findings collectively support a role for nuclear Lyn in the response of cells to DNA damage. The present studies have addressed the involvement of other Lyn-associated signals in the DNA damage response. The results demonstrate that treatment with ara-C induces Lyn-dependent phosphorylation of the SHPTP1 protein-tyrosine phosphatase (21Pei D. Neel B.G. Walsh C.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1092-1096Crossref PubMed Scopus (67) Google Scholar, 22Pei D. Lorenz U. Klingmüller U. Neel B.G. Walsh C.T. Biochemistry. 1994; 33: 15483-15493Crossref PubMed Scopus (186) Google Scholar). We show that Lyn activates SHPTP1, and in a potential feedback mechanism, SHPTP1 down-regulates the Lyn kinase function. The results also demonstrate that SHPTP1 attenuates Lyn-dependent induction of apoptosis in the response to DNA damage. Human U-937 myeloid leukemia cells (ATCC, Manassas, VA) were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mml-glutamine. 293T embryonal kidney and HeLa cells were grown in DMEM containing 10% fetal bovine serum and antibiotics. Cells were treated with the indicated concentrations of ara-C (Sigma). Irradiation was performed at room temperature using a Gammacell 1000 (Atomic Energy of Canada, Ottawa, Ontario, Canada) under aerobic conditions with a137Cs source emitting at a fixed dose rate of 0.76 gray/min as determined by dosimetry. The Lyn(K-R) cDNA that encodes the kinase-negative mutant (the lysine residue at position 275 in the putative ATP-binding site replaced with arginine) and the SHPTP1 mutants were generated by site-directed mutagenesis. Mutations were confirmed by sequencing. Lyn wild-type and mutant cDNAs were subcloned into pSRα (23Takebe Y. Seiki M. Fujisawa J. Hoy P. Yokota K. Arai K. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar) and pEF2 (24Datta R. Kojima H. Banach D. Bump N.J. Talanian R.V. Alnemri E.S. Weichselbaum R.R. Wong W.W. Kufe D.W. J. Biol. Chem. 1997; 272: 1965-1969Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) vectors. U-937 cells were resuspended at 107/ml and transfected with empty pEF2 vector or pEF2/Lyn(K-R) by electroporation (Gene Pulser; Bio-Rad; 0.25 V, 960 microfarads). HeLa cells were transfected with pSRα vector or pSRα/Lyn(K-R) by electroporation. Two days posttransfection, the cells were cultured in media containing 400 μg/ml Geneticin sulfate (Life Technologies, Inc.), and individual clones were selected by limiting dilution. 293T cells were transiently transfected with pSRα/Lyn, pSRα/Lyn(K-R), pcDNA3/SHPTP1, pcDNA3/SHPTP1(C-S) (25Kharbanda S. Bharti A. Pei D. Wang J. Pandey P. Ren R. Weichselbaum R. Walsh C.T. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6898-6901Crossref PubMed Scopus (84) Google Scholar), pcDNA3/ SHPTP1(Y564F), or pcDNA3/SHPTP1(C-S/Y564F) by the calcium phosphate method. At 48 h posttransfection, cells were harvested for preparing whole cell lysates. HeLa/neo cells or HeLa/Lyn(K-R) cells were transiently cotransfected with 5 μg of pEGFP-C1 vector (CLONTECH) and 10 μg of pcDNA3 vector, pcDNA3/SHPTP1, pcDNA3/SHPTP1(C-S), or pcDNA3/SHPTP1(Y564F) using SuperFect transfection reagent (Qiagen). At 40 h posttransfection, cells were left untreated or treated with 10 μm ara-C and then analyzed by flow cytometry. Cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed on ice for 30 min in lysis buffer (50 mm Tris-HCl, pH 7.6, 150 mm NaCl, 1% Nonidet P-40, 1 mm sodium vanadate, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 10 mm sodium fluoride, and 10 μg/ml aprotinin, leupeptin, and pepstatin A). Soluble proteins were incubated with anti-Lyn antibody (sc-15; Santa Cruz Biotechnology) or anti-SHPTP1 antibody (sc-287; Santa Cruz Biotechnology) for 1 h at 4 °C followed by 1 h of incubation with protein A-Sepharose beads (Amersham Pharmacia Biotech). The immune complexes were washed three times with lysis buffer, separated by 7.5% SDS-polyacrylamide gel electrophoresis (PAGE), and then transferred to nitrocellulose filters. The residual binding sites were blocked by incubating with 5% dry milk in PBS, 0.05% Tween 20 overnight at 4 °C. The filters were incubated with anti-Lyn (mouse monoclonal antibody; Transduction Laboratory), anti-SHPTP1, or anti-phosphotyrosine antibody (4G10; Upstate Biotechnology Inc.) for 1 h with shaking. After washing twice with 5% dry milk in PBS, 0.05% Tween 20, the filters were incubated with anti-rabbit or anti-mouse IgG peroxidase conjugate (Amersham Pharmacia Biotech). The antigen-antibody complexes were visualized by chemiluminescence (NEN Life Science Products). GlutathioneS-transferase (GST), GST-Lyn (1–243), GST-Lyn (1–131), GST-Lyn (30–130), and GST-Lyn (131–243) (26Kharbanda S. Saleem A. Shafman T. Emoto Y. Weichselbaum R. Woodgett J. Avruch J. Kyriakis J. Kufe D. J. Biol. Chem. 1995; 270: 18871-18874Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) were purified by affinity chromatography using glutathione-Sepharose beads and equilibrated in PBS, 0.1% sodium azide. Cell lysates were incubated with 2 μg of immobilized GST fusion protein for 2 h at 4 °C. The resulting protein complexes were washed three times with lysis buffer and boiled for 5 min in SDS sample buffer. The complexes were then separated by SDS-PAGE and subjected to immunoblot analysis with anti-Lyn antibody. For direct binding assays, recombinant Lyn (Upstate Biotechnology, Inc.) was incubated in lysis buffer with glutathione-Sepharose beads bound to GST-SHPTP1 (Upstate Biotechnology, Inc.) or GST for 1 h at 4 °C. The adsorbed material obtained by washing three times with lysis buffer was separated by SDS-PAGE and analyzed by immunoblotting with anti-Lyn antibody. GST, GST-SHPTP1 (5 μg), or His-tagged SHPTP1 proteins (2 μg) were incubated in kinase buffer (50 mm HEPES, pH 7.4, 10 mm MgCl2, 10 mm MnCl2, 2 mm dithiothreitol, 0.1 mm sodium vanadate) with recombinant Lyn and [γ-32P]ATP (3000 Ci/mmol; NEN Life Science Products) for 15 min at 30 °C. Anti-Lyn immune complexes were washed three times with lysis buffer and once with kinase buffer and resuspended in kinase buffer containing 2–5 μCi of [γ-32P]ATP and 20 μm ATP. Kinase reactions were also performed in the presence of enolase (Sigma). The reaction mixtures were incubated for 15 or 30 min at 30 °C and terminated by the addition of SDS sample buffer. Samples were analyzed by 10% SDS-PAGE and autoradiography. Tyrosine phosphatase assays were performed by the malachite green phosphatase assay method (Upstate Biotechnology, Inc.) using the phosphopeptide (RRLIEDAEpYAARG, where pY is phosphotyrosine) as a substrate. For in vivo phosphatase assays, cells were lysed with lysis buffer without phosphatase inhibitor. The lysates were incubated with anti-SHPTP1 antibody for 2 h at 4 °C followed by 1 h of incubation with protein A-Sepharose beads. The immune complexes were washed three times with lysis buffer without phosphatase inhibitor and once with phosphatase buffer (10 mm Tris-HCl, pH 7.4) and resuspended in phosphatase buffer containing the phosphopeptide. The reaction mixtures were incubated for 30 min at room temperature and terminated by the addition of malachite green solution. Absorbance was measured in a microtiter plate reader (Bio-Rad) at 655 nm. Phosphate release was determined by comparing absorbance to that obtained with the phosphate standard. DNA content was assessed by staining ethanol-fixed cells with propidium iodide and monitoring with a FACScan (Becton Dickinson). The numbers of cells positive for green fluorescence with sub-G1 DNA content were determined with a CELLQuest program (Becton Dickinson). The findings that c-Abl forms a nuclear complex with Lyn (27Kharbanda S. Yuan Z.-M. Weichselbaum R. Kufe D. Biochimica et Biophysica Acta. 1997; 1333: 1-7PubMed Google Scholar) and that c-Abl interacts with SHPTP1 (25Kharbanda S. Bharti A. Pei D. Wang J. Pandey P. Ren R. Weichselbaum R. Walsh C.T. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6898-6901Crossref PubMed Scopus (84) Google Scholar) prompted studies on a potential interaction between Lyn and SHPTP1. To determine whether Lyn associates with SHPTP1, U-937 cells were subjected to immunoprecipitation with anti-Lyn. Immunoblot analysis of the anti-Lyn immunoprecipitates with anti-SHPTP1 demonstrated reactivity at 70 kDa (Fig.1 A). Similar findings were obtained in cells treated with ara-C (Fig. 1 A). In a reciprocal experiment, analysis of anti-SHPTP1 immunoprecipitates with anti-Lyn confirmed a constitutive association of these proteins (Fig.1 B). Binding of Lyn and SHPTP1 was assessed by incubating U-937 cell lysates with GST fusion proteins prepared with Lyn fragments. Analysis of the adsorbates with anti-SHPTP1 demonstrated binding to Lyn (1–243), Lyn (1–131), and Lyn (30–130) but not Lyn (131–243) (Fig. 1 C). As Lyn (30–130) contains the SH3 and not the SH2 domain, these findings suggest that Lyn SH3 is responsible for binding to SHPTP1. To determine whether the interaction is direct, glutathione beads containing GST or GST-SHPTP1 were incubated with purified Lyn protein. The beads were washed, and protein was eluted by boiling in SDS. Analysis of the eluted protein by immunoblotting with anti-Lyn demonstrated a direct interaction between Lyn and SHPTP1 (Fig. 1 D). To determine whether Lyn phosphorylates SHPTP1 in vitro, purified Lyn was incubated with GST or GST-SHPTP1 and [γ-32P]ATP. These assays were performed in the presence of sodium vanadate at a concentration (0.1 mm) that completely inhibits SHPTP1 phosphatase activity (data not shown). Analysis of the reaction products by SDS-PAGE and autoradiography demonstrated phosphorylation of SHPTP1 (Fig.2 A). SHPTP1 contains two N-terminal SH2 domains, the protein-tyrosine phosphatase domain and a C terminus, which includes potential tyrosine phosphorylation sites (Tyr-536, Tyr-541, Tyr-564) (21Pei D. Neel B.G. Walsh C.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1092-1096Crossref PubMed Scopus (67) Google Scholar). Mutation of Tyr-536 to Phe had no apparent effect on Lyn-mediated phosphorylation of SHPTP1 (Fig.2 B). Similar results were obtained with the Y541F mutants (Fig. 2 B). By contrast, Lyn-mediated phosphorylation was abrogated with the Y564F mutant as substrate (Fig. 2 B). These findings demonstrate that Lyn phosphorylates the Tyr-564 site. To assess whether Lyn phosphorylates SHPTP1 in vivo, 293T cells were cotransfected with wild-type Lyn and a phosphatase-inactive SHPTP1, which expressed a C453S mutant (22Pei D. Lorenz U. Klingmüller U. Neel B.G. Walsh C.T. Biochemistry. 1994; 33: 15483-15493Crossref PubMed Scopus (186) Google Scholar). Analysis of anti-SHPTP1 immunoprecipitates with anti-Tyr(P) demonstrated tyrosine phosphorylation of SHPTP1 (Fig. 2 C). Cotransfection of SHPTP1(C-S) with the empty vector or kinase-inactive Lyn(K-R) was associated with little if any detectable tyrosine phosphorylation of SHPTP1 (Fig. 2 C). In addition, Lyn-mediated phosphorylation of SHPTP1(C-S) was substantially abrogated when the Tyr-564 site was mutated to F (Fig. 2 C). These findings demonstrate that Lyn phosphorylates SHPTP1 in vitro and in vivo. The functional significance of the interaction between Lyn and SHPTP1 was assessed by incubation of glutathione beads containing GST-SHPTP1 with recombinant Lyn and a synthetic phosphopeptide as substrate. The results demonstrate that Lyn stimulates the tyrosine phosphatase activity of SHPTP1 in a Lyn concentration-dependent manner (Fig. 3 A). To determine whether Lyn activates SHPTP1 in vivo, 293T cells were cotransfected with kinase-active Lyn or Lyn(K-R) and phosphatase-active SHPTP1. Anti-SHPTP1 immunoprecipitates were analyzed for tyrosine phosphatase activity. The results demonstrate that Lyn and not Lyn(K-R) stimulates SHPTP1 activity (Fig. 3 B). As a control, anti-SHPTP1 immunoprecipitates from cells transfected with SHPTP1(C-S) exhibited little if any phosphatase activity (data not shown). These findings demonstrate that Lyn stimulates SHPTP1 activity in vitro and in vivo. In reciprocal experiments, anti-Lyn immunoprecipitates were analyzed for Lyn kinase activity. In control cells transfected with Lyn alone, the anti-Lyn immunoprecipitates exhibited autophosphorylation of Lyn and phosphorylation of enolase (Fig. 3 C). Cotransfection of Lyn and 1 μg of SHPTP1 had little effect, whereas cotransfection of Lyn and 5 μg of SHPTP1 was associated with down-regulation of Lyn activity (Fig. 3 C). By contrast, cotransfection of Lyn and SHPTP1(C-S) had no apparent effect on Lyn and enolase phosphorylation (Fig. 3 C). These findings support a model in which Lyn stimulates the tyrosine phosphatase activity of SHPTP1, and in a potential feedback mechanism, SHPTP1 inhibits Lyn-mediated phosphorylation. Treatment of cells with ara-C and other DNA-damaging agents is associated with induction of Lyn activity (14Kharbanda S. Saleem A. Yuan Z.-M. Kraeft S. Weichselbaum R. Chen L.B. Kufe D. Cancer Res. 1996; 56: 3617-3621PubMed Google Scholar, 15Kharbanda S. Yuan Z.M. Weichselbaum R. Kufe D. J. Biol. Chem. 1994; 269: 20739-20743Abstract Full Text PDF PubMed Google Scholar, 16Kharbanda S. Yuan Z.-M. Taneja N. Weichselbaum R. Kufe D. Oncogene. 1994; 9: 3005-3011PubMed Google Scholar, 17Yuan Z.-M. Kharbanda S. Kufe D. Biochemistry. 1995; 34: 1058-1063Crossref PubMed Scopus (23) Google Scholar). To determine whether activation of Lyn is associated with SHPTP1 phosphorylation, we prepared U-937 cells that stably express a kinase-inactive Lyn(K-R) mutant. U-937 cells expressing the empty pEF2 vector (U-937/neo) or Lyn(K-R) were exposed to ara-C and harvested at 1 h. Anti-Lyn immunoprecipitates were analyzed for autophosphorylation and enolase phosphorylation in the presence of [γ-32P]ATP. Analysis of the reaction products by SDS-PAGE and autoradiography demonstrated induction of Lyn activity in ara-C-treated U-937/neo cells (Fig.4 A). By contrast, ara-C-induced Lyn activity was substantially abrogated in U-937/Lyn(K-R) cells (Fig. 4 A). To assess Lyn phosphorylation of SHPTP1, lysates from ara-C-treated cells were subjected to immunoprecipitation with anti-SHPTP1, and the precipitates were analyzed by immunoblotting with anti-Tyr(P). The results demonstrate tyrosine phosphorylation of SHPTP1 after ara-C treatment of U-937/neo but not U-937/Lyn(K-R) cells (Fig. 4 B). Similar studies were performed with irradiated cells to assess the effects of other DNA-damaging agents. The results demonstrate that ionizing radiation-induced tyrosine phosphorylation of SHPTP1 is abrogated in the cells expressing Lyn(K-R) (Fig. 4 B). To determine whether DNA damage is associated with induction of SHPTP1 activity, the anti-SHPTP1 immunoprecipitates from ara-C-treated cells were analyzed for dephosphorylation of the synthetic phosphopeptide. ara-C-induced increases in SHPTP1 activity were attenuated in U-937/Lyn(K-R), as compared with U-937/neo, cells (Fig. 4 C). These findings demonstrate that ara-C-induced activation of Lyn is associated with tyrosine phosphorylation of SHPTP1 and stimulation of SHPTP1 activity. To assess the significance of the interaction between Lyn and SHPTP1 in the cellular response to DNA damage, we prepared HeLa cells that stably express the empty neo vector or the kinase-inactive Lyn(K-R) mutant (Fig. 5 A). Treatment of HeLa/neo cells with ara-C was associated with an increase in the percentage with sub-G1 DNA (Fig. 5 B). By contrast, ara-C-induced apoptosis was abrogated in part in the HeLa/Lyn(K-R) cells (Fig. 5 B). These results support a role for Lyn in the apoptotic response to ara-C exposure. To define the role of Lyn in the context of SHPTP1, HeLa cells were transfected to express wild-type SHPTP1 or the phosphatase-inactive SHPTP1(C-S). Expression of SHPTP1, but not SHPTP1(C-S), attenuated ara-C-induced apoptosis of the HeLa/neo cells (Fig. 5 C). In addition, expression of SHPTP1 or SHPTP1(C-S) had little if any effect on induction of ara-C-treated HeLa/Lyn(K-R) cells with sub-G1 DNA (Fig. 5 C). These findings indicate that activation of Lyn contributes to the apoptotic response to DNA damage and that SHPTP1 attenuates the Lyn-mediated response. To confirm the functional interaction between Lyn and SHPTP1, we expressed the SHPTP1(Y564F) mutant in 293T cells. Analysis of anti-SHPTP1 immunoprecipitates demonstrated that the tyrosine phosphatase activity of SHPTP1(Y564F) is comparable to that of wild-type SHPTP1 (Fig. 6 A). Cotransfection of Lyn and wild-type SHPTP1 was associated with an increase in tyrosine phosphatase activity (Fig. 6 A). By contrast and in concert with the demonstration that Lyn phosphorylates SHPTP1 on Tyr-564, cotransfection of Lyn and SHPTP1(Y564F) resulted in no significant activation of the tyrosine phosphatase function (Fig.6 A). In addition, whereas expression of wild-type SHPTP1 attenuated ara-C-induced apoptosis of HeLa/neo cells, transfection of SHPTP1(Y564F) was significantly less effective in abrogating the apoptotic response to ara-C (Figs. 5 C and 6 B). As a control, ara-C treatment of HeLa/Lyn(K-R) cells expressing SHPTP1(Y564F) resulted in a percentage of apoptotic cells similar to that obtained when these cells were transfected with pcDNA3, wild-type SHPTP1, or SHPTP1(C-S) (Figs. 5 C and6 B). These findings demonstrate that the SHPTP1(Y564F) mutant is not activated by Lyn and that this mutant is less effective than wild-type SHPTP1 in attenuating Lyn-mediated apoptosis. The finding that activation of Lyn by DNA-damaging agents contributes to the down-regulation of Cdc2 has indicated that Lyn is an effector of cell cycle progression in the response to DNA damage (14Kharbanda S. Saleem A. Yuan Z.-M. Kraeft S. Weichselbaum R. Chen L.B. Kufe D. Cancer Res. 1996; 56: 3617-3621PubMed Google Scholar, 15Kharbanda S. Yuan Z.M. Weichselbaum R. Kufe D. J. Biol. Chem. 1994; 269: 20739-20743Abstract Full Text PDF PubMed Google Scholar, 16Kharbanda S. Yuan Z.-M. Taneja N. Weichselbaum R. Kufe D. Oncogene. 1994; 9: 3005-3011PubMed Google Scholar, 17Yuan Z.-M. Kharbanda S. Kufe D. Biochemistry. 1995; 34: 1058-1063Crossref PubMed Scopus (23) Google Scholar). Moreover, the interaction between Lyn and DNA-PK has supported a function for Lyn in the regulation of DNA repair (18Kumar S. Pandey P. Bharti A. Jin S. Weichselbaum R. Weaver D. Kufe D. Kharbanda S. J. Biol. Chem. 1998; 273: 25654-25658Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The present studies demonstrate that Lyn phosphorylates and activates SHPTP1 in response to genotoxic stress. To our knowledge, this is the first demonstration that DNA damage is associated with activation of a tyrosine phosphatase. Previous work has shown that Lyn forms a nuclear complex with c-Abl (27Kharbanda S. Yuan Z.-M. Weichselbaum R. Kufe D. Biochimica et Biophysica Acta. 1997; 1333: 1-7PubMed Google Scholar) and that c-Abl interacts with SHPTP1 (25Kharbanda S. Bharti A. Pei D. Wang J. Pandey P. Ren R. Weichselbaum R. Walsh C.T. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6898-6901Crossref PubMed Scopus (84) Google Scholar). c-Abl phosphorylates SHPTP1 on the C-terminal Tyr-536 and Tyr-564 sites (25Kharbanda S. Bharti A. Pei D. Wang J. Pandey P. Ren R. Weichselbaum R. Walsh C.T. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6898-6901Crossref PubMed Scopus (84) Google Scholar). Although the effects of c-Abl on SHPTP1 activity were not defined (25Kharbanda S. Bharti A. Pei D. Wang J. Pandey P. Ren R. Weichselbaum R. Walsh C.T. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6898-6901Crossref PubMed Scopus (84) Google Scholar), more recent studies have demonstrated that c-Abl stimulates SHPTP1 at least in vitro. 2K. Yoshida, S. Kharbanda, and D. Kufe, unpublished data. Thus, both Lyn and c-Abl are activated by DNA damage and phosphorylate SHPTP1 as a downstream effector. The present results also demonstrate that SHPTP1, in a potential feedback mechanism, down-regulates Lyn-mediated phosphorylation. The functional significance of the Lyn-SHPTP1 interaction is further supported by the findings that the activity of Lyn is responsible in part for DNA damage-induced apoptosis and that SHPTP1 attenuates the Lyn-dependent proapoptotic signals. Taken together with the previous demonstration that SHPTP1 down-regulates c-Abl-dependent activation of the stress-activated protein kinase cascade (25Kharbanda S. Bharti A. Pei D. Wang J. Pandey P. Ren R. Weichselbaum R. Walsh C.T. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6898-6901Crossref PubMed Scopus (84) Google Scholar), the present findings support a model in which activation of SHPTP1 in the response to DNA damage functions as a negative regulator of events activated by Lyn- and c-Abl-mediated phosphorylation. We thank John Combier for the GST-Lyn constructs, Tadashi Yamamoto for the Lyn cDNA, and Chris Walsh for the SHPTP1 cDNA and proteins.
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TIMP‐1, a well‐known MMP inhibitor, displays other biological activities such as cell survival, proliferation and differentiation in hematopoietic cells. In this report, we investigated the role of the Src‐related kinase Lyn in TIMP‐1 induced UT‐7 erythroleukemic cell survival. We showed that (i) tyrosine 507 of Lyn was dephosphorylated and Lyn kinase activity enhanced by TIMP‐1, (ii) Lyn silencing suppressed TIMP‐1 anti‐apoptotic activity and (iii) Lyn was activated upstream the JAK2/PI 3‐kinase/Akt pathway. Our data suggest a novel role for Lyn in erythroid cell survival.
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Colorectal cancer is the second leading cause of death from cancer in the United States. Metastases in the liver, the most common metastatic site for colorectal cancer, are found in one-third of the patients who die of colorectal cancer. Currently, the genes and molecular mechanisms that are functionally critical in modulating colorectal cancer hepatic metastasis remain unclear. Here, we report our studies using functional selection in an orthotopic mouse model of colorectal cancer to identify a set of genes that play an important role in mediating colorectal cancer liver metastasis. These genes included APOBEC3G, CD133, LIPC, and S100P. Clinically, we found these genes to be highly expressed in a cohort of human hepatic metastasis and their primary colorectal tumors, suggesting that it might be possible to use these genes to predict the likelihood of hepatic metastasis. We have further revealed what we believe to be a novel mechanism in which APOBEC3G promotes colorectal cancer hepatic metastasis through inhibition of miR-29-mediated suppression of MMP2. Together, our data elucidate key factors and mechanisms involved in colorectal cancer liver metastasis, which could be potential targets for diagnosis and treatment.
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Abstract Engagement of FcεRI causes its phosphorylation by Lyn kinase. Two alternatively spliced variants, Lyn A and B, are expressed in mast cells, and both isoforms interact with FcεRI. Unlike Lyn A, Lyn B lacks a 21-aa region in the N-terminal unique domain. In this study, we investigated the role of Lyn A and B isoforms in mast cell signaling and responses. Lyn B was found to be a poor inducer of mast cell degranulation and was less potent in both inositol 1,4,5-triphosphate production and calcium responses. Expression of Lyn B alone showed reduced phosphorylation of both phospholipase Cγ-1 and -2 and decreased interaction of phospholipase Cγ-1 with the phosphorylated linker for activation of T cells. Lyn B also showed increased binding of tyrosine-phosphorylated proteins, which included the negative regulatory lipid phosphatase SHIP-1. In contrast, both Lyn A and B caused similar total cellular tyrosine phosphorylation and FcεRI phosphorylation and neither Lyn A nor Lyn B alone could completely restore mast cell degranulation or dampen the excessive cytokine production seen in the absence of Lyn. However, expression of both isoforms showed complementation and normalized responses. These findings demonstrate that Lyn B differs from Lyn A in its association with SHIP-1 and in the regulation of calcium responses. However, complementation of both isoforms is required in mast cell activation.
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