Báo cáo khoa học: Alpha 1,3-fucosyltransferase-VII regulates the signaling molecules of the insulin receptor pathway potx

Alpha 1,3-fucosyltransferase-VII regulates the signaling molecules of the insulin receptor pathway Qiu-yan Wang1, Ying Zhang1, Hai-jiao Chen2, Zong-hou Shen1 and Hui-li Chen1 Key Laboratory of Glycoconjugate Research, Shanghai Medical College, Fudan University, Shanghai, China Department of Urology, Zhong-shan Hospital, Fudan University, Shanghai, China Keywords epidermal growth factor receptor; a1,3fucosyltransferase-VII; human hepatocarcinoma cell line; insulin receptor; signaling molecules Correspondence H Chen, Key Laboratory of Glycoconjugate Research, Ministry of Health, Department of Biochemistry, Shanghai Medical College, Fudan University, Shanghai 200032, China Fax: + 86 21 6416 4489 Tel: + 86 21 5423 7223 E-mail: hlchen@shmu.edu.cn (Received 19 July 2006, revised 13 November 2006, accepted 17 November 2006) doi:10.1111/j.1742-4658.2006.05599.x Two H7721 human hepatocarcinoma cell lines showing moderate and high expression of a1,3-fucosyltransferase (FucT)-VII cDNA were established and designated FucTVII-M and FucTVII-H, respectively In a1,3-FucTVII-transfected cells, expression of insulin receptor (InR) a- and b subunits and epidermal growth factor receptor (EGFR) on the cell surface and in cells, as well as the sialyl Lewis X (SLex, the product of a1,3-FucT-VII) content of the EGFR were unchanged However the level of SLex on the InR a subunit (InR-a) was increased dramatically Tyrosine autophosphorylation of InR-b , but not EGFR, was elevated Concomitantly, tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), Ser ⁄ Thr phosphorylation of protein kinase B (PKB; Akt), p42 ⁄ 44 mitogen-activated protein kinase (MAPK), MAPK kinase (MEK), and the protein of some other signaling molecules, such as phosphoinositide-dependent kinase-1 (PDK-1), novel protein kinase (PKN), c-Raf-1 and b-catenin were also upregulated The activities of PKB and transcription factor TCF were concomitantly stimulated Upregulation of InR signaling molecules and their phosphorylation was correlated with the level of SLex on InR-a and a1,3-FucT-VII expression in cells In addition, the phosphorylation intensity and difference in phosphorylation intensity between cells with different levels of a1,3-FucT-VII expression were attenuated significantly by the inhibitor of InR tyrosine kinase and by the mAb to SLex Furthermore, insulin-induced signaling was facilitated in a1,3-FucT-VII-transfected cells, particularly FucTVII-H These findings provide strong evidence that a1,3-FucT-VII may affect insulin signaling by upregulating the phosphorylation and expression of some signaling molecules involved in the InR-signaling pathway These effects are likely mediated by its product, SLex, on the glycans of the InR This is the first study to report that changes in the terminal structure of glycans on a surface receptor can modify cell signaling Glycosylation is important and the most common form of post-translational modification that regulates many aspects of protein function [1,2] In recent years, increased attention has been paid to the relationship between structural changes in surface glycans and surface receptor signaling It has been reported [3] that overexpression of N-acetylglucosaminyltransferase (GnT)-III introducing a bisecting N-acetylglucosamine Abbreviations CDK, cyclin-dependent kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FucT, fucosyltransferase; GlcNAc, N-acetylglucosamine; GnT, N-acetylglucosaminyltransferase; InR, insulin receptor; IRS-1, insulin receptor substrate-1; MAPK, mitogenactivated protein kinase; MEK, MAPK kinase; NGF, nerve growth factor; PDK-1, phosphoinositide-dependent kinase-1; PKB, protein kinase B (Akt); PKN, novel protein kinase; TGF, transforming growth factor 526 FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS Q Wang et al (GlcNAc) into the N-glycans of epidermal growth factor receptor (EGFR) in U373 MG glioma cells led to decreased epidermal growth factor (EGF) binding and autophosphorylation of EGFR, as well as reduced cell proliferation upon EGF stimulation It has also been reported [4] that overexpression of GnT-III in pheochromocytoma PC12 cells inhibited transient tyrosine phosphorylation and dimerization of the nerve growth factor (NGF) receptor (Trk) upon stimulation with NGF and resulted in blockage of the neurite outgrowth during differentiation We previously found [5] that transfection of the sense cDNA of GnT-V, an enzyme associated with cancer progression and metastasis, into human H7721 hepatocarcinoma cells resulted in an increase in the level of GlcNAcb1,6Mana1,6-branch (GnT-V product) on the N-glycans of EGFR; this promoted EGF binding and tyrosine autophosphorylation, but had little effect on expression of the EGFR protein The phosphorylation (at T308, S473 and tyrosine residues) and activity of protein kinase B (PKB; Akt), as well as the phosphorylation of p42 ⁄ 44 mitogen-activated protein kinase (MAPK; ERK-1 ⁄ 2) and MAPK kinase (MEK) before and after EGF stimulation, were also upregulated Conversely, in H7721 cells expressing antisense GnT-V (GnTV-AS), the results were the opposite of those seen in GnT-V sense cDNA (GnTV-S)transfected cells After GnT-V-transfected H7721 cells were treated with 1-deoxymannojirimycin, an inhibitor of N-glycan processing at the high mannose stage, or antibody against the extracellular glycan domain of EGFR, the increase in PKB activity and MAPK phosphorylation were significantly blocked, and the differences in PKB activity and MAPK phosphorylation among GnTV-S, GnTV-AS and mock-transfected cells (cells transfected with empty vector) were attenuated significantly These findings indicated that the altered signaling after GnTV-S or GnTV-AS transfection was mediated by a structural change in N-glycans on the EGFR [5] Furthermore, Guo et al [6] reported that transfection of GnT-V into human fibrosarcoma HT1080 and mouse NIH 3T3 cells to increase the GlcNAcb1,6-branch on N-cadherin inhibited signaling between N-cadherin and ERK1 ⁄ 2, and consequently reduced calcium-dependent cell–cell adhesion mediated by N-cadherin These results provide evidence that the N-cadherin signaling pathway is also influenced by the glycan structures on N-cadherin Wang et al [7] reported that in embryonic fibroblast cells deprived of a1,6fucosyltransferase (FucT-VIII), an enzyme responsible for the synthesis of core fucose on N-glycans, EGFinduced phosphorylation of EGFR and EGFRmediated JNK or ERK activation were suppressed Taniguchi [8] also discovered that signal transduction Fucosyltransferase-VII regulates insulin signaling of the transforming growth factor b1 (TGF b1) receptor was deficient in FucT-VIII knockout mice, leading to emphysema-like phenotypes in the lung These results show that the core fucose on N-glycans is essential for EGF and TGF b1 signaling However, all the above-mentioned structural changes in receptor glycans are located in the core portion of N-glycan, and whether alteration of the terminal residue on the outer chain of either N- or O-glycan can also modify surface receptor signaling remains unclear It has been documented that sialyl Lewis antigens (SLe) expressed on the surface of cancer cells, such as SLex [SAa2,3 Galb1,4 (Fuca1,3) GlcNAc-] and SLea [SAa2,3 Galb1,3 (Fuca1,4) GlcNAc-], are another kind of glycan structure involved in metastasis, and which can serve as the ligands for E- or P-selectin expressed on the surface of vascular endothelial cells and mediate the adhesion of malignant cells to vascular endothelium [9–11] The final fucosylation step in Lewis antigen synthesis is catalyzed by a1,3-fucosyltransferase (a1,3-FucT) To date, six a1,3-FucTs (III to VII and IX) have been identified Each enzyme has a unique acceptor–substrate binding pattern, and each generates a unique range of fucosylated products [12,13] Among these, a1,3-FucT-VII, which is expressed mainly in leukocytes, catalyzes sialylated substrate and produces SLex as its only product [14] SLex is always located at the terminus of sugar chains, and a1,3-FucT-VII may be considered a terminal glycosyltransferase that catalyzes the final step in sugar-chain processing In our laboratory, it has been found that the surface SLex and cellular a1,3-FucT-VII of H7721 cells is up- and downregulated by transfection of the metastasis-promoting gene c-erbB2 ⁄ neu and the metastasissuppressive gene nm23-H1, respectively [15–17] In addition, surface SLex was increased when H7721 cells were treated with proliferative inducers, and decreased after treatment with differentiative inducers [18] The change in SLex level was proportional to a1,3-FucTVII expression Moreover, the ex vivo metastatic potential was positively correlated with surface SLex and cellular a1,3-FucT-VII levels, and could be inhibited by a mAb (KM93) against surface SLex [15,18] Further studies have shown that insulin also enhanced expression of SLex and a1,3-FucT-VII and the metastatic potential of H7721 cells [19] In addition, our group recently found that expression of cyclin-dependent kinase (CDK) inhibitor, p27Kip1 protein, was decreased in H7721 cells transfected with a1,3-FucTVII cDNA Uninhibited CDK2 resulted from a reduction in the p27Kip1-stimulated phosphorylation of retinoblastoma protein, facilitating G1 ⁄ S transition and increasing the growth rate in the cells These effects FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS 527 Fucosyltransferase-VII regulates insulin signaling Q Wang et al were correlated with an increase in surface SLex on H7721 cells expressing different a1,3-FucT-VII intensities, and could be blocked by SLex antibody in a dosedependent manner, indicating that p27Kip1 expression was influenced by a1,3-FucT-VII and its product SLex [20] Therefore, it is interesting to study whether transfection of a1,3-FucT-VII can also affect the function of some other surface receptors and subsequently result in altered receptor signaling Insulin receptor (InR) was selected to study the effects of a1,3FucT-VII on its expression, SLex content and tyrosine autophosphorylation InR contains two extracellular carbohydrate-containing a subunits and two b subunits with cytoplasmic tyrosine kinase activity [21] InR results were compared with those from EGFR, which also contains N-glycans on its extracellular domain and tyrosine autophosphorylation sites at its intracellular domain [22] Furthermore, insulin receptor substrate-1 (IRS-1), PKB, phosphoinositidedependent kinase-1 (PDK-1), novel protein kinase (PKN), p42 ⁄ 44 MAPK and MEK were analyzed as the signaling molecules involved in InR signaling [19,23] Expression of b catenin and its downstream transcription factor TCF in the Wnt signaling pathway [24], which cross-talks with the InR pathway was Mock FucTVII-M 497 bp β-actin As shown in Fig 1A,B, a1,3-FucT-VII mRNA was increased significantly in H7721 cells transfected with a1,3-FucT-VII cDNA In FucTVII-M (moderate expression) and FucTVII-H (high expression) cells, it was upregulated to 373.3 and 613.3% of the mocktransfected cell level, respectively (both P < 0.01) Consequently, expression of SLex, the product of a1,3FucT-VII, was also elevated on the cell surface, to 171 and 284% of the mock-transfection value in FucTVII-M and FucTVII-H cells, respectively (both P < 0.01; Fig 1C,D) Expression of InR-a, EGFR and their SLex in a1,3-FucT-VII-transfected H7721 cells Expression of cell-surface InR-a and EGFR were analyzed using specific antibodies and flow cytometry The 1.2 Mock * FucTVII-M FucTVII-H 0.8 * 0.4 Counts Counts 200 200 789 bp Mock M1 D 0 M1 100 101 102 103 FL1-H Mock Counts Counts 200 200 ( - ) Control M1 FucTVII-H 160 * 120 * 80 40 0 M1 FucTVII-M 104 Expression of Slex 100 101 102 103 104 FL1-H 100 Characterization of two a1,3-FucT-VII-transfected cell lines B FucTVII-H FucT-VII C Results Fuc T -V I I / be t a -a c t in A also studied Mock cells transfected with the vector pcDNA3.1 were used as controls 101 102 103 FL1-H FucTVII-M 104 100 101 102 103 FL1-H FucTVII-H 104 Mock FucTVII-M FucTVII-H Fig Characterization of a1,3-FucT-VII cDNA-transfected H7721 cells (A) RT-PCR profiles of a1,3-FucT-VII mRNA in mock- and a1,3-FucTVII-transfected cells (B) Relative expressions of a1,3-FucT-VII mRNA in mock- and a1,3-FucT-VII transfected cells (n ¼ 3) (C) Fluorescenceactivated cell spectra of cell-surface SLex on mock- and a1,3-FucT-VII transfected cell lines (D) Relative expressions of surface SLex on mock- and a1,3-FucT-VII transfected cells (n ¼ 3) Mock, H7721 cells transfected with pcDNA3.1 vector; FucTVII-M, H7721 cell line with moderate expression of transfected a1,3-FucT-VII; FucTVII-H, H7721 cell line with high expression of transfected a1,3-FucT-VII (A) and (C) are representative of three reproducible experiments *P < 0.01 compared with ‘Mock’ RT-PCR and flow cytometry are described in the Experimental procedures 528 FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS Fucosyltransferase-VII regulates insulin signaling B 150 M1 0 M1 101 102 103 FL1-H 100 104 200 Counts 200 M1 10 10 10 FL1-H 10 10 10 10 10 FL1-H 101 102 103 FL1-H 104 Mock-EGFR C 60 30 10 10 FL1-H 100 101 102 103 FL1-H 104 10 D FucTVII-M 101 102 103 FL1-H Mock FucTVII-M FucTVII-H IP: InR-α WB: SLeX M1 100 EGFR M1 FucTVII-M-EGFR Mock 10 Counts Counts 200 100 FucTVII-H-InR Counts M1 90 InR 10 10 FucTVII-M-InR 200 Mock-InR 200 10 M1 120 Mock FucTVII-M FucTVII-H 0 M1 104 102 103 FL1-H ( - ) Control-EGFR Counts Counts 200 ( - ) Control-InR 101 104 FucTVII-H-EGFR IP: InR-α WB: InR-α IP: EGFR WB: SLeX IP: EGFR WB: EGFR E FucTVII-H Relative expression 100 Mean fluorescence intensity Counts A Counts 200 200 Q Wang et al InR α EGFR β-actin 500 450 400 350 300 250 200 150 100 50 Mock FucTVII-M FucTVII-H InR-α InR-α SLeX EGFR EGFR SLeX Fig Effects of a1,3-FucT-VII transfection on expression of InR-a and EGFR and the SLex content of the glycans of InR-a and EGFR (A) Fluorescence-activated cell spectra of InR-a and EGFR on the cell surface (B) Relative expression of surface InR-a and EGFR (n ¼ 3) (C) Western immunoblot profiles of InR-a and EGFR after staining with an antibody to InR-a or EGFR and horseradish peroxidase-labeled secondary antibody (D) Western immunoblot profiles of immunoprecipited InR-a and EGFR (precipitated by CF4) after staining with antibody to SLex (KM93), InR-a or EGFR (antibody 528) and horseradish peroxidase-labeled secondary antibody to determine the SLex content of InR-a and EGFR (E) Densitometric quantification of (D) (n ¼ 3) Mock, H7721 cells transfected with pcDNA3.1 vector; FucTVII-M, H7721 cell line with moderate expression of transfected a1,3-FucT-VII; FucTVII-H, H7721 cell line with high expression of transfected a1,3-FucT-VII; InR, insulin receptor a-subunit; EGFR: epidermal growth factor receptor; IP, immunoprecipitation by the antibody to the protein indicated at the right; WB, western immunoblot with the antibody to the compound indicated at the right (A), (C) and (D) are representative of three reproducible experiments *P < 0.01 compared with ‘Mock’ Flow cytometry, immunoprecipitation and western immunoblot are described in the Experimental procedures results in Fig 2A,B show that their expression was not obviously changed in a1,3-FucT-VII-transfected cells compared with mock-transfected cells Results from western immunoblots indicated that protein expression in InR-a and EGFR was also unchanged following transfection with a1,3-FucT-VII (Fig 2C) However, after immunoprecipitation and western blotting of these receptors, and using KM93 as the probe for SLex, it was found that expression of SLex on InR-a of FucTVII-M and FucTVII-H cells was increased to 248 and 409% of the mock-transfection level, respectively (both P < 0.01), whereas expression of SLex on EGFR remained unchanged (Fig 2D,E) Tyrosine phosphorylation of InR-b, EGFR and IRS-1 in a1,3-FucT-VII-transfected H7721 cells As shown in Fig 3A,B, the amount of immunoprecipitated InR-b was also unchanged following a1,3-FucT-VII transfection The relative intensity of tyrosine autophosphorylation in immunoprecipatated InR-b or EGFR was calculated from the intensity ratio of the phosphorylated band to the unphosphorylated band Figure 3B also shows that tyrosine autophosphorylation of InR-b was increased to 186 and 352% of the mock-transfection value in FucTVII-M and FucTVII-H cells, respectively FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS 529 Fucosyltransferase-VII regulates insulin signaling A Mock FucTVII-M Q Wang et al FucTVII-H B 450 Mock FucTVII-M FucTVII-H 400 Relative intensity IP: InR-β WB: PT66 IP: InR-β WB: InR-β IP: EGFR WB: PT66 350 300 250 200 150 100 50 IP: EGFR WB: EGFR D InR-β 600 C Mock FucTVII-M FucTVII-H IP: IRS-1 WB: PT66 Relative intensity 500 InR-β-Tyr-p EGFR EGFR-Tyr-p Mock FucTVII-M FucTVII-H 400 300 200 100 IP: IRS-1 WB: IRS-1 IRS-1 IRS-Tyr p Fig Effects of a1,3-FucT-VII transfection on the tyrosine autophosphorylation of two receptors and of IRS-1 (A) Western immunoblot profiles of immunoprecipitated InR-b and EGFR (precipitated by CF4) after staining with antibody to phosphotyrosine (PT66), InR-b or EGFR (CF4) and horseradish peroxidase-labeled secondary antibody (B) Densitometric quantification of (A) (n ¼ 3) (C) Western immunoblot profiles of immunoprecipitated IRS-1 after staining with phosphotyrosine antibody (PT66), IRS-1 antibody and horseradish peroxidase-labeled secondary antibody (D) Densitometric quantification of (C) (n ¼ 3) Mock, H7721 cells transfected with pcDNA3.1 vector; FucTVII-M, H7721 cell line with moderate expression of transfected a1,3-FucT-VII; FucTVII-H, H7721 cell line with high expression of transfected a1,3-FucT-VII; WB, western immunoblot with the antibody to phosphotyrosine (PT66) or to the protein indicated on the right; IRS-1, insulin receptor substrate-1; Tyr-p, tyrosine phosphorylated (A) and (C) are representative of three reproducible experiments *P < 0.01 compared with ‘Mock’ Immunoprecipitation and western immunoblotting are described in the Experimental procedures (both P < 0.01), whereas that of EGFR was unchanged Tyrosine phosphorylation of IRS-1 occurs earlier in insulin signaling The IRS-1 protein was decreased in a1,3-FucT-VII-transfected cells, although the change was not statistically significant When relative tyrosine phosphorylation was calculated as above, it was found that the level of phosphorylated IRS-1 was increased to 2.8 and 8.5 times that of the mock-transfection value in FucTVII-M and FucTVII-H cells, respectively (both P < 0.01; Fig 3C,D) Phosphorylation and activity of PKB, expression of PDK-1, PKN and phospho-PKN in a1,3-FucT-VII-transfected H7721 cells In insulin signaling, activation of PKB has been implicated as a key step and it also has a major role in the physiological effects of insulin [25] As shown in Fig 4A,B, expression of PKB protein was not obviously altered in a1,3-FucT-VII-transfected H7721 cells, but relative phosphorylation at T308 and S473 in PKB (calculated from the ratio of the staining intensity of phosphorylated protein to unphosphorylated protein after normalization with b-actin) was apparently elevated when compared with mock-transfected cells After densitometric quantification, relative T308 phosphorylation was 149 and 205% of the mock-transfection 530 level in FucTVII-M and FucTVII-H cells, respectively (both P < 0.01) Meanwhile, relative S473 phosphorylation was 170 and 315% of the mock-transfection value, respectively (both P < 0.01) Increased phosphorylation of PKB at both T308 and S473 resulted in an upregulation of PKB activity, measured as the amount of phosphorylated GSK3a ⁄ b product As indicated in Fig 4C,D, phosphorylated GSK3a ⁄ b was elevated to 165 and 270% of the mock-transfected value in FucTVII-M and FucTVII-H cells, respectively (both P < 0.01) PDK-1 is the enzyme responsible for PKB phosphorytion [26], and PKN is another substrate of PDK-1 related to cytoskeleton and transcription factor [27] Figure 4E,F shows that the PDK-1 protein was upregulated to 162 and 198% of the control value in FucTVIIM and FucTVII-H cells, respectively (both P < 0.01) Protein expression of PKN and Ser ⁄ Thr phosphorylation of PKN (p-PKN) were also increased to a similar degree Therefore, the relative phosphorylation of p-PKN (pPKN ⁄ PKN protein) was generally unchanged Expression and phosphorylation of c-Raf-1, MEK and p42 ⁄ 44 MAPK in a1,3-FucT-VII-transfected H7721 cells The Ras–Raf–MEK–MAPK pathway is another important signaling pathway in addition to the FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS Q Wang et al Fucosyltransferase-VII regulates insulin signaling FucTVII-M FucTVII-H PKB-T308 PKB-S473 PKB β-actin D 400 350 300 Relative PKB kinase activity Mock β-actin 200 150 100 50 E PKB-T308p Mock PKB-S473p FucTVII-M PDK-1 200 PKN 100 p-PKN β-actin Mock FucTVII-M FucTVII-M FucTVII-H Phosphorylated GSK3 α/β 250 400 300 C Mock FucTVII-M FucTVII-H FucTVII-H F Relative intensity B Mock Relative phosphorylation of PKB A 600 500 400 300 200 100 FucTVII-H Mock FucT-VII-M FucT-VII-H PDK-1 PKN p-PKN Fig Effects of a1,3-FucT-VII transfection on the phosphorylation, protein expression and activity of some signaling molecules (A) Western blot profiles of PKB and T308-, S473-phosphorylated PKB after staining with specific antibodies and horseradish peroxidase-labeled secondary antibody (B) Densitometric quantification of (A) (n ¼ 3) (C) Determination of PKB activity as the amount of phosphorylated GSK3a ⁄ b product (D) Densitometric quantification of (C) (n ¼ 3) (E) Western immunoblot profiles of PDK-1, PKN and p-PKN after staining with specific antibodies and horseradish peroxidase-labeled secondary antibody (F) Densitometric quantification of E (n ¼ 3) Mock, H7721 cells transfected with pcDNA3.1 vector; FucTVII-M, H7721 cell line with moderate expression of transfected a1,3-FucT-VII; FucTVII-H, H7721 cell line with high expression of transfected a1,3-FucT-VII; PKB-T308p, phosphorylated PKB at T308; PKB-S473p, phosphorylated PKB at S473 (A), (C) and (E) are representative of three reproducible experiments *P < 0.01 compared with ‘Mock’ Western blotting, western immunoblotting and assay of PKB activity are described in the Experimental procedures PDK-1 ⁄ PKB pathway in the insulin receptor [23] Figure 5A,B shows that expression of MEK and p42 ⁄ 44 MAPK proteins was not apparently altered in a1, 3-FucT-VII-transfected H7721 cells However, expression of c-Raf-1 increased significantly following a1,3FucT-VII transfection, being 168 and 325% of the mock-transfection value in FucTVII-M and FucTVIIH cells, respectively (both P < 0.01) Moreover, the relative phosphorylation of MEK, as determined by the ratio of p-MEK to MEK, was upregulated to 207 and 425% in FucTVII-M and FucTVII-H cells, respectively (both P < 0.01), and the relative phosphorylation of p42 ⁄ 44 MAPK (the ratio of p-p42 ⁄ 44 MAPK to p42 ⁄ 44 MAPK) was also increased in FucTVII-M and FucTVII-H cells, being 2.82 and 6.01 times the mock-transfection value (both P < 0.01) Effect of HNMPA-(AM)3 and KM93 on the phosphorylation of PKB and p42/44 MAPK In order to study whether the alteration in the phosphorylation of PKB and p42 ⁄ 44 MAPK was mediated by InR kinase and surface SLex, phosphorylation of these two signaling molecules was determined before and after cultured cells were treated with 50 lm HNMPA-(AM)3 (a specific inhibitor of InR tyrosine kinase) [28] or 30 lgỈmL)1 KM93 (SLex antibody) for 24 h; corresponding untreated cells were used as the control It was found that the results from the untreated cells were the same as those shown in Figs and When H7721 cells were treated with HNMPA-(AM)3, phosphorylation of PKB at both T308 and S473, and of p42 ⁄ 44 MAPK was apparently decreased in mock- and a1,3-FucT-VII- transfected cells (Fig 6A) The decrease in phosphorylation of PKB and p42 ⁄ 44 MAPK was ~ 40.9–76.5% (P < 0.01) in a1,3-FucT-VII-transfected cells, compared with the corresponding untreated cells (Fig 6B) By contrast, differences in phosphorylation intensity for PKB and MAPK among mock, FucTVIIM and FucTVII-H cell groups were attenuated in HNMPA-(AM)3-treated cells (Fig A,B) Similarly, in the presence of KM93, phosphorylation of both PKB and p42 ⁄ 44 MAPK, and the differences in their phosphorylation intensities among the three cell lines were also decreased significantly (Fig 6C,D) The reduction in phosphorylation of PKB and p42 ⁄ 44 MAPK in a1,3-FucT-VII-transfected cells was  41.1–89.7% (P < 0.01) In the presence of HNMPA-(AM)3 or KM93, the rate of inhibition of phosphorylation was correlated with expression of a1,3-FucT-VII, which was FucTVII-H > FucTVII-H > mock-transfected cells However, some differences in the phosphorylation intensities of PKB and MAPK were observed in mockand a1,3-FucT-VII-transfected cells in the presence of both inhibitors, but the differences were either not statistically significant or P < 0.05 FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS 531 Fucosyltransferase-VII regulates insulin signaling A Mock FucTVII-M Q Wang et al FucTVII-H Modification of insulin signaling in a1,3-FucT-VII-transfected cells c-Raf p-MEK MEK p-p42/44 MAPK p42/44 MAPK β-actin Relative intensity B 800 Mock FucTVII-M 600 FucTVII-H 400 200 c-Raf MEK p42/44 MAPK p-MEK p-p42/44 MAPK Fig Effects of a1,3-FucT-VII transfection on the expression of c-Raf-1 and phosphorylation of MEK and p42 ⁄ 44 MAPK (A) Western immunoblot profiles of c-Raf-1, p-MEK, MEK, p-p42 ⁄ 44 MAPK and p42 ⁄ 44 MAPK after staining with specific antibodies and horseradish peroxidase-labeled second antibody (B) Densitometric quantification of (A) (n ¼ 3) Mock, H7721 cells transfected with pcDNA3.1 vector; FucTVII-M, H7721 cell line with moderate expression of transfected a1,3-FucT-VII; FucTVII-H, H7721 cell line with high expression of transfected a1,3-FucT-VII; p-MEK, phosphorylated MEK; p-p42 ⁄ 44 MAPK, phosphorylated p42 ⁄ 44 MAPK (A) is representative of three reproducible experiments *P < 0.01 compared with ‘Mock’ Western immunoblotting is described in the Experimental procedures Expression of b-catenin and TCF in a1,3-FucT-VII-transfected H7721 cells Beta-catenin is a substrate of GSK-3 and a key molecule in the Wnt and TGF-b signaling pathways [29– 31] As shown in Fig 7A,B, the level of b-catenin was upregulated in a1,3-FucT-VII-transfected cells, to 242% in FucTVII-M cells and 504% in FucTVII-H cells (both P < 0.01) Luciferase activity was measured as an indicator of the activity of transcription factor TCF As shown in Fig 7C, TCF activity was also increased in FucTVIIM and FucTVII-H cells, being 239 and 333% of the mock-transfection level, respectively (both P < 0.01) 532 The effect of the above-mentioned changes in insulinsignaling molecules on transduction of the insulin signal was further studied in a1,3-FucT-VII-transfected cells following serum starvation Phosphorylation at T308 and S473 of PKB and p42 ⁄ 44 MAPK was also selected as an indicator of signaling efficiency It was found that phospho-PKB-S473 was barely seen in insulin-untreated and serum-starved cells, but was expressed in insulin-treated cells By contrast, phosphoPKB-T308 and phospho-p42 ⁄ 44 MAPK were expressed in both insulin-untreated and insulin-treated cells The intensity levels for phospho-PKB-T308 and phospho-MAPK in both insulin-untreated and -treated cells, as well as the phospho-PKB-S473 in insulin-treated cells, were FucTVII-H > FucTVII-M > mock (Fig 8A) In the presence of insulin, phosphorylation of PKB and MAPK was obviously upregulated, and was significantly higher than in the corresponding control cells cultured in the absence of insulin The response to insulin stimulation was proportional to the expression of a1,3-FucT-VII In insulin-stimulated FucTVII-M and FucTVII-H cells, phospho-PKB-T308 was upregulated to 215 and 398% of the mock-transfction level (both P < 0.01), and phospho-PKB-S473 was upregulated to 192 and 354% of the mock-transfection level, respectively (both P < 0.01) Similarly, phospho-MAPK was 184 and 345% of the mocktransfection level, respectively (both P < 0.01) (Fig 8B) Discussion The results shown in Fig 1A,B indicate that two a1,3FucT-VII-transfected cell lines were established with moderate and high expression of the exogenous cDNA Expression of SLex, the product of a1,3-FucTVII, was positively correlated with expression of a1,3FucT-VII mRNA (Fig 1C,D) After transfection of the a1,3-FucT-VII cDNA, protein expression of InR (including the a- and b-subunits) and EGFR both on the cell surface and in cells (Fig 2A,C), as well as the SLex content of the glycans of EGFR were unchanged, but the SLex content of the glycans of InR-a was increased significantly (Fig 2D,E) If the SLex of InR-a glycans is compared with that of EGFR in mock-transfected cells, it is observed that the SLex content of EGFR is far greater than that of InR-a (Fig 2D), suggesting that the SLex on EGFR is high enough and cannot be upregulated further by overexpression of a1,3-FucT-VII This may FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS Q Wang et al Fucosyltransferase-VII regulates insulin signaling Mock-1 FucTVII-M FucTVII-H A Mock-2 FucTVII-M FucTVII-H B PKB-T308p PKB-S473p * 700 600 (+) HNMPA-(AM)3 Relative intensity ( ) HNMPA-(AM)3 500 PKB-T308 PKB-S473 p-p42/44MAPK 400 300 * 200 * * * * # # # # # 100 p-p42/44 MAPK Mock-1 β-actin Mock-1 FucTVII-M FucTVII-H ( ) KM93 Mock-2 FucTVII-M FucTVII-H D PKB-T308p PKB-S473p p-p42/44 MAPK β-actin 800 700 (+) KM93 Relative intensity C FucTVII-M FucTVII-H Without HNMPA-(AM)3 600 PKB-T308p PKB-S473p p-p42/44MAPK FucTVII-M FucTVII-H Mock-2 With HNMPA-(AM)3 * 500 400 300 ** 200 * * * # # # # 100 Mock-1 FucTVII-M FucTVII-H Without KM93 Mock-2 FucTVII-M FucTVII-H With KM93 Fig Effect of HNMPA-(AM)3 and KM93 on the phosphorylation of PKB and p42 ⁄ 44 MAPK (A) Western immunoblot profiles of phosphorylated PKB and p42 ⁄ 44 MAPK in the absence and presence of HNMPA-(AM)3 (B) Densitometric quantification of (A) (n ¼ 3) (C) Western immunoblot profiles of phosphorylated PKB and p42 ⁄ 44 MAPK in the absence and presence of KM93 (D) Densitometric quantification of (C) (n ¼ 3) Mock, H7721 cells transfected with pcDNA3.1 vector; FucTVII-M, H7721 cell line with moderate expression of transfected a1,3FucT-VII; FucTVII-H, H7721 cell line with high expression of transfected a1,3-FucT-VII; PKB-T308p, phosphorylated PKB at T308; PKB-S473p, phosphorylated PKB at S473; p-p42 ⁄ 44 MAPK, phosphorylated p42 ⁄ 44 MAPK; Mock-1, mock cells without HNMPA-(AM)3 or KM93 treatment; Mock-2, mock cells with HNMPA-(AM)3 or KM93 treatment (A) and (C) are representative of three reproducible experiments *P < 0.01 compared with ‘Mock-1’ #P < 0.05 compared with ‘Mock-2 Western immunoblotting is described in the Experimental procedures Samples without and with HNMPA-(AM)3 or KM93 were examined simultaneously on the same electrophoresis gel be one reasons why a1,3-FucT-VII did not increase the amount of SLex on EGFR Alternatively, the composition and structure of EGFR glycans probably differ from those of InR-a, and the EGFR glycans are not suitable substrates for fucosylation by exogenous a1,3-FucT-VII In other words, SLex on EGFR is probably not synthesized by a1,3-FucT-VII, but by other a1,3-FucTs Our findings showed that transfection of a1,3-FucTVII promoted the functional activity of InR, as verified by increased tyrosine phosphorylation of InR-b and IRS-1 (Fig 3) Moreover, Ser ⁄ Thr phosphorylation of InR signaling molecules, including PKB (Fig 4A,B), MEK, p42 ⁄ 44 MAPK (Fig 5A,B) and the activity of PKB (Fig 4C,D) was stimulated concomitantly Expression of some other signaling proteins, such as PDK-1, PKN (Fig 4E,F), c-Raf-1 (Fig 5A,B) and b-catenin (Fig 7A,B), was also upregulated by a1,3FucT-VII Elevation of Ser ⁄ Thr phosphorylation in downstream signaling molecules was presumed to be mediated by increased tyrosine phosphorylation of InR and IRS-1; the latter resulting from the increased SLex content of InR-a This speculation was evidenced by the following First, the intensity of Ser ⁄ Thr phos- phorylation in downstream signaling molecules was positively correlated with the intensity of tyrosine phosphorylation in InR and IRS-1, and tyrosine phosphorylation was proportional to the SLex content of InR-a and also the mRNA expression of a-1,3-FucTVII in mock, FucTVII-M and FucTVII-H cells Second, inhibition of InR tyrosine autophosphorylation by HNMPA-(AM)3, which inhibits EGFR tyrosine kinase slightly [28], led to a dramatic reduction in the Ser ⁄ Thr phosphorylation of PKB and p42 ⁄ 44 MAPK, and obvious attenuation of the difference in phosphorylation intensity among three cell lines with different a1,3-FucT-VII expression levels (Fig 6A) Third, blockage of cell surface SLex by KM93 also resulted in significant attenuation of the phosphorylation of PKB and p42 ⁄ 44 MAPK, as well as the difference in phosphorylation intensity among three cell lines (Fig 6B) However, in the presence of HNMPA-(AM)3 and KM93, some differences in the phosphorylation intensities of PKB and p42 ⁄ 44 MAPK were still observed in mock- and a1,3-FucT-VII-transfected cells, indicating that the SLex on InR contributes a large proportion, though not all, of the increased phosphorylation caused by the overexpression of a1,3-FucT-VII It FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS 533 Fucosyltransferase-VII regulates insulin signaling A Mock FucTVII-M Q Wang et al A FucTVII-H Mock FT7M FT7H Mock FT7M FT7H ( ) Insulin β-Catenin PKB-T308p β-actin PKB-S473p Relative intensity B 600 * p-p42/44 MAPK 400 β-actin * 200 B FucTVII-H Relative intensity FucTVII-M C 30 * 25 Luciferase activity 450 * 400 Mock 20 * 15 350 Mock FT7-M FT7-H * * 300 250 * * * PKB-S473p p-p42/44 MAPK 200 150 100 50 10 PKB-T308p Mock FucTVII-M FucTVII-H Fig Effects of a1,3-FucT-VII cDNA transfection on the expression of b-catenin and TCF activity (A) Western blot profile of b-catenin (B) Densitometric quantification of (A) (n ¼ 3) (C) Transactivation activity of TCF measured as luciferase activity (n ¼ 3) Mock, H7721 cells transfected with pcDNA3.1 vector; FucTVII-M, H7721 cell line with moderate expression of transfected a1,3-FucTVII; FucTVII-H, H7721 cell line with high expression of transfected a1,3-FucT-VII; TCF, T-cell factor (transcription factor) (A) is representative of three reproducible experiments *P < 0.01 compared with ‘Mock’ Western blotting and luciferase assay are described in the Experimental procedures appears that upregulation of phosphorylation and protein expression was not mediated by EGFR, because the SLex content and tyrosine autophosphorylation of EGFR remained constant following a1,3-FucT-VII transfection In a previous insulin stimulation experiment, it was found that H7721 cells were prone to die in serumfree (0%) medium; therefore, 2% fetal bovine serumdeficient medium was used The results showed that phospho-PKB-S473 barely appeared in cells cultured in the serum-deficient medium (Fig 8A) As shown in Fig 4A, however, there was basal expression of both phospho-PKB-T308 and phospho-PKB-S473 in 534 (+) Insulin Fig Facilitation of insulin signaling in a1,3-FucT-VII-transfected cells (A) Western profiles of phosphorylated PKB and p22 ⁄ 24 MAPK in insulin-untreated and -treated cells cultured in 2% fetal bovine serum medium (B) Quantification of phosphorylated PKB and p22 ⁄ 24 MAPK in the presence of insulin (n ¼ 3) Mock, H7721 cells transfected with pcDNA3.1 vector; FucTVII-M, H7721 cell line with moderate expression of transfected a1,3-FucT-VII; FucTVII-H, H7721 cell line with high expression of transfected a1,3-FucT-VII; PKB-T308p, phosphorylated PKB at T308; PKB-S473p, phosphorylated PKB at S473; p-p42 ⁄ 44 MAPK, phosphorylated p42 ⁄ 44 MAPK; FT7M, H7721 cell line with moderate expression of transfected a1,3-FucT-VII; FT7H, H7721 cell line with high expression of transfected a1,3-FucT-VII (A) is representative of three reproducible experiments *P < 0.01 compared with ‘Mock’ Cell culture, insulin treatment and western immunoblotting are described in the Experimental procedures Samples without and with insulin treatment were examined simultaneously on the same electrophoresis gel mock- and a1,3-FucT-VII-transfected cells These observations suggest that phospho-PKB-T308 and phospho-PKB-S473 are regulated by different mechanisms It has been documented that phosphoPKB-T308 is regulated by phosphatidyl inositol-3kinase ⁄ PDK-1 [25,26], but the signal for PKB-S473 phosphorylation comes from the integrin ⁄ integrinlinked kinase signaling pathway [32] Sarbassov et al reported that PKB-S473 can be phosphorylated directly by a kinase, named target of rapamycin (TOR) kinase and its associated protein rictor, FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS Q Wang et al because a reduction in rictor or mammalian TOR (mTOR) expression inhibited the signaling of PKB Rictor–mTOR complex can also facilitate the phosphorylation of PKB-T308 by PDK-1 [33] Basal expression of phospho-PKB-S473 in serum-containing medium may result from the stimulation of insulin or growth factors in the 10% fetal bovine serum Our finding that the cell response to insulin was correlated with expression of a1,3-FucT-VII and SLex (Fig 8) reveals that insulin signaling was facilitated in a1,3-FucT-VII-transfected cells Expression of PDK-1, PKN, c-Raf-1 and b-catenin was upregulated after a1,3-FucT-VII transfection The mechanism is not well understood However, it is reasonable to speculate that upregulation may be caused by the promotion of InR signaling, because activated PKB can stimulate the phosphorylation of GSK-3 and inhibit the activity of GSK-3 GSK-3 phosphorylates b-catenin and stimulates the ubiquitination and proteasomal proteolysis of b-catenin During activation of the InR pathway, GSK-3 is inactivated, which leads to the accumulation and nuclear translocation of cytoplasmic unphosphorylated b-catenin In nuclei, b-catenin binds to transcription factor TCF (T-cell factor, also called LEF, leukocyte enhancer factor) to form a heterodimer, and transactivates the transcription of target genes [29–31] This model of mechanism is supported by the increased activity of TCF in this study (Fig 7C) Recently, we discovered that a1,3-FucT-VII can upregulate expression of the integrin-a5 subunit at both the mRNA and protein levels (unpublished) The latter finding supports the suggestion that transfection of a1,3-FucT-VII might affect the transcription of some genes It would be better to use antisense a1,3-FucT-VII, iRNA or a gene-knockout method to suppress endogenous a1,3-FucT-VII to confirm the above results Unfortunately, we found that suppression of a1,3-FucT-VII expression after transfection of antisense a1,3-FucT-VII cDNA was not apparent, because parent H7721 cells express a low level of endogenous a-1,3-FucT-VII Sometimes antisense cDNA even led to cell death When a gene of a1,3FucT-VII was knocked-out, almost all cells died within 24 h This suggests a1,3FucT-VII is essential for the survival of H7721 cells Therefore, construction of a plasmid containing a mutant at the catalytic domain of a1,3-FucT-VII with deletion of enzyme activity of its coding protein is very critical if we are to determine whether the changed phosphorylation of signaling molecules was mediated by the altered amount of SLex on InR This is being investigated in our laboratory Fucosyltransferase-VII regulates insulin signaling It would be of interest to study whether the SLex level of InR on insulin-responsive cells in diabetic patients was changed This may reveal the role of the sugar chains on InR in the pathogenesis of diabetes In summary, the cDNA of a1,3-FucT-VII is able to regulate the phosphorylation and expression of some signaling molecules in the InR pathway, and these effects of a1,3-FucT-VII are probably mediated by its product, SLex, on the glycans of cell-surface receptors Increased expression and phosphorylation of insulinsignaling molecules leads to the facilitation of insulin signaling These findings provide evidence that modification of the terminal structure of glycans on surface receptors may also affect cell signaling The detailed mechanism requires further study Experimental procedures Materials H7721 human hepatocarcinoma cell line was obtained from the Institute of Cell Biology RPMI-1640 and liposome LipofectamineTM were purchased from Gibco ⁄ BRL (Rockville, MD) Rabbit polyclonal antibodies against human insulin receptor a- and b-subunit, IRS-1, Raf-1, PDK-1, PKN, b-catenin and mouse mAb 528 (against human EGFR extracellular domain), b-actin and Protein G plusagarose were from Santa Cruz Technology (Santa Cruz, CA) Rabbit polyclonal antibodies against human PKB, phospho-PKB, phospho-PKN, MEK1 ⁄ 2, phosphoMEK1 ⁄ 2, p42 ⁄ 44 MAPK, and mAb against phosphop42 ⁄ 44 MAPK were from Cell Signaling Technology (Beverly, MA) The PKB assay kit was from New England Biolabs (NEB Ltd., Beijing, China) KM93 was from Seikagaku Co (Tokyo, Japan) The InR kinase inhibitor, HNMPA-(AM)3, [28] was from Calbiochem (San Diego, CA) Polyvinylidene difluoride membrane was from BioRad Laboratories (Hercules, CA) Phosphotyrosine antibody (PT66), Mes, Hepes, leupeptin, pepstatin, human EGFR mAb CF4 (against intracellular domain), fluorescein isothiocyanate-conjugated and horseradish peroxidase-labeled secondary antibodies (goat anti-mouse and anti-rabbit IgG) were from Sigma (St Luois, MO) Trizol, AMV reverse transcriptase, transcription factor TCF analysis kit (Dual-luciferaseR reporter assay system) and Renilla luciferase reporter plasmid (pRL-TK) were from Promega (Madison, WI) The TCF reporter plasmid (TK-luciferase reporter) was the product of Upstate Biotechnology (Lake Placid, NY) The RT-PCR primer of a1,3-FucT-VII was provided by TaKaRa Co (Tokyo, Japan) Other reagents were commercially available in China a1,3-FucT-VII transfected human hepatocarcinoma H7721 cell lines were established as previously reported [15] FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS 535 Fucosyltransferase-VII regulates insulin signaling Q Wang et al Cell culture and treatment Immunoprecipitation of InR-a, -b, EGFR and IRS-1 Cells were cultured at 37 °C, 5%CO2 in RPMI-1640 containing 10% fetal bovine serum, penicillin and streptomycin as described previously [5,20] In the treatment of InR kinase inhibitor and SLex mAb, the final concentration of HNMPA-(AM)3, and KM93 were 50 lm and 30 lgỈmL)1, respectively The duration of treatment was 24 h In the experiments using insulin, the cells were precultured in 2% fetal bovine serum (serum deficiency) medium for 24 h, and then 10 nm of insulin was added for 10 incubation Washed monolayer cells were lyzed with 200 lL lysis buffer (50 mm pH 7.4 Hepes, 150 mm NaCl, 100 mm NaF, mm MgCl2, 1.5 mm EGTA, 1% Nonidet P-40, mm phenykmethylsulfonyl fluoride and mg % of leupeptin and pepstatin) After protein determination, cell lysate containing 500 lg protein was incubated with lg of one of the following antibodies (antibody to InR-a or InR-b, IRS-1, CF4 antibody against EGFR intracellular domain), and incubated at °C for h Protein G plus-agarose was added and the samples were incubated at °C for h for immunoprecipitation Determination of a1,3 FucT-VII mRNAs with RT-PCR Total-cell RNA was extracted with Trizol and the cDNA was synthesized with oligo(dT)18 primer and AMV reverse transcriptase from lg total RNA The RT-PCR was performed in 50 lL of reaction mixture containing lL cDNA, 0.2 lm of the primer pair of a1,3FucT-VII or b-actin (internal standard), 0.2 m d-NTP and 1unit Taq DNA polymeraseas described previously [34] The primer for a1,3FucT-VII was F: 5¢-CACCTCCGAGGCATCTTC AACTG-3¢, R: 5¢-CGTTGGTATCGGCTC TCATTCA TG-3¢ The primer for b-actin was F: 5¢-GATATCGCC GCGCTCGTCGTCGAC-3¢, R: 5¢-CAGGAAGGAAGG CTGGAAGAGTGC-3¢ [35] The cDNA was subjected to denaturation at 94 °C for min, followed by 28 cycles (94 °C, 61.5 °C and 72 °C, for each) of PCR, and incubated at 72 °C for 10 and °C for Then 10 lL products were applied to agarose gel electrophoresis The amplified DNA bands were scanned and analyzed with nih image software The quantitative data were obtained by the intensity ratios of a1,3FucT-VII ⁄ b-actin band Detection of the expression of SLex, InR-a subunit and EGFR on the cell surface using flow cytometry After being washed with NaCl ⁄ Pi and blocked with 1% BSA, the EDTA-detected cells (1 · 106) were incubated with : 50 SLex antibody KM93, 2.5 lgỈmL)1 polyclonal antibody against InR-a subunit or 528 mAb against the EGFR extracellular domain for 45 at °C In the ‘(–) Control’ sample the primary antibody was omitted Washed cells were incubated with : 128 fluorescein isothiocyanate-conjugated secondary antibody for 30 at °C cells were then suspended in NaCl ⁄ Pi and subjected to flow cytometry Fluorescence-activated cell spectra were drawn automatically, and the relative amount of surface SLex was expressed as mean fluorescence intensity 536 Analysis of SLex expression on InR-a and EGFR using western immunoblotting In brief, immunoprecipitated InR-a and EGFR were subjected to SDS ⁄ PAGE, then transferred to a poly(vinylidene difluoride) membrane and treated with : 500 diluted antiSLex (KM93) and anti-InR-a or anti-EGFR (528) sera in Tris-buffered saline with 5% fat-free dry milk, followed by : 500 HRP-labeled secondary antibody Finally, the color was developed with enhanced chemiluminescence reagents, and followed by densitometric scanning Determination of tyrosine phosphorylation of InR-b, EGFR or IRS-1 using western immunoblotting Monolayer cells were lysed with 200 lL lysis buffer as described above Immunoprecipitated InR-b or EGFR was divided into two (for different probes) and subjected to 8% SDS ⁄ PAGE and western blotting, the membranes were probed with : 1000 phosphotyrosine mAb (PT66) and InR-b antibody or : 500 EGFR antibody (CF4) in Tris-buffered saline with 5% fat-free dry milk, followed by incubation with : 500 diluted horseradish peroxidaselabeled secondary antibody The color was also developed with an enhanced chemiluminescence reagent The measurement of tyrosine phosphorylation of IRS-1 was similar to that of InR-b and EGFR, but the primary antibodies used in western immunoblotting were anti-PT66 and antiIRS-1 sera Analysis of the proteins or phosphorylated proteins of PKB, PDK-1, PKN, Raf-1, MEK, p42/44 MAPK and b-catenin using western immunoblotting Briefly, cells were homogenized in Mes buffer (0.1 m pH 6.5, 150 mm NaCl, 2% Triton X-100, 25% glycerol, mm phenylmethylsulfonyl fluoride, mg % leupeptin FEBS Journal 274 (2007) 526–538 ª 2006 The Authors Journal compilation ª 2006 FEBS Q Wang et al and pepstatin), and 50 lg supernatant protein after centrifugation were subjected to 10% SDS ⁄ PAGE The membranes were treated with one of the : 500-diluted primary antibodies of the determined proteins or phosphorylated proteins in Tris-buffered saline with 5% fatfree dry milk, followed by incubation with : 500-diluted horseradish peroxidase-labeled secondary antibody and stained with enhanced chemiluminescence reagent b-Actin was used as loading control and stained with : 800 diluted mAb and : 500 horseradish peroxidase-labeled secondary antibody The protein bands were also quantified with densitometric analysis Assay of PKB activity PKB activity was performed with assay kit according to the instruction manual In short, the washed cells were lyzed with the buffer provided by the kit After protein determination, 500 lg of cell lysate was mixed with 20 lL suspension of immobilized PKB antibody and incubated at °C for h with shaking After centrifugation, the washed pellet was suspended in 40 lL kinase buffer (20 mm Tris ⁄ HCl, pH 7.5, mm b-glycerolphosphate, mm dithiothreitol, 0.1 mm Na3VO4, 10 mm MgCl2) and supplemented with substrates, including 0.8 lL of 10 mm ATP and lg GSK-3a ⁄ b fusion protein After incubated at 30 °C for 60 min, the phosphorylated GSK-3a ⁄ b fusion protein was detected with western blotting using the antibody to phospho-GSK-3a ⁄ b, followed by the addition of secondary antibody and enhanced chemiluminescence reagents [5] Determination of TCF transcription factor activity with dual luciferase reporter system The method was performed according to the protocol of ‘dual-luciferaseÒ reporter assay’ in the manual In brief, 0.5 lg TCF reporter plasmid and 0.4 lg pRL-TK plasmid was mixed in 25 lL antibiotic- and serum-free RPMI-1640 (ASF-RPMI), and lL LipofectamineTM was added to 23 lL of the above medium Two separate preparations were mixed within After standing at room temperature for 30 and adding 100 lL ASF-RPMI, the 150 lL plasmid ⁄ Lipofectamine mixture was added to the ASF-RPMI washed cells already cultured in 12-well plate for 24 h according the LipofectamineTM manual Cells were further cultured at 37 °C, 5% CO2 for h, and transferred to 300 lL antibiotic-free RPMI-1640 containing 20% serum for 24 h incubation Finally, the cells were incubated in normal RPMI-1640 for 48 h After washing with NaCl ⁄ Pi, cells were lyzed and luciferase activity was assayed according to the manual provided with the kit The firefly luminescence intensity of TK-luciferase was normalized against the Renilla luminescence intensity of pRL-TK [36] Fucosyltransferase-VII regulates insulin signaling Acknowledgements This work was supported by the grant from National Natural Science Foundation of China no 30670467 References Rudd PM & Dwek RA (1997) 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able to regulate the phosphorylation and expression of some signaling molecules in the InR pathway, and these effects of a1,3-FucT-VII... level, respectively (both P < 0.01) 532 The effect of the above-mentioned changes in insulinsignaling molecules on transduction of the insulin signal was further studied in a1,3-FucT-VII-transfected