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TEL-Syk fusion constitutively activates PI3-K/Akt, MAPK and JAK2-independent STAT5 signal pathways
T Kanie, A Abe, T Matsuda, Y Kuno, M Towatari, T Yamamoto, H Saito, N Emi and T Naoe
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Figure 1.
Schematic representation of constructed TEL-Syk and deletion mutant TEL
PNT-Syk (deletion of nt 222–461). For high expression in BaF3 cells, TEL-Syk and TELPNT-Syk were cloned into the pBabeNeo construct, a retroviral vector based on an MMLV-expressing neomycin-resistant gene. For immunofluorescence microscopy, TEL-Syk cDNA cloned into pFlag (the Flag epitope tagged) was used. LTR, long-terminal repeat; SV40, SV40 promoter; SV40pA, SV40 polyadenylation signal; CMV, CMV promoter.
Figure 2.
TEL-Syk fusion proteins were associated with PI3-K and constitutively activated Akt. (a) BaF3 cells were deprived of IL-3 for 12 h and then restimulated with 5 ng/ml of IL-3 for 30 min. Cell lysates were immunoblotted with antibody against phospho-Akt and reprobed with antibody against Akt. Akt was activated in TEL-Syk-transformed BaF3 cells in the absence of IL-3. (b) Cell lysates were immunoprecipitated with antibody against the 85K subunit of PI3-K and immunoblotted with human-specific Syk mAb. The same membrane was stripped and reprobed with antibody against the 85K subunit of PI3-K. TEL-Syk fusion products (lane 2) were co-precipitated with 85K subunit of PI3-K, and BaF3 cell lysates (lane 1) and TELPNT-Syk lysates (lane 3) were not. Lane 4 consisted of whole-cell lysates of TEL-Syk and lane 5 consisted of TELPNT-Syk.
Figure 3.
Constitutive kinase activation in TEL-Syk-transformed BaF3 cell. (a) BaF3 cell lysates were immunoprecipitated with antibody against Vav, PLC-
1 and PLC-2, and immunoblotted with the mAb against phosphotyrosine. Vav and PLC-2 were tyrosine phosphorylated in TEL-Syk-transformed BaF3 cells in the absence of IL-3. These tyrosine kinase activations were not observed in the BaF3 cells with TELPNT-Syk. (b) BaF3 cells were deprived of IL-3 for 12 h and then restimulated with 5 ng/ml of IL-3 for 30 min. Cell lysates were immunoblotted with the antibody against phospho-ERK1/2 (p42/44 MAPK) and phospho-MEK1/2 (ERK kinases), and reprobed with antibody against ERK1/2, MEK1/2, respectively. ERK and MEK were activated in TEL-Syk-transformed BaF3 cells in the absence of IL-3.
Figure 4.
TEL-Syk fusion protein constitutively activates STAT5 in the absence of JAK2 activation. (a) BaF3 cell lysates were immunoprecipitated with antibody against STAT1, STAT3 and STAT5, and immunoblotted with the mAb against phosphotyrosine. STAT5 was tyrosine phosphorylated in TEL-Syk-transformed BaF3 cells in the absence of IL-3. (b) BaF3 cells were deprived of IL-3 for 12 h and then restimulated with 5 ng/ml of IL-3 for 30 min. The cell lysates were immunoblotted with the antibody against phospho-STAT5 and phospho-JAK2, and reprobed with antibody against STAT5A, JAK2, respectively. The BaF3 cell transformed by TEL-Syk was constitutively tyrosine phosphorylated of STAT5 in the absence of JAK2 activation.
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Figure 5.
LY294002 inhibited the growth of TEL-Syk-transformed BaF3 cells and phosphorylation of Akt. (a) BaF3 cells and TEL-Syk-transformed BaF3 cells were added to 96-well plates with varying concentrations of LY294002 or carrier (DMSO) alone in the presence or absence of IL-3. The plates were incubated 37°C for 72 h before the addition of a mixture of tetrazolium and electron carrier. Cell proliferation assay was performed in triplicate, and mean absorption values with standard deviation were graphed. (b) BaF3 cells were deprived of IL-3 for 12 h, pretreated with 20 
M of LY294002 (+) or without LY294002 (-) (DMSO alone) for 30 min and then stimulated in the presence (+) or absence (-) of IL-3. The cell lysates were immunoblotted with the antibody against phospho-Akt, phospho-ERK1/2 and phospho-STAT5, and reprobed with antibody against Akt, ERK1/2, STAT5A, respectively. Phosphorylation of Akt by TEL-Syk and IL-3 are PI3-K dependent.
Figure 6.
The MAPK signal pathway is required for TEL-Syk-mediated cell proliferation. (a) BaF3 cells and TEL-Syk-transformed BaF3 cells were added to 96-well plates with varying concentrations of U0126 or carrier (DMSO) alone in the presence or absence of IL-3. Cell proliferation assay was performed in triplicate, and mean absorption values with standard deviation were graphed. TEL-Syk-mediated cell proliferation was inhibited by U0126. (b) BaF3 cells were deprived of IL-3 for 12 h, pretreated with 20
M of U0126 (+) or without U0126 (-) (DMSO alone) for 4 h and then stimulated in the presence (+) or absence (-) of IL-3. Cell lysates were immunoblotted with the antibody against phospho-ERK1/2, phospho-Akt and phospho-STAT5, and reprobed with antibody against ERK1/2, Akt, STAT5A, respectively. Phosphorylation of ERK1/2 by TEL-Syk was inhibited by U0126.
Figure 7.
Immunofluorescence localization of TEL-Syk fusion protein. (a) COS-7 cells transfected with pF-TS was stained with anti-Flag mAb and visualized with goat anti-mouse IgG (FITC). (b) COS-7 cells transfected with pF-TS was stained with goat anti-mouse IgG (FITC) (secondary antibody alone). Propidium iodide (Sigma Aldrich) was used for the fluorescent staining of the nucleic acid.
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