Article Figures & Data
Figures
- Fig. 1.
Activation of JNK2 by MKK7 mutants. HEK-293 cells were transiently transfected with 2.5 μg of plasmids encoding Myc-tagged forms of the indicated kinases (pCS3MT-MKK7, pCS3MT-MKK73E, pCS3MT-MKK73A, and pCS3MT-MKK7K149M) and with peVHA-JNK2. Empty pCS3MT vector was added to a total of 7.5 μg of DNA per transfection; 48 h later, cells were lysed in whole-cell lysis buffer. (A) A 100-μg aliquot of protein from each cell extract was separated by SDS-PAGE on a 10% gel, and expression of Myc epitope-tagged MKK7 proteins and HA-JNK2 was analyzed by Western blotting using anti-Myc and anti-HA antibodies, respectively. (B) GST-Jun kinase activity of cell extracts was assayed in vitro, using GST-Jun (amino acids 1 to 135) and [γ-32P]-ATP as the substrate. GST-Jun was then purified from the reaction mixture and resolved by SDS-PAGE on a 10% gel, and phosphorylation was visualized by autoradiography. See Materials and Methods for details. MKK7wt, wild-type MKK7.
- Fig. 2.
Transient expression of active MKK7 is sufficient to induce IL-8 secretion. KB (A) or HEK-293 (B) cells were transfected with 5 μg of empty vector pCS3MT-MKK73E or left untransfected; 24 h after transfection, the medium was changed. Cells were incubated for a further 24 h, and the amount of IL-8 in the medium was determined by specific ELISA as described in Materials and Methods. Shown is the fold increase (mean ± SEM) of IL-8 secretion compared to untransfected cells from three (A) or eight (B) independent experiments, each performed in triplicate (P < 0.01 for comparison of MKK73E versus vector). (C) HEK-293 cells were transfected with increasing amounts of the expression plasmid pCS3MT-MKK73E or pCS3MT-MKK73A or empty vector. The total DNA amount in each transfection (5 μg) was kept constant by adding empty pCS3MT. Supernatants were collected, and IL-8 protein concentrations were determined as for panel A. (C) The cells were lysed in whole-cell lysis buffer as described in Materials and Methods; 100-μg aliquots of proteins from lysates were separated by SDS-PAGE on 10% gels, and expression of Myc-tagged MKK7 protein kinases was analyzed by Western blotting using anti-Myc antibodies.
- Fig. 3.
Transient expression of NIK activates NF-κB and induces IL-8 secretion. (A) HEK-293 cells were transfected with 2.5 (lanes 1 and 3) or 5 (lanes 2 and 4) μg of expression plasmid pCDNA3flagNIK or pCDNA3flagNIK(KK429-430AA) or 5 μg of empty pCDNA3 vector (lane 5). In lanes 1 and 3, 2.5 μg of pCDNA3 was added to keep the DNA amount constant. At 48 h after transfection, cells were lysed in whole-cell lysis buffer. Activation of NF-κB in whole-cell extracts was analyzed by binding to a radiolabeled NF-κB consensus oligonucleotide as described in Materials and Methods. Protein-DNA complexes were resolved by nondenaturing PAGE on a 5% gel and visualized by autoradiography. The migration position of NF-κB complexed with labeled DNA, which was competed by excess unlabeled oligonucleotide (cold oligo; lanes 6 and 7), is indicated. (B) Expression of NIK was analyzed in extracts from transfected cells by immunoprecipitation (IP) with anti-Flag antibodies followed by Western blotting with polyclonal anti-NIK antiserum (SAK14). (C) IL-8 concentrations were determined by ELISA in supernatants of cells 48 h after transfection with pCDNA3flagNIK, pCDNA3flagNIK(KK429-430AA), or pCDNA3 (5 μg of each) or with 50 ng pFcMEKK1 plus 4.95 μg of pCDNA3. Shown are the means values ± SEM from eight transfection experiments performed in triplicate (P < 0.01 for comparison of all kinases to vector).
- Fig. 4.
Synergistic activation of IL-8 secretion by coexpression of NIK and MKK73E. (A) HEK-293 cells were transfected with 5 μg of empty vector, pCS3MT-MKK73E, pCDNAflag3NIK, or both; 48 h later, IL-8 secretion into the cell culture supernatant was determined by ELISA. Shown are means ± SEM from three independent experiments performed in triplicate. (B) Expression levels of MKK7 and NIK from one experiment were analyzed by immunoprecipitation (IP) from 1 mg of cell extract protein followed by Western blotting using antibodies against the Myc and Flag epitope tags, respectively (IgG hc, IgG heavy chain).
- Fig. 5.
Activation of SAPK/JNK or NF-κB by overexpression of MKK73E or NIK. HEK-293 cells were cotransfected with 2.5 μg of expression plasmids for the indicated kinases and empty vector (7.5 μg of total DNA in each sample); 48 h later, cells were lysed, and cytosolic and nuclear extracts were prepared. (A) Ectopically expressed proteins were detected in the lysates (100 μg of protein) by Western blotting using antibodies against the epitope tags. (B) Activation of SAPK/JNK was assayed in cytosolic extracts as described for Fig. 1. (C) Activation of NF-κB was tested by EMSA in nuclear extracts as described for Fig. 3. Shown are the data for one of four independent experiments with essentially similar results. NIKwt, wild-type NIK.
- Fig. 6.
MKK73E and NIK each require NF-κB and AP-1cis elements and synergize to activate a minimal IL-8 promoter. (A) Schematic representation of the minimal IL-8 promoter cloned 5′ of the luciferase cDNA in pUHC13-3-IL- 8pr. (B) HEK-293 cells were cotransfected with 0.25 μg of pUHC13-3-IL-8pr and with 5 μg of pCS3MT-MKK73E, pCS3MT-MKK73A, pCS3MT-NIK, or pCS3MT-NIK(KK429-430AA), 0.1 μg of pFcMEKK1 plus 4.9 μg of pCS3MT, or pCS3MT alone; 48 h after transfection, cells were lysed and luciferase (luc.) reporter gene activity was determined as described in Materials and Methods. Results are expressed as relative light units [RLU; mean ± SEM from four independent experiments performed in triplicate; P < 0.01 for comparing vector versus MKK73E and NIK). (C) HEK-293 cells were cotransfected with 0.25 μg of pUHC13-3-IL-8pr and either pCS3MT-NIK or pCS3MT-NIK(KK429-430AA) (0.5 μg of each), 5 μg pCS3MT-MKK73E alone, or a combination thereof as indicated. Amounts of DNA were kept equal by adding empty pCS3MT. Reporter gene activity (mean ± SEM from three independent experiments) was determined as for panel B. (D) HEK-293 cells were cotransfected with 0.25 μg of pUHC13-3-IL-8pr (open bars) or of mutants thereof in which the AP-1 (black bars) or NF-κB (hatched bars) site or both sites (cross-hatched bars) were mutated and a combination of pCS3MT-MKK73E (5 μg) plus pCS3MT-NIK (0.5 μg) or pFcMEKK1 (0.1 μg) plus pCS3MT (4.9 μg). Reporter gene activity (mean ± SEM from four independent experiments) was determined as for panel B. For each experiment, equal expression of protein kinases was confirmed by Western blotting (not shown).
- Fig. 7.
MKK73E-induced activation of the IL-8 promoter is suppressed by coexpression of dominant negative NIK. HEK-293 cells were transfected with 5 μg of pCS3MT-MKK73E, pCS3MT-MKK73A, pCS3MT-NIK, or pCS3MT-NIK(KK429-430AA) in the indicated combinations together with pUHC13-3-IL-8pr. Where required, empty pCS3MT was added to keep total DNA amounts constant. (A) At 48 h after transfection, cells were lysed and luciferase reporter gene activity was determined as for Fig.6 (mean ± SEM from three independent experiments); (B) 100 μg of lysate proteins from one experiment was separated by SDS-PAGE and Western blotted to confirm equal expression of Myc-epitope tagged protein kinases MKK7 and NIK. RLU, relative light units.
- Fig. 8.
Weak induction of IL-8 secretion by EGF correlates with its lack of SAPK/JNK activation. Human KB epithelial cells stably transfected with vector (KB-vector) or with antisense RNA to SAPKβ (KB-SAPKβas64, clone 64) as described in detail in reference24 were left untreated (control [C]) or stimulated for 24 h with IL-1α (IL-1; 10 ng/ml) or with EGF (50 ng/ml). (A) IL-8 concentrations in the cell culture supernatant were determined by ELISA (means ± SEM from two independent experiments performed in duplicate). (B) The cells were stimulated with IL-1α (10 ng/ml) or EGF (50 ng/ml) or left untreated for 15 min and lysed, and SAPK/JNK activity was determined as described for Fig. 1. Shown is a result representative for three independent experiments.
- Fig. 9.
MEKK1 and MKK6, but not NIK or MKK7, induce IL-8 mRNA stabilization. HeLa cells stably expressing the tettransactivator protein were cotransfected with 4 μg of the IL-8 mRNA reporter plasmid pUHD10-3-CATIL-8 and 12 μg of expression plasmids for the indicated kinases or empty vector. After 24 h, doxycyline (Dox; 3 μg/ml) was added to stop tettransactivator-dependent transcription. (A) Total RNA was prepared at the indicated times thereafter, and CAT-tagged IL-8 mRNA was detected by Northern blotting as described in Materials and Methods. (B) Intensity of CAT–IL-8 mRNA bands (as shown in panel A and from additional experiments) was quantified, and values are expressed relative to CAT–IL-8 mRNA amount measured at the time of doxycycline addition (=100%).
- Fig. 10.
MKK62E potentiates NIK and MKK73E-induced IL-8 protein secretion. HEK-293 cells were transfected with 0.5 μg of pCS3MT-NIK, 2.5 μg of pCS3MT-MKK73E, 2.5 μg of peVHA-MKK62E, and 2.5 μg of peVHA-MKK6K82A in the combinations indicated; 0.25 μg of pUHC13-3-IL-8pr was co-transfected. Total amount of DNA was kept constant by adding empty pCS3MT. The cell culture medium was changed 24 h after transfection; 24 h later, cell culture supernatants were analyzed for IL-8 protein content by ELISA. Cells were lysed, and IL-8 promoter (prom.) activity was determined as described in Materials and Methods. Results are expressed as fold increase (mean ± SEM from two independent experiments).
