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First published online May 28, 2005
doi: 10.1242/10.1242/jcs.02357

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ERK-mediated phosphorylation of Thr735 in TNF-converting enzyme and its potential role in TACE protein trafficking

¹ University of Cambridge, Department of Oncology, Cambridge Institute of Medical Research, Hills Road, Cambridge, CB2 2XY, UK
² Center for Experimental Therapeutics, University of Pennsylvania School of Medicine, 421 Curie Blvd, Philadelphia, PA 19104, USA
³ National Jewish Medical and Research Center, Department of Pediatrics, 1400 Jackson Street, Colorado 80206, USA

View larger version (35K): [in a new window]   Fig. 1. TACE phosphorylation at Thr735 in intact COS-7 cells is ERK activation-dependent. (A) COS-7 cells were transfected with pcDNA3.1+ (1 µg), pWT.TACE (0.5 µg), pCA.MEK-1.HA (0.25 µg) and/or pWT.ERK-HA (0.25 µg) for 24 hours. Amounts of transfected DNA were kept equivalent to 1 µg using pcDNA3.1+/Zeo. Cells were incubated in the presence of 2 mCi [33P]orthophosphate for 6 hours before the cells were lysed and TACE immunoprecipitated with the anti-TACE ICD antibody. Immune complexes were washed, resolved using SDS-PAGE and western transfer followed by autoradiography (top panel). The same membrane was probed with the anti-TACE ICD antibody to reveal immunoprecipitated TACE protein (-TACE IP panel). Whole cell lysates (WCL) were also analysed by western blotting to determine the expression levels of TACE, CA.MEK-1.HA and WT.ERK2-HA (lower three panels). (B) Cells were transfected with expression plasmids for WT- and T735A-TACE and TACE protein as in A, and isolated 24 hours later by immunoprecipitation. Immune complexes were washed and probed with an anti-phosphothreonine monoclonal antibody (-pThr, top panel) and anti-TACE (second panel). WCL were also analysed for expression of TACE, MEK-1 and ERK2 (lower three panels). Arrows highlight the detected TACE species.

View larger version (71K): [in a new window]   Fig. 2. WT.TACE-eGFP and T735A.TACE-eGFP proteins reside in the ER. (A) HeLa cells were transfected with 0.3 µg pWT.TACE-eGFP and pT735A.TACE-eGFP, fixed using methanol and stained for the ER-specific marker, calregulin (C-17) and Alexa 546 donkey anti-goat secondary antibodies. The coverslips were mounted and visualised using laser-scanning confocal microscopy. The TACE-eGFP protein, ER and nuclei are green, red and blue, respectively. (B) HeLa cells stably expressing WT.TACE (WT.HeLa) were seeded on coverslips and the following day fixed using methanol, stained for TACE using anti-TACE antibody (C-15) and Texas Red-conjugated donkey anti-goat secondary antibodies and the ER stained using anti-calregulin (H-170) and Alexa 488 donkey anti-goat secondary antibodies. Cells were visualised using laser-scanning confocal microscopy with the ER in green, TACE in red, nuclei (stained using DAPI) in blue and colocalisation highlighted with arrowheads (top panels). Untransfected HeLa cells were treated identically and stained using anti-TACE (C-15) primary antibody followed by Alexa 546 donkey anti-goat secondary antibodies. Red and blue channels for TACE and nuclei are shown, respectively (bottom panels). Bar, 10 µm.

View larger version (57K): [in a new window]   Fig. 3. Subcellular distribution of TACE-eGFP changes with the coexpression of active ERK2. (A) HeLa cells were transfected with either 0.3 µg pWT.TACE-GFP or pT735A.TACE-eGFP, with or without 0.1 µg of pCA.MEK-1.HA and/or pWT.ERK2-HA. DNA amounts were kept equivalent using pcDNA3.1+/Zeo. Cells were fixed in methanol and stained for constitutively active MEK-1 (-MEK-1), WT.ERK-HA (-HA), or active phosphor-ERK2 (-pERK2 in B), before being visualised using laser-scanning confocal microscopy. TACE-eGFP, CA.MEK-1 and/or ERK2 are green and red respectively, whereas the nuclei are blue. The arrowheads highlight a change in the subcellular location of TACE-eGFP protein. Bar, 10 µm.

View larger version (72K): [in a new window]   Fig. 4. T735E.TACE-eGFP colocalises with components of the cells protein secretory pathway. HeLa cells were transfected with 3 µg pT735E.TACE-eGFP and incubated at 37°C for 24 hours. Cells were fixed in methanol, followed by immunostaining with antibodies specific for the ER (calregulin), COPII vesicles (Sec-23) and trans-Golgi network (TGN-46) and visualised using laser-scanning confocal microscopy. T735E.TACE-eGFP and the subcellular markers are highlighted in green and red (respectively) whereas the nuclei are shown in blue. Positive colocalisation is highlighted with white arrowheads. Bar, 10 µm.

View larger version (45K): [in a new window]   Fig. 5. The molecular mass of T735E.TACE corresponds to mature TACE and is processed to maturity quicker than pro-WT.TACE. (A) COS-7 cells were transfected with 3 µg pcDNA3.1+ (lane U) or 3 µg pWT.TACE (lane NIgG and WT.TACE), pT735A.TACE (lane T735A.TACE) and pT735E.TACE (lane T735E.TACE). Cells transfected with pT735E.TACE were treated with 50 µM leupeptin for 18 hours after 6 hours of transfection. Twenty-four hours after transfection, the cells were lysed and TACE was immunoprecipitated using non-immune IgG antibodies (lane NIgG) or the anti-TACE ICD specific antibody. Immune complexes were washed with NP-40LB, and analysed by western blotting with the anti-TACE ICD antibody (C-15). (B) Similarly, 2x106 WT.HeLa or untransfected HeLa cells were seeded overnight and TACE immunoprecipitated using the anti-TACE antibody (C-15). After extensively washing the immune complexes using NP-40LB they were analysed by western blotting using an anti-TACE antibody (C-15). (C) COS-7 cells were transfected with pWT.TACE, pT735A.TACE and pT735E.TACE and treated with 50 µM leupeptin for 18 hours after 6 hours of transfection. Cells were radiolabelled for 3 hours with [35S]methionine/cysteine in the presence of 50 µM leupeptin, washed and chased in complete medium (lacking leupeptin) for the time indicated. TACE was purified and analysed by SDS-PAGE and autoradiography.

View larger version (32K): [in a new window]   Fig. 6. PMA stimulation of cells causes the translocation of TACE to the cell surface in an ERK phosphorylation-dependent fashion. (A) HeLa cells transfected with 1 µg pcDNA3.1+ (Lane U) or pWT.TACE-HA were pre-incubated with 10 µM U0126 or carrier (DMSO) for 30 minutes, 24 hours after transfection. Cells were stimulated with 1 µM PMA at 37°C for the time points shown (in minutes) before cell surface proteins were biotinylated and TACE immunoprecipitated using anti-HA antibodies (C-15). Immune complexes were washed and analysed by western blotting with streptavidin-HRP (top panel). Equal volumes of whole cell lysates (3% input) were also analysed following biotinylation by western blotting using an anti-HA antibody to detect pro-TACE (lower panel). HeLa cells were transfected with 1 µg pWT.TACE-HA (B) and pT735A.TACE-HA (C) and 24 hours after transfection treated with 10 µM U0126 or carrier (DMSO) at 37°C for 30 minutes. Cells were stimulated at 37°C with 1 µM PMA for the times stated (or carrier for 10 minutes) before cell surface proteins were biotinylated and TACE immunoprecipitated then analysed by western blotting with streptavidin-HRP. Whole cell lysates (3% input) were also analysed by western blotting for the expression of pro-TACE (lower panels in B and C). The arrowheads indicate mature TACE (100 kDa).

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