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

JCS02357 Supplementary Material

Files in this Data Supplement:

• Supplementary Methods (Adobe PDF)
• Supplemental Figure 1 -

  Fig. S1. GST-TACE ICD is phosphorylated by a component transiently activated in U937 and THP-1 cells in vitro. (A) U937 and THP-1 cells seeded in 35 mm wells were stimulated with vehicle (DMSO) or 1 mM PMA for 20 minutes. At timed intervals, cells were lysed and supernatants incubated with GST-TACE ICD or GST sepharose beads. Following incubation, the beads were washed and subjected to an in vitro kinase assay. Reaction products were resolved using SDS-PAGE followed by autoradiography. (B) U937 and THP-1 cells were pre-treated with DMSO, 1 mM SB203580 or 10 mM U0126 for 30 minutes before being stimulated for 10 minutes using PMA. Cells were harvested, lysed in NP-40LB and supernatants assayed for kinase activity to GST-TACE ICD sepharose. Reaction products were resolved and analysed using SDS-PAGE and autoradiography. GST fusion proteins were also analysed by SDS-PAGE to evaluate loading quantities (lower panels). Data collected from three independent experiments are shown.

• Supplemental Figure 2 -

  Fig. S2. GST-TACE ICD is phosphorylated in an ERK dependent manner in vitro. U937 (A) and THP-1 (B) cells were stimulated in duplicate with PMA, TNF-a, IL-6, MCP-1, EGF stimulation of U937 and THP-1 cells at 10, 10, 5, 10 and 10 minutes, respectively. Cells were lysed and in one set active ERK1 and 2 were immuno-depleted from the lysates using a phospho-ERK specific antibody and the other set of lysates left untreated. GST-TACE ICD or GST beads were incubated with all lysates and subjected to an in vitro kinase assay. Reaction products were resolved using SDS-PAGE and autoradiography. GST fusion proteins were also analysed by SDS-PAGE to evaluate loading quantities (lower panel). Equal volumes of cell lysates (after immunodepletion) were also analysed by western blotting using an anti-ERK2 specific antibody.

• Supplemental Figure 3 -

  Fig. S3. GST-TACE ICD is phosphorylated by catalytically active mouse ERK2 at Thr735. GST-TACE ICD deletion and point mutants used are shown schematically. The ERK consensus phosphorylation site is shown in grey and the precise sequence between residues 733 and 736 highlighted in which Thr735 is underlined. (B) Constructs were incubated in the presence of catalytically active ERK2 in a radioactive in vitro kinase assay, before the products were resolved and analysed by autoradiography. Myelin basic protein (MBP) and GST alone were used as positive and negative controls, respectively. MBP and GST fusion proteins were also analysed by SDS-PAGE to evaluate loading quantities (lower panel).

• Supplemental Figure 4 -

  Fig. S4. T735E-eGFP can colocalise with the endosomal marker EEA1 and the lysosomal marker LAMP1. HeLa cells were transfected with 3 mg pT735E.TACE-eGFP and 6 hours after transfection leupeptin was added to a concentration of 50 mM (for LAMP1 staining) and the cells incubated at 37°C for a further 18 hours. Cells were methanol fixed and stained using the endosomal (anti-EEA1) and lysosomal (anti-LAMP1) specific antibodies (red channel) and Texas Red-conjugated secondary antibodies. T735E.TACE-eGFP is highlighted in green and nuclei (stained with DAPI) are shown in blue. In both panels, positive colocalisation is highlighted with white arrowheads. Bar, 10 mm.

• Supplemental Figure 5 -

  Fig. S5. The treatment of WT.TACE-eGFP expressing cells with PMA alters the subcellular localisation of TACE. HeLa cells seeded on coverslips were transfected with 300 ng WT.TACE-eGFP and T735A.TACE-eGFP for 18 hours after which they were treated with carrier (DMSO) for 30 minutes and 1 mM PMA for the time periods indicated. Cells were fixed using 4% PFA and the coverslips mounted and visualised using immunofluorescence microscopy. TACE-eGFP and nuclei (stained using DAPI) are shown in green and blue, respectively. White arrowheads highlight an intracellular punctate staining pattern. Bar, 10 mm.