To determine the sorbitol-dependent activation of MEKK3, serum starve the cells of interest for 3 h and then treat with vehicle or 0.2 M sorbitol for 5 to 15 min. Rinse the cells twice in PBS and lyse in ice-cold MEKK3 lysis buffer (20 mMTris-HCl, pH 7.4, 1% Nonidet P-40 [NP-40], 135 mMNaCl, 10% glycerol, 0.1 mM EDTA, 0.1 mM EGTA, plus protease inhibitors). For MEKK3 assays, Triton X-100 cannot be substituted for NP-40 as MEKK3 is inactive in this detergent (Widmann et al., 2001). Immunoprecipitate MEKK3 with the anti-MEKK3 antibody (BD Biosciences, San Jose, CA) from 1 mg of lysate in 1 ml of MEKK3 lysis buffer for 1 to 2 h and then add 0.03 ml of recombinant protein G—Sepharose 4B (Invitrogen). Alternatively, epitope-tagged MEKK3 subcloned into a mammalian expression vector such as pcDNA3 or pCMV5 can be transfected into cells 24 to 48 h prior to treatment of cells with sorbitol and immunoprecipitated with antibodies against the epitope tag or MEKK3. Collect the immunoprecipitated complex by centrifugation and wash three times with cold buffer B and two times with MEKK3 kinase buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2, 1 mM dithiothreitol [DTT], 0.1 mM Na3VO4, and 10 mM ^-glycerophosphate). Remove all liquid from the immunoprecipitates, and begin the assay by adding 0.05 ml of MEKK3 kinase buffer containing 0.5 mg His-MKK6 K/M (kinase inactive MKK6 purified from Escherichia coli using standard methods) and 1 ml 10 mCi/ml [g-32P]ATP (MP Biomedicals, Irvine, CA). Incubate the assay at 30° for 20 min and stop the assay by adding 2 x SDS-PAGE buffer. Load the assay on a gel; after running, stain the gel with Coomassie or silver, dry, and expose to X-ray film. Quantitate the amount of phosphate incorporated into the substrate by excising the His-MKK6 band from the dried gel and subjecting it to liquid scintillation counting. This assay as described was used to demonstrate that sorbitol induces a twofold increase in MEKK3 kinase activity following a 15-min sorbitol treatment (Uhlik et al., 2003).
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