Cells (30×106 ml-1), handled as in FIGS. 12A and 12B, were stimulated with IL-1β (10 ng ml-1) and homogenized at 4° C. with a Dounce homogenizer in buffer (25 mMHEPES, pH 7.4, 5 mM EGTA, 50 mM NaF and 10 μg/ml each of leupeptin and soybean trypsin inhibitor). Homogenates were centrifuged at 500×g for 5 minutes to remove nuclei and at 200,000×g for 30 minutes to prepare microsomal membranes. Membranes were resuspended into homogenizing buffer (2.2 μg membrane protein μl-1). For assay of kinase activity, the reaction mixture contained 20 μl of microsomal membrane, 40 μl EGFR peptide (4 mg ml-1 in 25 mM Hepes, pH 7.4) and 100 μl buffer (50 mM HEPES, pH 7.4, 20 mM MgCl2) (40). Phosphorylation was initiated at 22° C. by addition of 40 μl [γ-32P] ATP (100 μM final concentration) and terminated at the indicated times by addition of 40 μl of 0.5M ATP in 90% formic acid. Phosphorylated peptide was eluted from a C18Sep pak cartridge (Millipore), lyophilized, and resolved by C18 reverse phase HPLC using a linear gradient of acetonitrile. The peptide eluted at 30% acetonitrile as determined by measuring Cerenkov radiation in 1 ml fractions. All assays were performed under conditions determined as linear for time and enzyme concentration. Enzyme activity was determined from the percent conversion of substrate to product and the specific radioactivity of [γ-32P] ATP. Baseline kinetic analyses revealed a maximum reaction velocity of 12.5 pmol min-1 mg-1 of microsomal membrane protein and Michaelis constants (Km) of 70 μM ATP and 0.15 mg/ml for EGFR peptide. For most studies, 100 μM ATP was used to maintain a high 32 p specific radioactivity (4000 dpm pmol-1), although qualitatively similar results were obtained with 500 μM ATP. Ceramide and sphingosine (10 nM to 1 μM) enhanced kinass activity to 1.5-2.5 of control. Values (mean ± range) represent duplicate determinations from two experiments.
