Neurofilament Phosphorylation in Cultured Bovine Adrenal Chromaffin Cells Is Stimulated by Phorbol Ester

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Abstract
Primary cultures of bovine adrenal chromaffin cells contain neurofilament proteins that are hypophosphorylated. When the cells were grown in medium containing 32Pi and 0.1 μM 12-O-tetradecanoyl-phorbol 13-acetate (TPA), 32P-labelling of the three neurofilament subunits was increased 6- to 20-fold relative to controls, the highest level of stimulation occurring for the mid-sized subunit. Addition of the protease inhibitor leupeptin to the growth medium had no effect on TPA-stimulated phosphorylation. The increased 32P incorporation was accompanied by a marked reduction in the gel electrophoretic mobilities of the two largest subunits. The augmented phosphorylation was observed 10 min after addition of TPA to a concentration of 0.1 μM or after 1 h of incubation in the presence of 0.01 μM TPA. One-dimensional peptide mapping and phosphoamino acid analysis indicated that TPA stimulated the phosphorylation of seryl residues at new sites in the mid-sized subunit. All of the latter subunit contained in the cytoskeletal fraction of chromaffin cells was converted to a more highly phosphorylated state after the cells were grown in the presence of TPA for 1 h.