Neurite Outgrowth and Protein Phosphorylation in Chick Embryonic Sensory Ganglia Induced by a Brief Exposure to 12-O-Tetradecanoylphorbol 13-Acetate

Abstract: An exposure to 12- O -tetradecanoylphorbol 13-acetate (TPA) at 20 n M for as short as 30 min was sufficient to elicit neurite outgrowth from explanted chick embryonic sensory ganglia. Attachment of the ganglia to the collagencoated substratum during exposure to TPA was essential for subsequent neurite outgrowth. Pulse-labeling with ³⁵S]-methionine indicated no significant difference in protein synthesis between control and TPA-treated ganglia. In vitro phosphorylation assay revealed a prominent protein kinase C substrate with an apparent molecular mass of 66,000 dalton (66 kDa) in chick embryo ganglia extracts. Treatment of intact ganglia with TPA for 30 min also specifically stimulated the phosphorylation of the same protein. When staurosporine, a potent inhibitor of protein kinase C, was present during TPA treatment, both neurite outgrowth and the phosphorylation of the 66-kDa protein were blocked. Biochemical analysis of the phosphorylated 66-kDa protein indicated that (1) phosphorylation was only in serine residue, (2) the pI value was 4.5, (3) after V8 protease digestion, two phosphorylated peptide fragments, 6.0 and 7.5 kDa in size, were produced, and (4) it cross-reacted with an antiserum raised against a 66-kDa neurofilament subunit from rat spinal cord. These results suggest that early activation of protein kinase C and the phosphorylation of the 66-kDa protein may be involved in neuritogenesis.