Distribution and phosphorylation of the growth-associated protein GAP- 43 in regenerating sympathetic neurons in culture

Abstract

Sympathetic neurons regenerating in culture were studied in order to gain further insight into the intracellular distribution and phosphorylation of GAP-43, a protein that has been suggested to have a role in axonal outgrowth and neuronal plasticity (Willard et al., 1987). Superior cervical ganglion neurons from embryonic rats were highly reactive with a polyclonal antibody against the growth- associated protein GAP-43 soon after they were placed in culture on a laminin substrate. As these neurons extended neurites, the distribution of GAP-43 reactivity changed. The cell body became progressively less reactive, whereas the growth cone at the tip of the growing neurite reacted strongly. The pattern of immunofluorescence was punctate both in the growth cone and the adjacent neurite, but appeared more diffusely distributed in the cell body. The antibody reacted only with cells that had been subjected to treatment that permeabilized the plasma membrane. When antibody was supplied in the medium of growing neurons, it neither bound to the cells nor altered normal neurite initiation or elongation. Of the different types of cells in these cultures, the antibody reacted only with neurons; it did not react with Schwann cells or fibroblasts. The stimulation of protein kinase C in these cultures resulted in a 7-fold stimulation of the phosphorylation of a protein of similar electrophoretic mobility to GAP-43. These observations demonstrate that GAP-43 is neuron-specific, is present throughout the neuron but at higher levels in the growth cone, and is a major substrate of protein kinase C. The high concentration of GAP-43 in the growth cones may necessitate its increased synthesis in neurons with elongating axons. Its location and phosphorylation by kinase C suggest that it could perform a function in the growth cone that is modulated by extracellular signals, such as those used in pathfinding or in the control of axonal elongation.