Protein kinase C substrates in corneal epithelium during wound healing: the phosphorylation of growth associated protein-43 (GAP-43).

Protein kinase C (PKC) plays an important role in regulating cell growth. In the cornea, alpha-PKC activity increases during wound-healing. This activation of PKC will result in the increased phosphorylation of specific PKC substrates. In this study, several PKC substrates of relative low molecular weight were identified and characterized in cytosol and membrane preparations obtained from rabbit corneal epithelium before and during wound-healing. Corneal epithelium proteins were phosphorylated by endogenous PKC and by alpha-PKC isolated from rabbit brain, and then analysed using SDS-PAGE. In cytosol, PKC substrates with apparent molecular weights of 20, 25, 30, 35, 50 and 55 kDa were phosphorylated by PKC. The phosphorylation of the substrates increased 3 and 7 days after total de-epithelialization, due to the increase in alpha-PKC activity after wounding. However, when brain alpha-PKC was used as the exogenous source of PKC, there was a protein concentration-related decrease in the phosphorylation of corneal epithelium substrate after injury. This decreased phosphorylation of PKC substrates was inhibited by okadaic acid, a specific phosphatase inhibitor. The results suggest that an activated protein phosphatase takes part in controling the phosphorylation of PKC substrates during wound-healing. In the membrane fraction, a 60 kDa protein was phosphorylated by alpha-, beta- and gamma-PKC isoenzymes and was identified by Western blot as growth associated protein-43 (GAP-43), a protein kinase C substrate involved in axon regeneration. GAP-43 concentration increased 3 and 7 days after wounding as did its phosphorylation by alpha-PKC. These findings suggest a role for the protein in the innervation process in corneal epithelium after injury.