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 grow th. In the cornea, alpha-PKC activity increases during wound-healing. This activtion of PKC will result in the increased phosphorylation of specific PKC substrates. In this study, several PKC substrates of rela tive low molecular weight were identified and characterized in cytosol and membrane preparations obtained from rabbit corneal epithelium bef ore and during wound-healing. Corneal epithelium proteins were phospho rylated by endogenous PKC and by alpha-PKC isolated from rabbit brain, and then analysed using SDS-PAGE. In cytosol, PKC substrates with app arent molecular weights of 20, 25, 30, 35, 50 and 55 kDa were phosphor ylated by PKC. The phosphorylation of the substrates increased 3 and 7 days after total deepithelialization, due to the increase in alpha-PK C activity after wounding. However, when brain alpha-PKC was used as t he 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 sugges t that an activated protein phosphatase takes part in controling the p hosphorylation of PKC substrates during wound-healing, In the membrane fraction, a 60 kDa protein was phosphorylated by alpha-, beta- and ga mma-PKC isoenzymes and was identified by Western blot as growth associ ated protein-43 (GAP-43), a protein kinase C substrate involved in axo n regeneration. GAP-43 concentration increased 3 and 7 days after woun ding as did its phosphorylation by alpha-PKC. These findings suggest a role for the protein in the innervation process in corneal epithelium after injury. (C) 1995 Academic Press Limited