Exp. Eye Res. (1995) 61. 451-460 Protein Kinase C Substrates in Corneal Epithelium During Wound Healing" the Phosphorylation of Growth Associated Protein-43 (GAP-43) NAIGANG LIN AND HAYDEE E. P. BAZAN* Louisiana State University Eye Center, New Orleans, LA, U.S.A. (Received Columbia 16 December 1994 and accepted in revised form 30 April 1995) Protein kinase C (PKC) plays an important role in regulating cell growth. In the cornea. ~z-PKCactivity increases during wound-healing. This activtion of PKC will result in the increased phosphorylation of specific PKC substrates. In this study, several PKC substrates or 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 c~-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 --PKC activity after wounding, However, when brain c~-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 c~-,/3- and y-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 ~-PKC. These findings suggest a role for the protein in the innervation process in corneal epithelium after injury. © 1995 Academic Press Limited Key words: protein kinase C substrates: corneal epithelium protein phosphorylation : PKC activity and wound-healing; corneal epithelium phosphatase; GAP-43 in corneal epithelial layer. 1. Introduction Phospholipid-dependent protein kinase C (PKC) com- prises a family of at least ten isoenzymes which play a crucial role in cell signal transduction (Nishizuka, [992). The activation of these enzymes is involved in ~hort-term responses such as neurotransmitter release ~md hormone secretion, and in long-term responses ~uch as gene expression, cell proliferation and differen- tiation. In the rabbit cornea, PKC activity has been Ibund mainly in the cytosolic fraction of the epithelial layer (Bazan et al., 1987). The predominant Ca 2÷- ,lependent PKC isoenzyme in the corneal epithelium is :-PKC, and its activity increases during wound- ]~ealing, which suggests that this isoenzyme plays a ~ole in cellular responses to injury, such as corneal cell migration and proliferation (Lin and Bazan, 1992). I~ecent studies have also shown that down-regulation ,,f PKC activity reduces clonal proliferation of basal ~'pithelium (Kruse and Tseng, 1993). However, very little is known about the molecular steps between the ;tctivation of PKC and the resultant biological re- ,oonses. One important approach to reveal the molecular pathways is to study the cellular substrates " For correspondence at: Louisiana State University Medical t'enter, LSU Eye Center, 2020 Gravier Street, Suite B, New Orleans, I.,\ 70112, U.S.A. ~)014 4835/95/100451+09 $12.00/0 of PKC. Many efforts have been made to identify and characterize PKC substrates from different tissues. PKC is most enriched in brain tissue, but, so far, only two PKC substrates have been formally identified and characterized: neuromodulin (also known as GAP-43, F-l. B-50, p57, etc.) and a myristoylated alanine-rich substrate (known as MARCKS protein or p87) (Coggins and Zwiers, 1991: Blackshear, 1993). Studies of the distribution of PKC isoenzymes have also shown a distinct cellular location of the different subspecies. These suggest that each PKC isoenzyme is involved in an independent cellular function that is characterized by its substrate specificity within the same cell (Nishizuka, 1992). In this study, we have identified endogenous PKC substrates in normal corneal epithelium and have characterized the specific profiles Of protein phosp- horylation by using endogenous corneal PKC as well as exogenous ~z-PKC purified from rabbit brain. The changes in these profiles during epithelial wound- healing have also been studied. Furthermore, one of the PKC substrates in the membrane fraction of corneal epithelium was identified as GAP-43. We have also discussed a mechanism through which PKC phosphorylation could be regulated based on our preliminary finding of phosphatase activity in the corneal epithelium. © 1995 Academic Press Limited