Lin N, Bazan H E
Louisiana State University Eye Center, New Orleans, USA.
Exp Eye Res. 1995 Oct;61(4):451-9. doi: 10.1016/s0014-4835(05)80140-x.
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.
蛋白激酶C（PKC）在调节细胞生长中起重要作用。在角膜中，α-PKC活性在伤口愈合过程中增加。PKC的这种激活将导致特定PKC底物的磷酸化增加。在本研究中，在伤口愈合前和愈合过程中从兔角膜上皮获得的胞质溶胶和膜制剂中鉴定并表征了几种相对低分子量的PKC底物。角膜上皮蛋白被内源性PKC和从兔脑分离的α-PKC磷酸化，然后使用SDS-PAGE进行分析。在胞质溶胶中，表观分子量为20、25、30、35、50和55 kDa的PKC底物被PKC磷酸化。在完全去上皮化后3天和7天，底物的磷酸化增加，这是由于受伤后α-PKC活性增加所致。然而，当使用脑α-PKC作为PKC的外源来源时，损伤后角膜上皮底物的磷酸化存在与蛋白质浓度相关的降低。PKC底物磷酸化的这种降低被冈田酸（一种特异性磷酸酶抑制剂）抑制。结果表明，活化的蛋白磷酸酶参与了伤口愈合过程中PKC底物磷酸化的控制。在膜部分，一种60 kDa的蛋白质被α-、β-和γ-PKC同工酶磷酸化，并通过蛋白质印迹法鉴定为生长相关蛋白-43（GAP-43），一种参与轴突再生的蛋白激酶C底物。受伤后3天和7天，GAP-43浓度增加，其被α-PKC磷酸化的情况也增加。这些发现表明该蛋白在损伤后角膜上皮的神经支配过程中起作用。
