Uratsuji Y, DiCorleto P E
Department of Brain and Vascular Research, Cleveland Clinic Research Institute, Ohio 44195.
J Cell Physiol. 1988 Sep;136(3):431-8. doi: 10.1002/jcp.1041360306.
We have previously observed major differences in the phosphorylation of membrane proteins in sparse, proliferating versus confluent, quiescent pig aortic endothelial cells (EC) (Kazlauskas and DiCorleto, 1987). In the present study we examined whether EC growth state can influence the activity of a specific phosphorylating enzyme, protein kinase C (PKC) in cytosolic and membrane fractions of pig aortic EC. Levels of PKC were measured using two methods: 1) Ca2+ and phospholipid-dependent phosphorylation of exogenous histones using gamma-labeled [32P]ATP, and 2) [3H]phorbol-12,13-dibutyrate (PDBu) binding activity. The total amount of PKC activity in the quiescent versus proliferating cells was similar but the percentage of PKC activity in the membrane fraction correlated with the proliferative index of the cells: confluent, quiescent cultures exhibited a majority of PKC activity in the cytosolic fraction (67%), whereas sparse, proliferating cultures contained principally membrane-bound PKC (70%). We also examined the role of PKC in the mitogenic response of pig aortic EC to fetal calf serum. Following serum stimulation of sparse, serum-deprived pig aortic EC, PKC activity redistributed from the cytosolic to the membrane fraction in a rapid process that correlated with subsequent DNA synthesis. A potent activator of PKC, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced a minimal mitogenic response in pig aortic EC when added alone but acted synergistically with low concentrations of fetal calf serum to greatly stimulate DNA synthesis. Furthermore, pig aortic EC treated with TPA for 24 h to down-regulate PKC exhibited only 25% of the serum-stimulated mitogenic activity of control cultures. These results suggest a role for PKC activation and translocation in the proliferation of pig aortic EC.
我们之前观察到，稀疏、增殖状态的猪主动脉内皮细胞（EC）与汇合、静止状态的猪主动脉内皮细胞相比，膜蛋白磷酸化存在显著差异（Kazlauskas和DiCorleto，1987）。在本研究中，我们检测了内皮细胞的生长状态是否会影响猪主动脉内皮细胞胞质和膜组分中一种特定的磷酸化酶——蛋白激酶C（PKC）的活性。使用两种方法测量PKC水平：1）使用γ标记的[32P]ATP对外源组蛋白进行Ca2+和磷脂依赖性磷酸化，以及2）[3H]佛波醇-12,13-二丁酸酯（PDBu）结合活性。静止细胞与增殖细胞中PKC活性的总量相似，但膜组分中PKC活性的百分比与细胞的增殖指数相关：汇合、静止培养物中大部分PKC活性存在于胞质组分中（67%），而稀疏、增殖培养物中主要是膜结合型PKC（70%）。我们还研究了PKC在猪主动脉内皮细胞对胎牛血清的促有丝分裂反应中的作用。用血清刺激稀疏、血清饥饿的猪主动脉内皮细胞后，PKC活性以快速过程从胞质重新分布到膜组分，这一过程与随后的DNA合成相关。PKC的强效激活剂12-O-十四酰佛波醇-13-乙酸酯（TPA）单独添加时在猪主动脉内皮细胞中诱导的促有丝分裂反应最小，但与低浓度胎牛血清协同作用可极大地刺激DNA合成。此外，用TPA处理24小时以下调PKC的猪主动脉内皮细胞，其血清刺激的促有丝分裂活性仅为对照培养物的25%。这些结果表明PKC激活和易位在猪主动脉内皮细胞增殖中发挥作用。
