Rais S, Combadiere C, Hakim J, Perianin A
Laboratoire d'Hématologie, INSERM Unité 294, Hôpital Bichat, Paris, France.
Biochem Pharmacol. 1994 May 18;47(10):1797-804. doi: 10.1016/0006-2952(94)90308-5.
Tumor-promoting phorbol esters bind to and activate protein kinase C (PKC). Staurosporine, a potent PKC inhibitor, interferes with PKC catalytic activity without altering phorbol ester binding sites in cell-free systems. We found that, unlike cell-free systems, treatment of intact platelets with staurosporine enhances the expression of phorbol 12, 13-dibutyrate (PDBu) binding sites. Incubation of platelets at 37 degrees with staurosporine (25 nM to 1 microM and 2 nM tritiated PDBu ([3H]PDBu) increased the amount of [3H]PDBu specifically bound to intact platelets by approximately 10 to 200% of control values. This effect was rapid and plateaued after 10 min of cell treatment. Scatchard analysis of the data showed that staurosporine (500 nM) significantly increased the total binding capacity Bmax from 42.9 +/- 15.4 x 10(3) to 78 +/- 7.3 x 10(3) sites per platelet and reduced the apparent dissociation constant value Kd from 30.8 +/- 8.6 nM to 9.4 +/- 3.4 nM. Enhanced PDBu binding capacity and affinity were also observed with human mononuclear and polymorphonuclear leukocytes. Fractionation of staurosporine-treated platelets showed an increased binding capacity of the particulate fraction (102%) and decreased binding capacity of the soluble fraction (60%) compared to controls, with no change in the affinity of PDBu binding to these fractions. Chelation of internal calcium with BAPTA did not significantly attenuate the staurosporine-mediated rise in PBDu binding but prevented the platelet-activating factor-induced response, indicating that cytosolic calcium does not play an important role in these staurosporine effects. These results show that, in addition to interfering with PKC protein-phosphorylating activity, staurosporine enhances PDBu binding affinity and capacity in intact platelets. This latter effect appears to be due to translocation of soluble PDBu binding sites, presumably PKC units.
促肿瘤佛波酯可结合并激活蛋白激酶C（PKC）。星形孢菌素是一种有效的PKC抑制剂，在无细胞系统中，它可干扰PKC催化活性而不改变佛波酯结合位点。我们发现，与无细胞系统不同，用星形孢菌素处理完整血小板可增强佛波醇12,13 - 二丁酸酯（PDBu）结合位点的表达。将血小板与星形孢菌素（25 nM至1 μM）在37℃孵育，并加入2 nM氚标记的PDBu（[3H]PDBu），可使特异性结合到完整血小板上的[3H]PDBu量增加至对照值的约10%至200%。这种效应迅速出现，并在细胞处理10分钟后达到平台期。对数据进行Scatchard分析表明，星形孢菌素（500 nM）可使每个血小板的总结合能力Bmax从42.9±15.4×10³显著增加至78±7.3×10³个位点，并使表观解离常数Kd值从30.8±8.6 nM降低至9.4±3.4 nM。在人单核细胞和多形核白细胞中也观察到PDBu结合能力和亲和力增强。与对照相比，对用星形孢菌素处理的血小板进行分级分离显示，颗粒部分的结合能力增加（102%），可溶性部分的结合能力降低（60%），而PDBu与这些部分结合的亲和力没有变化。用BAPTA螯合细胞内钙不会显著减弱星形孢菌素介导的PBDu结合增加，但可阻止血小板活化因子诱导的反应，这表明胞质钙在这些星形孢菌素效应中不发挥重要作用。这些结果表明，除了干扰PKC蛋白磷酸化活性外，星形孢菌素还可增强完整血小板中PDBu的结合亲和力和能力。后一种效应似乎是由于可溶性PDBu结合位点（可能是PKC单位）的易位所致。
