Combadière C, Pedruzzi E, Hakim J, Périanin A
Laboratoire d'Hématologie, INSERM U 294, Hôpital Bichat, Paris, France.
Biochem J. 1993 Feb 1;289 ( Pt 3)(Pt 3):695-701. doi: 10.1042/bj2890695.
Staurosporine, a potent protein kinase C (PKC) inhibitor, was studied for its effects on the binding of phorbol 12,13-dibutyrate (PDBu) to human polymorphonuclear leucocytes (PMNs). Treatment of PMNs with staurosporine concentrations in the range 50 nM-2 microM at 37 degrees C (but not at 4 degrees C) and with 1 nM [3H]PDBu at both temperatures enhanced specific PDBu binding to PMNs by approx. 10-600% relative to control values. The potentiation was rapid (detectable within 1 min) and reached a plateau after 10 min of cell treatment. Scatchard analysis of the binding showed that staurosporine increased the total number of PDBu-binding sites (Bmax) from (178 +/- 9) x 10(3) (control) to (324 +/- 15) x 10(3) sites per PMN and lowered the apparent dissociation constant (Kd) from 9.6 +/- 0.8 (control) to 3.3 +/- 0.3 nM. In Ca(2+)-depleted cells, staurosporine induced similar changes in Kd values, whereas the Bmax. increased by 60%. Treatment of PMNs with 500 nM staurosporine enhanced PDBu binding in the particulate fraction by 120 +/- 7% and decreased PDBu binding in the soluble fraction by 69 +/- 4%, whereas PKC histone-phosphorylating activity of both fractions was almost completely inhibited. Incubation of staurosporine-pretreated particulate fractions with soluble fractions enriched the particulate fraction in PDBu-binding sites at the expense of the soluble fraction, without altering the binding affinity of PDBu for either fraction. Stimulation of PMNs with chemotactic N-formyl peptides induced a transient increase in PDBu binding. This effect was potentiated by approx. 52% by staurosporine. These results show that, in addition to its interference with PKC protein-phosphorylating activity, staurosporine enhances both PDBu-binding capacity and affinity to PMNs, through a mechanism involving Ca(2+)-dependent and -independent processes. Alterations of PDBu binding to soluble and particulate fractions suggest that the enhanced binding capacity in intact PMNs may be due to translocation of PDBu receptors, presumably PKC units. This phenomenon may affect PKC-dependent cellular responses mediated by physiological stimulation.
星形孢菌素是一种有效的蛋白激酶C（PKC）抑制剂，研究了其对佛波醇12,13 - 二丁酸酯（PDBu）与人多形核白细胞（PMN）结合的影响。在37℃（而非4℃）下用浓度范围为50 nM - 2 μM的星形孢菌素处理PMN，并在两个温度下用1 nM [³H]PDBu处理，相对于对照值，特异性PDBu与PMN的结合增强了约10 - 600%。这种增强作用迅速（1分钟内可检测到），细胞处理10分钟后达到平台期。结合的Scatchard分析表明，星形孢菌素使每个PMN的PDBu结合位点总数（Bmax）从（178 ± 9）× 10³（对照）增加到（324 ± 15）× 10³个位点，并将表观解离常数（Kd）从9.6 ± 0.8（对照）降低到3.3 ± 0.3 nM。在缺钙细胞中，星形孢菌素诱导Kd值发生类似变化，而Bmax增加了60%。用500 nM星形孢菌素处理PMN使颗粒部分的PDBu结合增加了120 ± 7%，并使可溶性部分的PDBu结合减少了69 ± 4%，而两个部分的PKC组蛋白磷酸化活性几乎完全被抑制。将用星形孢菌素预处理的颗粒部分与富含可溶性部分的部分一起孵育，以可溶性部分为代价，使颗粒部分的PDBu结合位点增加，而不改变PDBu对任何一部分的结合亲和力。用趋化性N - 甲酰肽刺激PMN诱导PDBu结合短暂增加。这种效应被星形孢菌素增强了约52%。这些结果表明，除了干扰PKC蛋白磷酸化活性外，星形孢菌素还通过涉及钙依赖性和非依赖性过程的机制增强了PDBu与PMN的结合能力和亲和力。PDBu与可溶性和颗粒部分结合的改变表明，完整PMN中结合能力的增强可能是由于PDBu受体（可能是PKC单位）的转位。这种现象可能影响由生理刺激介导的PKC依赖性细胞反应。
