May W S, Sahyoun N, Jacobs S, Wolf M, Cuatrecasas P
J Biol Chem. 1985 Aug 5;260(16):9419-26.
Phorbol diesters are tumor-promoting agents that cause differentiation of HL60 human leukemic cells and concomitantly regulate surface transferrin receptors. Regulation of transferrin receptors by phorbol diesters involves receptor internalization in association with increased receptor phosphorylation (hyperphosphorylation). The intracellular mechanism of action of phorbol diester involves binding to and activation of the Ca2+-phospholipid-dependent protein kinase (protein kinase C). Present studies comparing results obtained with whole cells and those from a cell-free system reconstituted from purified protein kinase C and transferrin receptor components have revealed that the transferrin receptor is phosphorylated by protein kinase C activated by phorbol esters. Following tryptic digestion and two-dimensional separation of phosphopeptides of phosphorylated transferrin receptors, two major and several minor phosphoserine-containing fragments are resolved. These fragments are identical whether transferrin receptor is phosphorylated in whole cells incubated with phorbol diesters or following phosphorylation of affinity immobilized transferrin receptor in the in vitro reconstitution system. Phosphoamino acid analysis of these fragments indicates that serine is the only amino acid phosphorylated in whole cells or in the cell-free system. In addition, colchicine is shown to inhibit in a dose-dependent manner phorbol diester-induced internalization but not hyperphosphorylation of the surface transferrin receptor in whole cells. This inhibition is specific for colchicine since inactive beta- and gamma-Lumicolchicine have no such effect, while taxol reverses the inhibition. These results indicate that the phorbol diester-mediated process of down-regulation of the surface transferrin receptor is associated with phosphorylation of the receptor by activated protein kinase C and requires an intact cytoskeleton to affect receptor internalization.
佛波酯是肿瘤促进剂，可导致HL60人白血病细胞分化，并同时调节表面转铁蛋白受体。佛波酯对转铁蛋白受体的调节涉及受体内化以及受体磷酸化增加（过度磷酸化）。佛波酯的细胞内作用机制涉及与Ca2+ -磷脂依赖性蛋白激酶（蛋白激酶C）结合并激活该激酶。目前的研究比较了用全细胞获得的结果与从纯化的蛋白激酶C和转铁蛋白受体成分重建的无细胞系统获得的结果，结果表明转铁蛋白受体被佛波酯激活的蛋白激酶C磷酸化。对磷酸化转铁蛋白受体的磷酸肽进行胰蛋白酶消化和二维分离后，可分辨出两个主要的和几个次要的含磷酸丝氨酸片段。无论转铁蛋白受体是在与佛波酯孵育的全细胞中磷酸化，还是在体外重建系统中亲和固定化转铁蛋白受体磷酸化后，这些片段都是相同的。对这些片段的磷酸氨基酸分析表明，丝氨酸是全细胞或无细胞系统中唯一被磷酸化的氨基酸。此外，秋水仙碱被证明以剂量依赖性方式抑制佛波酯诱导的全细胞表面转铁蛋白受体的内化，但不抑制其过度磷酸化。这种抑制作用对秋水仙碱具有特异性，因为无活性的β-和γ-光秋水仙碱没有这种作用，而紫杉醇可逆转这种抑制作用。这些结果表明，佛波酯介导的表面转铁蛋白受体下调过程与活化的蛋白激酶C对受体的磷酸化有关，并且需要完整的细胞骨架来影响受体内化。
