Abstract
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.
Highlights
From $The Johns Hopkins Oncology Center, Baltimore, Maryland 21205 and
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 haverevealed that the transferrrineceptor is phosphorylat ~by protein kinase C activated by phorbol esters
Ated in whole cells incubated with phorbol diesters or Important membrane-bound substrates for protein kinase following phosphorylation of affinity immobilized C include receptors for growth factors such as EGF (9-ll), transferrin receptor in the in vitro reconstitution sys- insulin, and somatomedin C [12]
Summary
'251-TrartsferrinBinding and Affinity Isolation of Transjerrin Receptor-Binding of lZ5I-transferrinto HL60 cells and phosphorylation and isolation of the transferrin receptor in intact cells were performed as described previously [18].Protein kinase C was prepared from rat brain by anion exchange high performance liquid chromatography as describedpreviously [23]. The resuiting lysate supernatants were used for affinity isolation of transferrin receptor on Sepharose 4Bgel to which ferrotransferrinhad been covalently coupled as described [18].Following a 3-h incubation with gentle tumbling at 4 "C, the affinity gel with bound transferrin receptor waswashed three times in Tris buffer containing 0.1% Triton X-100 and was resuspended in this buffer. Aliquots of this suspension were used for substrate in thien vitro reconstitution assay described below. Comparison of the Effects of TPA, PDBuD, iolein, and sn1-0leoyl-2-acetylglycerol on Phosphorylationof Isolated Transferrin Receptor by Purified Protein Kinase C in an in Vitro
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