Abstract
The effect of the protein phosphatase inhibitor okadaic acid on transferrin receptor internalization and recycling was examined in HeLa and K562 cells. Okadaic acid inhibited receptor uptake by more than 85% in both cell lines, whereas it affected transferrin recycling to differing degrees: recycling in HeLa cells was inhibited by greater than 90%, compared with only 65% in K562 cells. Okadaic acid also caused a marked redistribution of receptors in each cell line, which was accounted for by the difference in the extent to which transferrin uptake and recycling were inhibited. These effects were most likely mediated by a protein kinase, as they were delayed by 10-15 min and could be suppressed by prior incubation with certain protein kinase inhibitors. In addition, it was found that specific kinase inhibitors affected basal rates of transferrin uptake and recycling, although the extent of these effects differed between cell lines. Together, these results suggest that a complex pattern of protein phosphorylation influences the flux of the endocytic pathway in interphase cells.
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Selected References
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- BURTON K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J. 1956 Feb;62(2):315–323. doi: 10.1042/bj0620315. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bar-Zvi D., Mosley S. T., Branton D. In vivo phosphorylation of clathrin-coated vesicle proteins from rat reticulocytes. J Biol Chem. 1988 Mar 25;263(9):4408–4415. [PubMed] [Google Scholar]
- Bennett M. K., Calakos N., Scheller R. H. Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science. 1992 Jul 10;257(5067):255–259. doi: 10.1126/science.1321498. [DOI] [PubMed] [Google Scholar]
- Betz W. J., Bewick G. S. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. Science. 1992 Jan 10;255(5041):200–203. doi: 10.1126/science.1553547. [DOI] [PubMed] [Google Scholar]
- Bialojan C., Takai A. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem J. 1988 Nov 15;256(1):283–290. doi: 10.1042/bj2560283. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bleil J. D., Bretscher M. S. Transferrin receptor and its recycling in HeLa cells. EMBO J. 1982;1(3):351–355. doi: 10.1002/j.1460-2075.1982.tb01173.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carpenter G. Receptors for epidermal growth factor and other polypeptide mitogens. Annu Rev Biochem. 1987;56:881–914. doi: 10.1146/annurev.bi.56.070187.004313. [DOI] [PubMed] [Google Scholar]
- Carter L. L., Redelmeier T. E., Woollenweber L. A., Schmid S. L. Multiple GTP-binding proteins participate in clathrin-coated vesicle-mediated endocytosis. J Cell Biol. 1993 Jan;120(1):37–45. doi: 10.1083/jcb.120.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen W. J., Goldstein J. L., Brown M. S. NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor. J Biol Chem. 1990 Feb 25;265(6):3116–3123. [PubMed] [Google Scholar]
- Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 1989;58:453–508. doi: 10.1146/annurev.bi.58.070189.002321. [DOI] [PubMed] [Google Scholar]
- Collawn J. F., Stangel M., Kuhn L. A., Esekogwu V., Jing S. Q., Trowbridge I. S., Tainer J. A. Transferrin receptor internalization sequence YXRF implicates a tight turn as the structural recognition motif for endocytosis. Cell. 1990 Nov 30;63(5):1061–1072. doi: 10.1016/0092-8674(90)90509-d. [DOI] [PubMed] [Google Scholar]
- Colombo M. I., Mayorga L. S., Casey P. J., Stahl P. D. Evidence of a role for heterotrimeric GTP-binding proteins in endosome fusion. Science. 1992 Mar 27;255(5052):1695–1697. doi: 10.1126/science.1348148. [DOI] [PubMed] [Google Scholar]
- Dautry-Varsat A., Ciechanover A., Lodish H. F. pH and the recycling of transferrin during receptor-mediated endocytosis. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2258–2262. doi: 10.1073/pnas.80.8.2258. [DOI] [PMC free article] [PubMed] [Google Scholar]
- De Matteis M. A., Santini G., Kahn R. A., Di Tullio G., Luini A. Receptor and protein kinase C-mediated regulation of ARF binding to the Golgi complex. Nature. 1993 Aug 26;364(6440):818–821. doi: 10.1038/364818a0. [DOI] [PubMed] [Google Scholar]
- Geuze H. J., Slot J. W., Schwartz A. L. Membranes of sorting organelles display lateral heterogeneity in receptor distribution. J Cell Biol. 1987 Jun;104(6):1715–1723. doi: 10.1083/jcb.104.6.1715. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goud B., Huet C., Louvard D. Assembled and unassembled pools of clathrin: a quantitative study using an enzyme immunoassay. J Cell Biol. 1985 Feb;100(2):521–527. doi: 10.1083/jcb.100.2.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Griffiths G., Back R., Marsh M. A quantitative analysis of the endocytic pathway in baby hamster kidney cells. J Cell Biol. 1989 Dec;109(6 Pt 1):2703–2720. doi: 10.1083/jcb.109.6.2703. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gruenberg J., Howell K. E. Membrane traffic in endocytosis: insights from cell-free assays. Annu Rev Cell Biol. 1989;5:453–481. doi: 10.1146/annurev.cb.05.110189.002321. [DOI] [PubMed] [Google Scholar]
- Haystead T. A., Sim A. T., Carling D., Honnor R. C., Tsukitani Y., Cohen P., Hardie D. G. Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature. 1989 Jan 5;337(6202):78–81. doi: 10.1038/337078a0. [DOI] [PubMed] [Google Scholar]
- Herskovits J. S., Burgess C. C., Obar R. A., Vallee R. B. Effects of mutant rat dynamin on endocytosis. J Cell Biol. 1993 Aug;122(3):565–578. doi: 10.1083/jcb.122.3.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huet C., Ash J. F., Singer S. J. The antibody-induced clustering and endocytosis of HLA antigens on cultured human fibroblasts. Cell. 1980 Sep;21(2):429–438. doi: 10.1016/0092-8674(80)90479-1. [DOI] [PubMed] [Google Scholar]
- Kase H., Iwahashi K., Nakanishi S., Matsuda Y., Yamada K., Takahashi M., Murakata C., Sato A., Kaneko M. K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. Biochem Biophys Res Commun. 1987 Jan 30;142(2):436–440. doi: 10.1016/0006-291x(87)90293-2. [DOI] [PubMed] [Google Scholar]
- Keen J. H. Clathrin and associated assembly and disassembly proteins. Annu Rev Biochem. 1990;59:415–438. doi: 10.1146/annurev.bi.59.070190.002215. [DOI] [PubMed] [Google Scholar]
- Klausner R. D., Harford J., van Renswoude J. Rapid internalization of the transferrin receptor in K562 cells is triggered by ligand binding or treatment with a phorbol ester. Proc Natl Acad Sci U S A. 1984 May;81(10):3005–3009. doi: 10.1073/pnas.81.10.3005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lucocq J., Warren G., Pryde J. Okadaic acid induces Golgi apparatus fragmentation and arrest of intracellular transport. J Cell Sci. 1991 Dec;100(Pt 4):753–759. doi: 10.1242/jcs.100.4.753. [DOI] [PubMed] [Google Scholar]
- Mahaffey D. T., Peeler J. S., Brodsky F. M., Anderson R. G. Clathrin-coated pits contain an integral membrane protein that binds the AP-2 subunit with high affinity. J Biol Chem. 1990 Sep 25;265(27):16514–16520. [PubMed] [Google Scholar]
- Myers M., Forgac M. The coated vesicle vacuolar (H+)-ATPase associates with and is phosphorylated by the 50-kDa polypeptide of the clathrin assembly protein AP-2. J Biol Chem. 1993 May 5;268(13):9184–9186. [PubMed] [Google Scholar]
- Nakanishi S., Yamada K., Iwahashi K., Kuroda K., Kase H. KT5926, a potent and selective inhibitor of myosin light chain kinase. Mol Pharmacol. 1990 Apr;37(4):482–488. [PubMed] [Google Scholar]
- Nurse P. Universal control mechanism regulating onset of M-phase. Nature. 1990 Apr 5;344(6266):503–508. doi: 10.1038/344503a0. [DOI] [PubMed] [Google Scholar]
- Pearse B. M., Robinson M. S. Clathrin, adaptors, and sorting. Annu Rev Cell Biol. 1990;6:151–171. doi: 10.1146/annurev.cb.06.110190.001055. [DOI] [PubMed] [Google Scholar]
- Pelchen-Matthews A., Boulet I., Littman D. R., Fagard R., Marsh M. The protein tyrosine kinase p56lck inhibits CD4 endocytosis by preventing entry of CD4 into coated pits. J Cell Biol. 1992 Apr;117(2):279–290. doi: 10.1083/jcb.117.2.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pypaert M., Mundy D., Souter E., Labbé J. C., Warren G. Mitotic cytosol inhibits invagination of coated pits in broken mitotic cells. J Cell Biol. 1991 Sep;114(6):1159–1166. doi: 10.1083/jcb.114.6.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Robinson P. J., Sontag J. M., Liu J. P., Fykse E. M., Slaughter C., McMahon H., Südhof T. C. Dynamin GTPase regulated by protein kinase C phosphorylation in nerve terminals. Nature. 1993 Sep 9;365(6442):163–166. doi: 10.1038/365163a0. [DOI] [PubMed] [Google Scholar]
- Rothman J. E., Schmid S. L. Enzymatic recycling of clathrin from coated vesicles. Cell. 1986 Jul 4;46(1):5–9. doi: 10.1016/0092-8674(86)90852-4. [DOI] [PubMed] [Google Scholar]
- Rotman B., Papermaster B. W. Membrane properties of living mammalian cells as studied by enzymatic hydrolysis of fluorogenic esters. Proc Natl Acad Sci U S A. 1966 Jan;55(1):134–141. doi: 10.1073/pnas.55.1.134. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sager P. R., Brown P. A., Berlin R. D. Analysis of transferrin recycling in mitotic and interphase HeLa cells by quantitative fluorescence microscopy. Cell. 1984 Dec;39(2 Pt 1):275–282. doi: 10.1016/0092-8674(84)90005-9. [DOI] [PubMed] [Google Scholar]
- Sandvig K., Olsnes S., Brown J. E., Petersen O. W., van Deurs B. Endocytosis from coated pits of Shiga toxin: a glycolipid-binding protein from Shigella dysenteriae 1. J Cell Biol. 1989 Apr;108(4):1331–1343. doi: 10.1083/jcb.108.4.1331. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schlossman D. M., Schmid S. L., Braell W. A., Rothman J. E. An enzyme that removes clathrin coats: purification of an uncoating ATPase. J Cell Biol. 1984 Aug;99(2):723–733. doi: 10.1083/jcb.99.2.723. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schmid S. L., Carter L. L. ATP is required for receptor-mediated endocytosis in intact cells. J Cell Biol. 1990 Dec;111(6 Pt 1):2307–2318. doi: 10.1083/jcb.111.6.2307. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smythe E., Carter L. L., Schmid S. L. Cytosol- and clathrin-dependent stimulation of endocytosis in vitro by purified adaptors. J Cell Biol. 1992 Dec;119(5):1163–1171. doi: 10.1083/jcb.119.5.1163. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smythe E., Pypaert M., Lucocq J., Warren G. Formation of coated vesicles from coated pits in broken A431 cells. J Cell Biol. 1989 Mar;108(3):843–853. doi: 10.1083/jcb.108.3.843. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sorkin A., Carpenter G. Interaction of activated EGF receptors with coated pit adaptins. Science. 1993 Jul 30;261(5121):612–615. doi: 10.1126/science.8342026. [DOI] [PubMed] [Google Scholar]
- Stanley P. E., Williams S. G. Use of the liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme. Anal Biochem. 1969 Jun;29(3):381–392. doi: 10.1016/0003-2697(69)90323-6. [DOI] [PubMed] [Google Scholar]
- Tamaoki T., Nomoto H., Takahashi I., Kato Y., Morimoto M., Tomita F. Staurosporine, a potent inhibitor of phospholipid/Ca++dependent protein kinase. Biochem Biophys Res Commun. 1986 Mar 13;135(2):397–402. doi: 10.1016/0006-291x(86)90008-2. [DOI] [PubMed] [Google Scholar]
- Tooze J., Hollinshead M. In AtT20 and HeLa cells brefeldin A induces the fusion of tubular endosomes and changes their distribution and some of their endocytic properties. J Cell Biol. 1992 Aug;118(4):813–830. doi: 10.1083/jcb.118.4.813. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tuomikoski T., Felix M. A., Dorée M., Gruenberg J. Inhibition of endocytic vesicle fusion in vitro by the cell-cycle control protein kinase cdc2. Nature. 1989 Dec 21;342(6252):942–945. doi: 10.1038/342942a0. [DOI] [PubMed] [Google Scholar]
- Ward J. H., Kushner J. P., Kaplan J. Regulation of HeLa cell transferrin receptors. J Biol Chem. 1982 Sep 10;257(17):10317–10323. [PubMed] [Google Scholar]
- Warren G., Davoust J., Cockcroft A. Recycling of transferrin receptors in A431 cells is inhibited during mitosis. EMBO J. 1984 Oct;3(10):2217–2225. doi: 10.1002/j.1460-2075.1984.tb02119.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Warren G., Woodman P., Pypaert M., Smythe E. Cell-free assays and the mechanism of receptor-mediated endocytosis. Trends Biochem Sci. 1988 Dec;13(12):462–465. doi: 10.1016/0968-0004(88)90229-0. [DOI] [PubMed] [Google Scholar]
- Watts C. Rapid endocytosis of the transferrin receptor in the absence of bound transferrin. J Cell Biol. 1985 Feb;100(2):633–637. doi: 10.1083/jcb.100.2.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Woodman P. G., Mundy D. I., Cohen P., Warren G. Cell-free fusion of endocytic vesicles is regulated by phosphorylation. J Cell Biol. 1992 Jan;116(2):331–338. doi: 10.1083/jcb.116.2.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Woodman P. G., Warren G. Fusion of endocytic vesicles in a cell-free system. Methods Cell Biol. 1989;31:197–206. doi: 10.1016/s0091-679x(08)61610-6. [DOI] [PubMed] [Google Scholar]
- Zerial M., Suomalainen M., Zanetti-Schneider M., Schneider C., Garoff H. Phosphorylation of the human transferrin receptor by protein kinase C is not required for endocytosis and recycling in mouse 3T3 cells. EMBO J. 1987 Sep;6(9):2661–2667. doi: 10.1002/j.1460-2075.1987.tb02557.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- van der Bliek A. M., Redelmeier T. E., Damke H., Tisdale E. J., Meyerowitz E. M., Schmid S. L. Mutations in human dynamin block an intermediate stage in coated vesicle formation. J Cell Biol. 1993 Aug;122(3):553–563. doi: 10.1083/jcb.122.3.553. [DOI] [PMC free article] [PubMed] [Google Scholar]