Abstract
The 3T3-TNR9 cell line is a variant of Swiss 3T3 cells which does not respond mitogenically to tumor promoters, but does respond mitogenically to epidermal growth factor, fibroblast growth factor, and serum. To elucidate differences between tumor promoters and polypeptide mitogens in the pathway(s) of mitogenesis which might be responsible for the nonresponsiveness of the 3T3-TNR9 cells, we have examined in these cells the early protein phosphorylation events known to be associated with mitogenesis in the parental 3T3 cells. We find that the 3T3-TNR9 cells display levels of tetradecanoyl phorbol acetate binding and of a calcium- and phospholipid-dependent protein kinase activity which are at least the equal of those seen in the parental 3T3 cells, implicating some postreceptor event in the nonmitogenic phenotype. In addition, we find that phosphorylation of the epidermal growth factor receptor and of 80-kDa and 22-kDa proteins, as well as the tyrosine phosphorylation of a 42-kDa protein, all proceed normally in the nonmitogenic variant, even though these phosphorylations must depend on the activation of different kinases. Thus, all these early phosphorylation reactions are intact in the 3T3-TNR9 cells. Although these phosphorylations may be necessary, they clearly are insufficient to trigger mitogenesis.
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- Ashendel C. L., Boutwell R. K. Direct measurement of specific binding of highly lipophilic phorbol diester to mouse epidermal membranes using cold acetone. Biochem Biophys Res Commun. 1981 Mar 31;99(2):543–549. doi: 10.1016/0006-291x(81)91779-4. [DOI] [PubMed] [Google Scholar]
- Ashendel C. L., Staller J. M., Boutwell R. K. Identification of a calcium- and phospholipid- dependent phorbol ester binding activity in the soluble fraction of mouse tissues. Biochem Biophys Res Commun. 1983 Feb 28;111(1):340–345. doi: 10.1016/s0006-291x(83)80157-0. [DOI] [PubMed] [Google Scholar]
- Berridge M. J., Irvine R. F. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature. 1984 Nov 22;312(5992):315–321. doi: 10.1038/312315a0. [DOI] [PubMed] [Google Scholar]
- Bishop R., Martinez R., Nakamura K. D., Weber M. J. A tumor promoter stimulates phosphorylation on tyrosine. Biochem Biophys Res Commun. 1983 Sep 15;115(2):536–543. doi: 10.1016/s0006-291x(83)80178-8. [DOI] [PubMed] [Google Scholar]
- Blackshear P. J., Nemenoff R. A., Avruch J. Insulin and growth factors stimulate the phosphorylation of a Mr-22000 protein in 3T3-L1 adipocytes. Biochem J. 1983 Jul 15;214(1):11–19. doi: 10.1042/bj2140011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blumberg P. M., Butler-Gralla E., Herschman H. R. Analysis of phorbol ester receptors in phorbol ester unresponsive 3T3 cell variants. Biochem Biophys Res Commun. 1981 Oct 15;102(3):818–823. doi: 10.1016/0006-291x(81)91611-9. [DOI] [PubMed] [Google Scholar]
- Butler-Gralla E., Herschman H. R. Glucose uptake and ornithine decarboxylase activity in tetradecanoyl phorbol acetate non-proliferative variants. J Cell Physiol. 1983 Mar;114(3):317–320. doi: 10.1002/jcp.1041140310. [DOI] [PubMed] [Google Scholar]
- Butler-Gralla E., Herschman H. R. Variants of 3T3 cells lacking mitogenic response to the tumor promoter tetradecanoyl-phorbol-acetate. J Cell Physiol. 1981 Apr;107(1):59–67. doi: 10.1002/jcp.1041070108. [DOI] [PubMed] [Google Scholar]
- Butler-Gralla E., Taplitz S., Herschman H. R. 12-O-tetradecanoylphorbol-13-acetate stimulates release of arachidonic acid, prostaglandin E2 and prostaglandin F2 alpha from TPA nonproliferative variants of 3T3 cells. Biochem Biophys Res Commun. 1983 Feb 28;111(1):194–199. doi: 10.1016/s0006-291x(83)80135-1. [DOI] [PubMed] [Google Scholar]
- Castagna M., Takai Y., Kaibuchi K., Sano K., Kikkawa U., Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem. 1982 Jul 10;257(13):7847–7851. [PubMed] [Google Scholar]
- Cochet C., Gill G. N., Meisenhelder J., Cooper J. A., Hunter T. C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity. J Biol Chem. 1984 Feb 25;259(4):2553–2558. [PubMed] [Google Scholar]
- Colburn N. H., Gindhart T. D., Hegamyer G. A., Blumberg P. M., Delclos K. B., Magun B. E., Lockyer J. Phorbol diester and epidermal growth factor receptors in 12-O-tetradecanoylphorbol-13-acetate-resistant and -sensitive mouse epidermal cells. Cancer Res. 1982 Aug;42(8):3093–3097. [PubMed] [Google Scholar]
- Colburn N. H., Wendel E. J., Abruzzo G. Dissociation of mitogenesis and late-stage promotion of tumor cell phenotype by phorbol esters: mitogen-resistant variants are sensitive to promotion. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6912–6916. doi: 10.1073/pnas.78.11.6912. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Colburn N. H., Wendel E., Srinivas L. Responses of preneoplastic epidermal cells to tumor promoters and growth factors: use of promoter-resistant variants for mechanism studies. J Cell Biochem. 1982;18(3):261–270. doi: 10.1002/jcb.1982.240180302. [DOI] [PubMed] [Google Scholar]
- Cooper J. A., Hunter T. Similarities and differences between the effects of epidermal growth factor and Rous sarcoma virus. J Cell Biol. 1981 Dec;91(3 Pt 1):878–883. doi: 10.1083/jcb.91.3.878. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cooper J. A., Sefton B. M., Hunter T. Diverse mitogenic agents induce the phosphorylation of two related 42,000-dalton proteins on tyrosine in quiescent chick cells. Mol Cell Biol. 1984 Jan;4(1):30–37. doi: 10.1128/mcb.4.1.30. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Davis R. J., Czech M. P. Tumor-promoting phorbol diesters mediate phosphorylation of the epidermal growth factor receptor. J Biol Chem. 1984 Jul 10;259(13):8545–8549. [PubMed] [Google Scholar]
- Dicker P., Rozengurt E. Phorbol ester stimulation of Na influx and Na-K pump activity in Swiss 3T3 cells. Biochem Biophys Res Commun. 1981 May 15;100(1):433–441. doi: 10.1016/s0006-291x(81)80115-5. [DOI] [PubMed] [Google Scholar]
- Driedger P. E., Blumberg P. M. The effect of phorbol diesters on chicken embryo fibroblasts. Cancer Res. 1977 Sep;37(9):3257–3265. [PubMed] [Google Scholar]
- Friedman B., Frackelton A. R., Jr, Ross A. H., Connors J. M., Fujiki H., Sugimura T., Rosner M. R. Tumor promoters block tyrosine-specific phosphorylation of the epidermal growth factor receptor. Proc Natl Acad Sci U S A. 1984 May;81(10):3034–3038. doi: 10.1073/pnas.81.10.3034. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gilmore T., Martin G. S. Phorbol ester and diacylglycerol induce protein phosphorylation at tyrosine. Nature. 1983 Dec 1;306(5942):487–490. doi: 10.1038/306487a0. [DOI] [PubMed] [Google Scholar]
- Hershko A., Mamont P., Shields R., Tomkins G. M. "Pleiotypic response". Nat New Biol. 1971 Aug;232(33):206–211. [PubMed] [Google Scholar]
- Iwashita S., Fox C. F. Epidermal growth factor and potent phorbol tumor promoters induce epidermal growth factor receptor phosphorylation in a similar but distinctively different manner in human epidermoid carcinoma A431 cells. J Biol Chem. 1984 Feb 25;259(4):2559–2567. [PubMed] [Google Scholar]
- Kikkawa U., Takai Y., Minakuchi R., Inohara S., Nishizuka Y. Calcium-activated, phospholipid-dependent protein kinase from rat brain. Subcellular distribution, purification, and properties. J Biol Chem. 1982 Nov 25;257(22):13341–13348. [PubMed] [Google Scholar]
- Kikkawa U., Takai Y., Tanaka Y., Miyake R., Nishizuka Y. Protein kinase C as a possible receptor protein of tumor-promoting phorbol esters. J Biol Chem. 1983 Oct 10;258(19):11442–11445. [PubMed] [Google Scholar]
- Kishimoto A., Takai Y., Mori T., Kikkawa U., Nishizuka Y. Activation of calcium and phospholipid-dependent protein kinase by diacylglycerol, its possible relation to phosphatidylinositol turnover. J Biol Chem. 1980 Mar 25;255(6):2273–2276. [PubMed] [Google Scholar]
- Kraft A. S., Anderson W. B. Phorbol esters increase the amount of Ca2+, phospholipid-dependent protein kinase associated with plasma membrane. Nature. 1983 Feb 17;301(5901):621–623. doi: 10.1038/301621a0. [DOI] [PubMed] [Google Scholar]
- Laszlo A., Radke K., Chin S., Bissell M. J. Tumor promoters alter gene expression and protein phosphorylation in avian cells in culture. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6241–6245. doi: 10.1073/pnas.78.10.6241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee L. S., Weinstein I. B. Mechanism of tumor promoter inhibition of cellular binding of epidermal growth factor. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5168–5172. doi: 10.1073/pnas.76.10.5168. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee L. S., Weinstein I. B. Membrane effects of tumor promoters: stimulation of sugar uptake in mammalian cell cultures. J Cell Physiol. 1979 Jun;99(3):451–460. doi: 10.1002/jcp.1040990319. [DOI] [PubMed] [Google Scholar]
- Nakamura K. D., Martinez R., Weber M. J. Tyrosine phosphorylation of specific proteins after mitogen stimulation of chicken embryo fibroblasts. Mol Cell Biol. 1983 Mar;3(3):380–390. doi: 10.1128/mcb.3.3.380. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Niedel J. E., Kuhn L. J., Vandenbark G. R. Phorbol diester receptor copurifies with protein kinase C. Proc Natl Acad Sci U S A. 1983 Jan;80(1):36–40. doi: 10.1073/pnas.80.1.36. [DOI] [PMC free article] [PubMed] [Google Scholar]
- O'Brien T. G., Diamond L. Ornithine decarboxylase induction and DNA synthesis in hamster embryo cell cultures treated with tumor-promoting phorbol diesters. Cancer Res. 1977 Nov;37(11):3895–3900. [PubMed] [Google Scholar]
- O'Brien T. G., Lewis M. A., Diamond L. Ornithine decarboxylase activity and DNA synthesis after treatment of cells in culture with 12-O-tetradecanoylphorbol-13-acetate. Cancer Res. 1979 Nov;39(11):4477–4480. [PubMed] [Google Scholar]
- Parker P. J., Stabel S., Waterfield M. D. Purification to homogeneity of protein kinase C from bovine brain--identity with the phorbol ester receptor. EMBO J. 1984 May;3(5):953–959. doi: 10.1002/j.1460-2075.1984.tb01913.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rozengurt E., Rodriguez-Pena M., Smith K. A. Phorbol esters, phospholipase C, and growth factors rapidly stimulate the phosphorylation of a Mr 80,000 protein in intact quiescent 3T3 cells. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7244–7248. doi: 10.1073/pnas.80.23.7244. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Saito M., Ueno I., Egawa K. Effect of retinoic acid and 12-O-tetradecanoyl phorbol-13-acetate on the binding of epidermal growth factor to its cellular receptors. Biochim Biophys Acta. 1982 Aug 6;717(2):301–304. doi: 10.1016/0304-4165(82)90183-0. [DOI] [PubMed] [Google Scholar]
- Takai Y., Kishimoto A., Kikkawa U., Mori T., Nishizuka Y. Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activated, phospholipid-dependent protein kinase system. Biochem Biophys Res Commun. 1979 Dec 28;91(4):1218–1224. doi: 10.1016/0006-291x(79)91197-5. [DOI] [PubMed] [Google Scholar]
- Thomas G., Siegmann M., Gordon J. Multiple phosphorylation of ribosomal protein S6 during transition of quiescent 3T3 cells into early G1, and cellular compartmentalization of the phosphate donor. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3952–3956. doi: 10.1073/pnas.76.8.3952. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weber M. J. Ribosomal RNA turnover in contact inhibited cells. Nat New Biol. 1972 Jan 12;235(54):58–61. doi: 10.1038/newbio235058a0. [DOI] [PubMed] [Google Scholar]
- Wenner C. E., Hackney J., Kimelberg H. K., Mayhew E. Membrane effects of phorbol esters. Cancer Res. 1974 Jul;34(7):1731–1737. [PubMed] [Google Scholar]
- Witt J. J., Roskoski R., Jr Rapid protein kinase assay using phosphocellulose-paper absorption. Anal Biochem. 1975 May 26;66(1):253–258. doi: 10.1016/0003-2697(75)90743-5. [DOI] [PubMed] [Google Scholar]