PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
 
Mol Cell Biol. 1994 February; 14(2): 1066–1074.
PMCID: PMC358462

Anisomycin and rapamycin define an area upstream of p70/85S6k containing a bifurcation to histone H3-HMG-like protein phosphorylation and c-fos-c-jun induction.

Abstract

Anisomycin, a translational inhibitor, synergizes with growth factors and phorbol esters to superinduce c-fos and c-jun by a number mechanisms, one of which is its ability to act as a potent signalling agonist, producing strong, prolonged activation of the same nuclear responses as epidermal growth factor or tetradecanoyl phorbol acetate. These responses include the phosphorylation of pp33, which exists in complexed and chromatin-associated forms, and of histone H3 and an HMG-like protein. By peptide mapping and microsequencing, we show here that pp33 is the phosphoprotein S6, present in ribosomes and in preribosomes in the nucleolus. Ablation of epidermal growth factor-, tetradecanoyl phorbol acetate-, or anisomycin-stimulated S6 phosphorylation by using the p70/85S6k inhibitor rapamycin has no effect on histone H3 and HMG-like protein phosphorylation or on the induction and superinduction of c-fos and c-jun. Further, [35S]methionine-labelling and immunoprecipitation studies show that the ablation of S6 phosphorylation has no discernible effect on translation in general or translation of newly induced c-fos transcripts. Finally, we show that anisomycin augments and prolongs S6 phosphorylation not by blocking S6 phosphatases but by sustained activation of p70/85S6k. These results suggest the possible use of anisomycin and rapamycin to define upstream and downstream boundaries of an area of signalling above p70/85S6k which contains a bifurcation that produces histone H3-HMG-like protein phosphorylation and c-fos-c-jun induction in the nucleus.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.2M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Alfageme CR, Zweidler A, Mahowald A, Cohen LH. Histones of Drosophila embryos. Electrophoretic isolation and structural studies. J Biol Chem. 1974 Jun 25;249(12):3729–3736. [PubMed]
  • Ballou LM, Luther H, Thomas G. MAP2 kinase and 70K S6 kinase lie on distinct signalling pathways. Nature. 1991 Jan 24;349(6307):348–350. [PubMed]
  • Blenis J, Chung J, Erikson E, Alcorta DA, Erikson RL. Distinct mechanisms for the activation of the RSK kinases/MAP2 kinase/pp90rsk and pp70-S6 kinase signaling systems are indicated by inhibition of protein synthesis. Cell Growth Differ. 1991 Jun;2(6):279–285. [PubMed]
  • Blenis J, Kuo CJ, Erikson RL. Identification of a ribosomal protein S6 kinase regulated by transformation and growth-promoting stimuli. J Biol Chem. 1987 Oct 25;262(30):14373–14376. [PubMed]
  • Cano E, Barratt MJ, Mahadevan LC. Which histone kinase? Nature. 1992 Nov 12;360(6400):116–116. [PubMed]
  • Cantley LC, Auger KR, Carpenter C, Duckworth B, Graziani A, Kapeller R, Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. [PubMed]
  • Chen RH, Sarnecki C, Blenis J. Nuclear localization and regulation of erk- and rsk-encoded protein kinases. Mol Cell Biol. 1992 Mar;12(3):915–927. [PMC free article] [PubMed]
  • Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. [PubMed]
  • Chung J, Kuo CJ, Crabtree GR, Blenis J. Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases. Cell. 1992 Jun 26;69(7):1227–1236. [PubMed]
  • Church GM, Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. [PubMed]
  • Cleveland DW, Fischer SG, Kirschner MW, Laemmli UK. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed]
  • Curran T, Peters G, Van Beveren C, Teich NM, Verma IM. FBJ murine osteosarcoma virus: identification and molecular cloning of biologically active proviral DNA. J Virol. 1982 Nov;44(2):674–682. [PMC free article] [PubMed]
  • Duncan R, McConkey EH. Preferential utilization of phosphorylated 40-S ribosomal subunits during initiation complex formation. Eur J Biochem. 1982 Apr;123(3):535–538. [PubMed]
  • Edwards DR, Mahadevan LC. Protein synthesis inhibitors differentially superinduce c-fos and c-jun by three distinct mechanisms: lack of evidence for labile repressors. EMBO J. 1992 Jul;11(7):2415–2424. [PubMed]
  • Erikson RL. Structure, expression, and regulation of protein kinases involved in the phosphorylation of ribosomal protein S6. J Biol Chem. 1991 Apr 5;266(10):6007–6010. [PubMed]
  • Feng JL, Villeponteau B. Serum stimulation of the c-fos enhancer induces reversible changes in c-fos chromatin structure. Mol Cell Biol. 1990 Mar;10(3):1126–1133. [PMC free article] [PubMed]
  • Ferrari S, Bannwarth W, Morley SJ, Totty NF, Thomas G. Activation of p70s6k is associated with phosphorylation of four clustered sites displaying Ser/Thr-Pro motifs. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7282–7286. [PubMed]
  • Franco R, Rosenfeld MG. Hormonally inducible phosphorylation of a nuclear pool of ribosomal protein S6. J Biol Chem. 1990 Mar 15;265(8):4321–4325. [PubMed]
  • Garrels JI. Quantitative two-dimensional gel electrophoresis of proteins. Methods Enzymol. 1983;100:411–423. [PubMed]
  • Gille H, Sharrocks AD, Shaw PE. Phosphorylation of transcription factor p62TCF by MAP kinase stimulates ternary complex formation at c-fos promoter. Nature. 1992 Jul 30;358(6385):414–417. [PubMed]
  • Greenberg ME, Hermanowski AL, Ziff EB. Effect of protein synthesis inhibitors on growth factor activation of c-fos, c-myc, and actin gene transcription. Mol Cell Biol. 1986 Apr;6(4):1050–1057. [PMC free article] [PubMed]
  • Greenberg ME, Ziff EB. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature. 1984 Oct 4;311(5985):433–438. [PubMed]
  • Greif H, Ben-Chaim J, Shimon T, Bechor E, Eldar H, Livneh E. The protein kinase C-related PKC-L(eta) gene product is localized in the cell nucleus. Mol Cell Biol. 1992 Mar;12(3):1304–1311. [PMC free article] [PubMed]
  • Hall FL, Braun RK, Mihara K, Fung YK, Berndt N, Carbonaro-Hall DA, Vulliet PR. Characterization of the cytoplasmic proline-directed protein kinase in proliferative cells and tissues as a heterodimer comprised of p34cdc2 and p58cyclin A. J Biol Chem. 1991 Sep 15;266(26):17430–17440. [PubMed]
  • Huang JT, Schneider RJ. Adenovirus inhibition of cellular protein synthesis is prevented by the drug 2-aminopurine. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7115–7119. [PubMed]
  • Kozma SC, Lane HA, Ferrari S, Luther H, Siegmann M, Thomas G. A stimulated S6 kinase from rat liver: identity with the mitogen activated S6 kinase of 3T3 cells. EMBO J. 1989 Dec 20;8(13):4125–4132. [PubMed]
  • Kyriakis JM, App H, Zhang XF, Banerjee P, Brautigan DL, Rapp UR, Avruch J. Raf-1 activates MAP kinase-kinase. Nature. 1992 Jul 30;358(6385):417–421. [PubMed]
  • Kyriakis JM, Avruch J. pp54 microtubule-associated protein 2 kinase. A novel serine/threonine protein kinase regulated by phosphorylation and stimulated by poly-L-lysine. J Biol Chem. 1990 Oct 5;265(28):17355–17363. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Lalanne JL, Lucero M, le Moullec JM. Complete sequence of mouse S6 ribosomal protein. Nucleic Acids Res. 1987 Jun 25;15(12):4990–4990. [PMC free article] [PubMed]
  • Lane HA, Fernandez A, Lamb NJ, Thomas G. p70s6k function is essential for G1 progression. Nature. 1993 May 13;363(6425):170–172. [PubMed]
  • Leevers SJ, Marshall CJ. Activation of extracellular signal-regulated kinase, ERK2, by p21ras oncoprotein. EMBO J. 1992 Feb;11(2):569–574. [PubMed]
  • Macfarlane DE, Gailani D. Identification of phosphoprotein NP33 as a nucleus-associated ribosomal S6 protein and its phosphorylation in hematopoietic cells. Cancer Res. 1990 May 15;50(10):2895–2900. [PubMed]
  • Mahadevan LC, Edwards DR. Signalling and superinduction. Nature. 1991 Feb 28;349(6312):747–748. [PubMed]
  • Mahadevan LC, Targett K, Heath JK. 2-Aminopurine abolishes epidermal growth factor-stimulated phosphorylation of complexed and chromatin-associated forms of a 33 kDa phosphoprotein. Oncogene. 1989 Jun;4(6):699–706. [PubMed]
  • Mahadevan LC, Willis AC, Barratt MJ. Rapid histone H3 phosphorylation in response to growth factors, phorbol esters, okadaic acid, and protein synthesis inhibitors. Cell. 1991 May 31;65(5):775–783. [PubMed]
  • Mahadevan LC, Wills AJ, Hirst EA, Rathjen PD, Heath JK. 2-Aminopurine abolishes EGF- and TPA-stimulated pp33 phosphorylation and c-fos induction without affecting the activation of protein kinase C. Oncogene. 1990 Mar;5(3):327–335. [PubMed]
  • Marais R, Wynne J, Treisman R. The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell. 1993 Apr 23;73(2):381–393. [PubMed]
  • Mukhopadhyay NK, Price DJ, Kyriakis JM, Pelech S, Sanghera J, Avruch J. An array of insulin-activated, proline-directed serine/threonine protein kinases phosphorylate the p70 S6 kinase. J Biol Chem. 1992 Feb 15;267(5):3325–3335. [PubMed]
  • Nishida E, Gotoh Y. The MAP kinase cascade is essential for diverse signal transduction pathways. Trends Biochem Sci. 1993 Apr;18(4):128–131. [PubMed]
  • Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature. 1988 Aug 25;334(6184):661–665. [PubMed]
  • O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed]
  • O'Neill RE, Racaniello VR. Inhibition of translation in cells infected with a poliovirus 2Apro mutant correlates with phosphorylation of the alpha subunit of eucaryotic initiation factor 2. J Virol. 1989 Dec;63(12):5069–5075. [PMC free article] [PubMed]
  • Pardee AB. G1 events and regulation of cell proliferation. Science. 1989 Nov 3;246(4930):603–608. [PubMed]
  • Price DJ, Grove JR, Calvo V, Avruch J, Bierer BE. Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase. Science. 1992 Aug 14;257(5072):973–977. [PubMed]
  • Reinhard C, Thomas G, Kozma SC. A single gene encodes two isoforms of the p70 S6 kinase: activation upon mitogenic stimulation. Proc Natl Acad Sci U S A. 1992 May 1;89(9):4052–4056. [PubMed]
  • Ryseck RP, Hirai SI, Yaniv M, Bravo R. Transcriptional activation of c-jun during the G0/G1 transition in mouse fibroblasts. Nature. 1988 Aug 11;334(6182):535–537. [PubMed]
  • Thomas G, Martin-Pérez J, Siegmann M, Otto AM. The effect of serum, EGF, PGF2 alpha and insulin on S6 phosphorylation and the initiation of protein and DNA synthesis. Cell. 1982 Aug;30(1):235–242. [PubMed]
  • Warner JR. The nucleolus and ribosome formation. Curr Opin Cell Biol. 1990 Jun;2(3):521–527. [PubMed]
  • Zinck R, Hipskind RA, Pingoud V, Nordheim A. c-fos transcriptional activation and repression correlate temporally with the phosphorylation status of TCF. EMBO J. 1993 Jun;12(6):2377–2387. [PubMed]
  • Zinn K, Keller A, Whittemore LA, Maniatis T. 2-Aminopurine selectively inhibits the induction of beta-interferon, c-fos, and c-myc gene expression. Science. 1988 Apr 8;240(4849):210–213. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)