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. 1995 Feb 28;92(5):1421–1425. doi: 10.1073/pnas.92.5.1421

Direct interaction and N-terminal phosphorylation of c-Jun by c-Mil/Raf.

G Radziwill 1, M Niehof 1, C Rommel 1, K Moelling 1
PMCID: PMC42531  PMID: 7877994

Abstract

c-Mil is the avian homologue of the mammalian serine/threonine kinase c-Raf-1. c-Mil/Raf is a mediator of signal transduction leading to gene expression via the c-Jun DNA-binding site, AP-1. Here we show that c-Mil immunopurified from MC29-virus-transformed quail fibroblasts phosphorylates c-Jun in vitro near its N terminus (Ser-63 and -73). Furthermore, the viral oncogene product Gag-Mil of the avian wild-type retrovirus MH2 phosphorylates c-Jun in vitro. A contribution by other known kinases phosphorylating c-Jun, such as the mitogen-activated protein kinases (MAPKs) and the c-Jun N-terminal kinases, was excluded by control reactions. c-Raf-1 and c-Jun directly interact in vitro as shown by various immobilized glutathione S-transferase-Raf fusion proteins which specify the cysteine-rich region of c-Mil/Raf as the major N-terminal binding site. An additional minor binding site is located in the C-terminal region. The biological relevance of these results is demonstrated by coimmunoprecipitation of c-Jun and c-Mil from 32P-labeled MC29- and MH2-transformed fibroblasts as well as normal quail embryo fibroblasts, whereby c-Jun was identified by tryptic phosphopeptide analysis. The complexed c-Jun exhibits a decreased electrophoretic mobility corresponding to a more highly phosphorylated state. Cell fractionation analyses indicate that the c-Mil/c-Jun complex is located in the cytoplasm. The data demonstrate that c-Jun can be a direct target of the protein kinase c-Mil/Raf, suggesting an alternative pathway, which leads to c-Jun phosphorylation independent of the MAPKs and MAPK-related proteins.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adler V., Polotskaya A., Wagner F., Kraft A. S. Affinity-purified c-Jun amino-terminal protein kinase requires serine/threonine phosphorylation for activity. J Biol Chem. 1992 Aug 25;267(24):17001–17005. [PubMed] [Google Scholar]
  2. Alvarez E., Northwood I. C., Gonzalez F. A., Latour D. A., Seth A., Abate C., Curran T., Davis R. J. Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation. Characterization of the phosphorylation of c-myc and c-jun proteins by an epidermal growth factor receptor threonine 669 protein kinase. J Biol Chem. 1991 Aug 15;266(23):15277–15285. [PubMed] [Google Scholar]
  3. Angel P., Hattori K., Smeal T., Karin M. The jun proto-oncogene is positively autoregulated by its product, Jun/AP-1. Cell. 1988 Dec 2;55(5):875–885. doi: 10.1016/0092-8674(88)90143-2. [DOI] [PubMed] [Google Scholar]
  4. Black E. J., Street A. J., Gillespie D. A. Protein phosphatase 2A reverses phosphorylation of c-Jun specified by the delta domain in vitro: correlation with oncogenic activation and deregulated transactivation activity of v-Jun. Oncogene. 1991 Nov;6(11):1949–1958. [PubMed] [Google Scholar]
  5. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  6. Bruder J. T., Heidecker G., Rapp U. R. Serum-, TPA-, and Ras-induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase. Genes Dev. 1992 Apr;6(4):545–556. doi: 10.1101/gad.6.4.545. [DOI] [PubMed] [Google Scholar]
  7. Catling A. D., Wyke J. A., Frame M. C. Mitogenesis of quiescent chick fibroblasts by v-Src: dependence on events at the membrane leading to early changes in AP-1. Oncogene. 1993 Jul;8(7):1875–1886. [PubMed] [Google Scholar]
  8. Chou S. Y., Baichwal V., Ferrell J. E., Jr Inhibition of c-Jun DNA binding by mitogen-activated protein kinase. Mol Biol Cell. 1992 Oct;3(10):1117–1130. doi: 10.1091/mbc.3.10.1117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clark-Lewis I., Sanghera J. S., Pelech S. L. Definition of a consensus sequence for peptide substrate recognition by p44mpk, the meiosis-activated myelin basic protein kinase. J Biol Chem. 1991 Aug 15;266(23):15180–15184. [PubMed] [Google Scholar]
  10. Cook S. J., McCormick F. Inhibition by cAMP of Ras-dependent activation of Raf. Science. 1993 Nov 12;262(5136):1069–1072. doi: 10.1126/science.7694367. [DOI] [PubMed] [Google Scholar]
  11. Denhez F., Heimann B., d'Auriol L., Graf T., Coquillaud M., Coll J., Galibert F., Moelling K., Stehelin D., Ghysdael J. Replacement of lys 622 in the ATP binding domain of P100gag-mil abolishes the in vitro autophosphorylation of the protein and the biological properties of the v-mil oncogene of MH2 virus. EMBO J. 1988 Feb;7(2):541–546. doi: 10.1002/j.1460-2075.1988.tb02843.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Drewes G., Lichtenberg-Kraag B., Döring F., Mandelkow E. M., Biernat J., Goris J., Dorée M., Mandelkow E. Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state. EMBO J. 1992 Jun;11(6):2131–2138. doi: 10.1002/j.1460-2075.1992.tb05272.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dérijard B., Hibi M., Wu I. H., Barrett T., Su B., Deng T., Karin M., Davis R. J. JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell. 1994 Mar 25;76(6):1025–1037. doi: 10.1016/0092-8674(94)90380-8. [DOI] [PubMed] [Google Scholar]
  14. Fabian J. R., Vojtek A. B., Cooper J. A., Morrison D. K. A single amino acid change in Raf-1 inhibits Ras binding and alters Raf-1 function. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5982–5986. doi: 10.1073/pnas.91.13.5982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gallego C., Gupta S. K., Heasley L. E., Qian N. X., Johnson G. L. Mitogen-activated protein kinase activation resulting from selective oncogene expression in NIH 3T3 and rat 1a cells. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7355–7359. doi: 10.1073/pnas.89.16.7355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hallberg B., Rayter S. I., Downward J. Interaction of Ras and Raf in intact mammalian cells upon extracellular stimulation. J Biol Chem. 1994 Feb 11;269(6):3913–3916. [PubMed] [Google Scholar]
  17. Hibi M., Lin A., Smeal T., Minden A., Karin M. Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev. 1993 Nov;7(11):2135–2148. doi: 10.1101/gad.7.11.2135. [DOI] [PubMed] [Google Scholar]
  18. Kolch W., Heidecker G., Kochs G., Hummel R., Vahidi H., Mischak H., Finkenzeller G., Marmé D., Rapp U. R. Protein kinase C alpha activates RAF-1 by direct phosphorylation. Nature. 1993 Jul 15;364(6434):249–252. doi: 10.1038/364249a0. [DOI] [PubMed] [Google Scholar]
  19. Kyriakis J. M., Banerjee P., Nikolakaki E., Dai T., Rubie E. A., Ahmad M. F., Avruch J., Woodgett J. R. The stress-activated protein kinase subfamily of c-Jun kinases. Nature. 1994 May 12;369(6476):156–160. doi: 10.1038/369156a0. [DOI] [PubMed] [Google Scholar]
  20. Lovrić J., Bischof O., Moelling K. Cell cycle-dependent association of Gag-Mil and hsp90. FEBS Lett. 1994 Apr 18;343(1):15–21. doi: 10.1016/0014-5793(94)80598-9. [DOI] [PubMed] [Google Scholar]
  21. Moelling K., Heimann B., Beimling P., Rapp U. R., Sander T. Serine- and threonine-specific protein kinase activities of purified gag-mil and gag-raf proteins. Nature. 1984 Dec 6;312(5994):558–561. doi: 10.1038/312558a0. [DOI] [PubMed] [Google Scholar]
  22. Moodie S. A., Wolfman A. The 3Rs of life: Ras, Raf and growth regulation. Trends Genet. 1994 Feb;10(2):44–48. doi: 10.1016/0168-9525(94)90147-3. [DOI] [PubMed] [Google Scholar]
  23. Morrison D. K., Kaplan D. R., Escobedo J. A., Rapp U. R., Roberts T. M., Williams L. T. Direct activation of the serine/threonine kinase activity of Raf-1 through tyrosine phosphorylation by the PDGF beta-receptor. Cell. 1989 Aug 25;58(4):649–657. doi: 10.1016/0092-8674(89)90100-1. [DOI] [PubMed] [Google Scholar]
  24. Niehof M., Beimling P., Moelling K. The aminoterminus of c-Raf-1 binds a 28-kD cellular protein. Biochem Biophys Res Commun. 1994 Sep 30;203(3):1560–1566. doi: 10.1006/bbrc.1994.2364. [DOI] [PubMed] [Google Scholar]
  25. Pulverer B. J., Kyriakis J. M., Avruch J., Nikolakaki E., Woodgett J. R. Phosphorylation of c-jun mediated by MAP kinases. Nature. 1991 Oct 17;353(6345):670–674. doi: 10.1038/353670a0. [DOI] [PubMed] [Google Scholar]
  26. Rapp U. R. Role of Raf-1 serine/threonine protein kinase in growth factor signal transduction. Oncogene. 1991 Apr;6(4):495–500. [PubMed] [Google Scholar]
  27. Schramm K., Niehof M., Radziwill G., Rommel C., Moelling K. Phosphorylation of c-Raf-1 by protein kinase A interferes with activation. Biochem Biophys Res Commun. 1994 Jun 15;201(2):740–747. doi: 10.1006/bbrc.1994.1763. [DOI] [PubMed] [Google Scholar]
  28. Siegel J. N., Klausner R. D., Rapp U. R., Samelson L. E. T cell antigen receptor engagement stimulates c-raf phosphorylation and induces c-raf-associated kinase activity via a protein kinase C-dependent pathway. J Biol Chem. 1990 Oct 25;265(30):18472–18480. [PubMed] [Google Scholar]
  29. Smeal T., Binetruy B., Mercola D., Grover-Bardwick A., Heidecker G., Rapp U. R., Karin M. Oncoprotein-mediated signalling cascade stimulates c-Jun activity by phosphorylation of serines 63 and 73. Mol Cell Biol. 1992 Aug;12(8):3507–3513. doi: 10.1128/mcb.12.8.3507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Vogt P. K., Bos T. J. jun: oncogene and transcription factor. Adv Cancer Res. 1990;55:1–35. doi: 10.1016/s0065-230x(08)60466-2. [DOI] [PubMed] [Google Scholar]
  31. Wartmann M., Davis R. J. The native structure of the activated Raf protein kinase is a membrane-bound multi-subunit complex. J Biol Chem. 1994 Mar 4;269(9):6695–6701. [PubMed] [Google Scholar]
  32. Wasylyk C., Wasylyk B., Heidecker G., Huleihel M., Rapp U. R. Expression of raf oncogenes activates the PEA1 transcription factor motif. Mol Cell Biol. 1989 May;9(5):2247–2250. doi: 10.1128/mcb.9.5.2247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Westwick J. K., Cox A. D., Der C. J., Cobb M. H., Hibi M., Karin M., Brenner D. A. Oncogenic Ras activates c-Jun via a separate pathway from the activation of extracellular signal-regulated kinases. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6030–6034. doi: 10.1073/pnas.91.13.6030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wood K. W., Sarnecki C., Roberts T. M., Blenis J. ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK. Cell. 1992 Mar 20;68(6):1041–1050. doi: 10.1016/0092-8674(92)90076-o. [DOI] [PubMed] [Google Scholar]
  35. Wu J., Dent P., Jelinek T., Wolfman A., Weber M. J., Sturgill T. W. Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate. Science. 1993 Nov 12;262(5136):1065–1069. doi: 10.1126/science.7694366. [DOI] [PubMed] [Google Scholar]

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