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. 1995 Aug;15(8):4125–4135. doi: 10.1128/mcb.15.8.4125

Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro.

P Dent 1, D B Reardon 1, D K Morrison 1, T W Sturgill 1
PMCID: PMC230651  PMID: 7623807

Abstract

The serine/threonine kinase Raf-1 functions downstream from Ras to activate mitogen-activated protein kinase kinase, but the mechanisms of Raf-1 activation are incompletely understood. To dissect these mechanisms, wild-type and mutant Raf-1 proteins were studied in an in vitro system with purified plasma membranes from v-Ras- and v-Src-transformed cells (transformed membranes). Wild-type (His)6- and FLAG-Raf-1 were activated in a Ras- and ATP-dependent manner by transformed membranes; however, Raf-1 proteins that are kinase defective (K375M), that lack an in vivo site(s) of regulatory tyrosine (YY340/341FF) or constitutive serine (S621A) phosphorylation, that do not bind Ras (R89L), or that lack an intact zinc finger (CC165/168SS) were not. Raf-1 proteins lacking putative regulatory sites for an unidentified kinase (S259A) or protein kinase C (S499A) were activated but with apparently reduced efficiency. The kinase(s) responsible for activation by Ras or Src may reside in the plasma membrane, since GTP loading of plasma membranes from quiescent NIH 3T3 cells (parental membranes) induced de novo capacity to activate Raf-1. Wild-type Raf-1, possessing only basal activity, was not activated by parental membranes in the absence of GTP loading. In contrast, Raf-1 Y340D, possessing significant activity, was, surprisingly, stimulated by parental membranes in a Ras-independent manner. The results suggest that activation of Raf-1 by phosphorylation may be permissive for further modulation by another membrane factor, such as a lipid. A factor(s) extracted with methanol-chloroform from transformed membranes or membranes from Sf9 cells coexpressing Ras and SrcY527F significantly enhanced the activity of Raf-1 Y340D or active Raf-1 but not that of inactive Raf-1. Our findings suggest a model for activation of Raf-1, wherein (i) Raf-1 associates with Ras-GTP, (ii) Raf-1 is activated by tyrosine and/or serine phosphorylation, and (iii) Raf-1 activity is further increased by a membrane cofactor.

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Selected References

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  1. Beimling P., Niehof M., Radziwill G., Moelling K. The aminoterminus of c-Raf-1 binds a protein kinase phosphorylating Ser259. Biochem Biophys Res Commun. 1994 Oct 28;204(2):841–848. doi: 10.1006/bbrc.1994.2536. [DOI] [PubMed] [Google Scholar]
  2. Cai H., Erhardt P., Troppmair J., Diaz-Meco M. T., Sithanandam G., Rapp U. R., Moscat J., Cooper G. M. Hydrolysis of phosphatidylcholine couples Ras to activation of Raf protein kinase during mitogenic signal transduction. Mol Cell Biol. 1993 Dec;13(12):7645–7651. doi: 10.1128/mcb.13.12.7645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carnero A., Lacal J. C. Activation of intracellular kinases in Xenopus oocytes by p21ras and phospholipases: a comparative study. Mol Cell Biol. 1995 Feb;15(2):1094–1101. doi: 10.1128/mcb.15.2.1094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Daum G., Eisenmann-Tappe I., Fries H. W., Troppmair J., Rapp U. R. The ins and outs of Raf kinases. Trends Biochem Sci. 1994 Nov;19(11):474–480. doi: 10.1016/0968-0004(94)90133-3. [DOI] [PubMed] [Google Scholar]
  5. Davis R. J. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem. 1993 Jul 15;268(20):14553–14556. [PubMed] [Google Scholar]
  6. Dent P., Chow Y. H., Wu J., Morrison D. K., Jove R., Sturgill T. W. Expression, purification and characterization of recombinant mitogen-activated protein kinase kinases. Biochem J. 1994 Oct 1;303(Pt 1):105–112. doi: 10.1042/bj3030105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dent P., Sturgill T. W. Activation of (His)6-Raf-1 in vitro by partially purified plasma membranes from v-Ras-transformed and serum-stimulated fibroblasts. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9544–9548. doi: 10.1073/pnas.91.20.9544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Diaz-Laviada I., Larrodera P., Diaz-Meco M. T., Cornet M. E., Guddal P. H., Johansen T., Moscat J. Evidence for a role of phosphatidylcholine-hydrolysing phospholipase C in the regulation of protein kinase C by ras and src oncogenes. EMBO J. 1990 Dec;9(12):3907–3912. doi: 10.1002/j.1460-2075.1990.tb07611.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fabian J. R., Daar I. O., Morrison D. K. Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase. Mol Cell Biol. 1993 Nov;13(11):7170–7179. doi: 10.1128/mcb.13.11.7170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fabian J. R., Morrison D. K., Daar I. O. Requirement for Raf and MAP kinase function during the meiotic maturation of Xenopus oocytes. J Cell Biol. 1993 Aug;122(3):645–652. doi: 10.1083/jcb.122.3.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Force T., Bonventre J. V., Heidecker G., Rapp U., Avruch J., Kyriakis J. M. Enzymatic characteristics of the c-Raf-1 protein kinase. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1270–1274. doi: 10.1073/pnas.91.4.1270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fu H., Xia K., Pallas D. C., Cui C., Conroy K., Narsimhan R. P., Mamon H., Collier R. J., Roberts T. M. Interaction of the protein kinase Raf-1 with 14-3-3 proteins. Science. 1994 Oct 7;266(5182):126–129. doi: 10.1126/science.7939632. [DOI] [PubMed] [Google Scholar]
  14. Gardner A. M., Lange-Carter C. A., Vaillancourt R. R., Johnson G. L. Measuring activation of kinases in mitogen-activated protein kinase regulatory network. Methods Enzymol. 1994;238:258–270. doi: 10.1016/0076-6879(94)38024-4. [DOI] [PubMed] [Google Scholar]
  15. Ghosh S., Bell R. M. Identification of discrete segments of human Raf-1 kinase critical for high affinity binding to Ha-Ras. J Biol Chem. 1994 Dec 9;269(49):30785–30788. [PubMed] [Google Scholar]
  16. Ghosh S., Xie W. Q., Quest A. F., Mabrouk G. M., Strum J. C., Bell R. M. The cysteine-rich region of raf-1 kinase contains zinc, translocates to liposomes, and is adjacent to a segment that binds GTP-ras. J Biol Chem. 1994 Apr 1;269(13):10000–10007. [PubMed] [Google Scholar]
  17. Jelinek T., Weber M. J. Optimization of the resolution of phosphoamino acids by one-dimensional thin-layer electrophoresis. Biotechniques. 1993 Oct;15(4):628–630. [PubMed] [Google Scholar]
  18. Kamps M. P., Sefton B. M. Acid and base hydrolysis of phosphoproteins bound to immobilon facilitates analysis of phosphoamino acids in gel-fractionated proteins. Anal Biochem. 1989 Jan;176(1):22–27. doi: 10.1016/0003-2697(89)90266-2. [DOI] [PubMed] [Google Scholar]
  19. Kanoh H., Sakane F., Imai S., Wada I. Diacylglycerol kinase and phosphatidic acid phosphatase--enzymes metabolizing lipid second messengers. Cell Signal. 1993 Sep;5(5):495–503. doi: 10.1016/0898-6568(93)90045-n. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Leevers S. J., Paterson H. F., Marshall C. J. Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature. 1994 Jun 2;369(6479):411–414. doi: 10.1038/369411a0. [DOI] [PubMed] [Google Scholar]
  22. Lin T. A., Kong X., Haystead T. A., Pause A., Belsham G., Sonenberg N., Lawrence J. C., Jr PHAS-I as a link between mitogen-activated protein kinase and translation initiation. Science. 1994 Oct 28;266(5185):653–656. doi: 10.1126/science.7939721. [DOI] [PubMed] [Google Scholar]
  23. Lowy D. R., Willumsen B. M. Function and regulation of ras. Annu Rev Biochem. 1993;62:851–891. doi: 10.1146/annurev.bi.62.070193.004223. [DOI] [PubMed] [Google Scholar]
  24. Marshall C. J. MAP kinase kinase kinase, MAP kinase kinase and MAP kinase. Curr Opin Genet Dev. 1994 Feb;4(1):82–89. doi: 10.1016/0959-437x(94)90095-7. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Morrison D. K., Heidecker G., Rapp U. R., Copeland T. D. Identification of the major phosphorylation sites of the Raf-1 kinase. J Biol Chem. 1993 Aug 15;268(23):17309–17316. [PubMed] [Google Scholar]
  27. Pumiglia K., Chow Y. H., Fabian J., Morrison D., Decker S., Jove R. Raf-1 N-terminal sequences necessary for Ras-Raf interaction and signal transduction. Mol Cell Biol. 1995 Jan;15(1):398–406. doi: 10.1128/mcb.15.1.398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stacey D. W., Tsai M. H., Yu C. L., Smith J. K. Critical role of cellular ras proteins in proliferative signal transduction. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 2):871–881. doi: 10.1101/sqb.1988.053.01.100. [DOI] [PubMed] [Google Scholar]
  29. Stancato L. F., Chow Y. H., Hutchison K. A., Perdew G. H., Jove R., Pratt W. B. Raf exists in a native heterocomplex with hsp90 and p50 that can be reconstituted in a cell-free system. J Biol Chem. 1993 Oct 15;268(29):21711–21716. [PubMed] [Google Scholar]
  30. Stokoe D., Macdonald S. G., Cadwallader K., Symons M., Hancock J. F. Activation of Raf as a result of recruitment to the plasma membrane. Science. 1994 Jun 3;264(5164):1463–1467. doi: 10.1126/science.7811320. [DOI] [PubMed] [Google Scholar]
  31. Troppmair J., Bruder J. T., Munoz H., Lloyd P. A., Kyriakis J., Banerjee P., Avruch J., Rapp U. R. Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation by oncogenes, serum, and 12-O-tetradecanoylphorbol-13-acetate requires Raf and is necessary for transformation. J Biol Chem. 1994 Mar 4;269(9):7030–7035. [PubMed] [Google Scholar]
  32. Turner B., Rapp U., App H., Greene M., Dobashi K., Reed J. Interleukin 2 induces tyrosine phosphorylation and activation of p72-74 Raf-1 kinase in a T-cell line. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1227–1231. doi: 10.1073/pnas.88.4.1227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Williams N. G., Roberts T. M., Li P. Both p21ras and pp60v-src are required, but neither alone is sufficient, to activate the Raf-1 kinase. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2922–2926. doi: 10.1073/pnas.89.7.2922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Williams N. G., Roberts T. M. Signal transduction pathways involving the Raf proto-oncogene. Cancer Metastasis Rev. 1994 Mar;13(1):105–116. doi: 10.1007/BF00690421. [DOI] [PubMed] [Google Scholar]
  35. Wolfman A., Macara I. G. Elevated levels of diacylglycerol and decreased phorbol ester sensitivity in ras-transformed fibroblasts. Nature. 1987 Jan 22;325(6102):359–361. doi: 10.1038/325359a0. [DOI] [PubMed] [Google Scholar]
  36. Zhang X. F., Settleman J., Kyriakis J. M., Takeuchi-Suzuki E., Elledge S. J., Marshall M. S., Bruder J. T., Rapp U. R., Avruch J. Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1. Nature. 1993 Jul 22;364(6435):308–313. doi: 10.1038/364308a0. [DOI] [PubMed] [Google Scholar]

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