Home LiteratureArticle Details
PMID: 7760835 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

14-3-3 is not essential for Raf-1 function: identification of Raf-1 proteins that are biologically activated in a 14-3-3- and Ras-independent manner.

Molecular and cellular biology ·Vol. 15 ·No. 6 ·1995-06-00 ·Pages 3390-7

Michaud NR, Fabian JR, Mathes KD, Morrison DK

Abstract

Recent reports have demonstrated the in vivo association of Raf-1 with members of the 14-3-3 protein family. To address the significance of the Raf-1-14-3-3 interaction, we investigated the enzymatic activity and biological function of Raf-1 in the presence and absence of associated 14-3-3. The interaction between these two molecules was disrupted in vivo and in vitro with a combination of molecular and biochemical techniques. Biochemical studies demonstrated that the enzymatic activities of Raf-1 were equivalent in the presence and absence of 14-3-3. Furthermore, mixing of purified Raf-1 and 14-3-3 in vitro was not sufficient to activate Raf-1. With a molecular approach, Cys-165 and Cys-168 as well as Ser-259 were identified as residues of Raf-1 required for the interaction with 14-3-3. Cys-165 and Cys-168 are located within the conserved cysteine-rich region of the CR1 domain, and Ser-259 is a conserved site of serine phosphorylation found within the CR2 domain. Mutation of either Cys-165 and Cys-168 or Ser-259 prevented the stable interaction of Raf-1 with 14-3-3 in vivo. Consistent with the model in which a site of serine phosphorylation is involved in the Raf-1-14-3-3 interaction, dephosphorylated Raf-1 was unable to associate with 14-3-3 in vitro. Phosphorylation may represent a general mechanism mediating 14-3-3 binding, because dephosphorylation of the Bcr kinase (known to interact with 14-3-3) also eliminated its association with 14-3-3. Finally, mutant Raf-1 proteins unable to stably interact with 14-3-3 exhibited enhanced enzymatic activity in human 293 cells and Xenopus oocytes and were biologically activated, as demonstrated by their ability to induced meiotic maturation of Xenopus oocytes. However, in contrast to wild-type Raf-1, activation of these mutants was independent of Ras. Our results therefore indicate that interaction with 14-3-3 is not essential for Raf-1 function.

MeSH Terms
14-3-3 Proteins Animals Cell Line Enzyme Activation Humans Mutation Phosphorylation Protein Serine-Threonine Kinases/chemistry,genetics,metabolism Proteins/metabolism Proto-Oncogene Proteins/chemistry,genetics,metabolism Proto-Oncogene Proteins c-raf Tyrosine 3-Monooxygenase Xenopus
Chemicals
14-3-3 Proteins Proteins Proto-Oncogene Proteins Tyrosine 3-Monooxygenase Protein Serine-Threonine Kinases Proto-Oncogene Proteins c-raf
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Michaud N R
Molecular Mechanisms of Carcinogenesis Laboratory, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702-1201, USA.
Fabian J R
Mathes K D
Morrison D K
References (49)
49 references, click to expand
  1. The Raf-1 kinase as a transducer of mitogenic signals.
    Cancer Cells. 1990 Dec;2(12):377-82 PMID: 2150916
  2. Expression, purification and characterization of recombinant mitogen-activated protein kinase kinases.
    Biochem J. 1994 Oct 1;303 ( Pt 1):105-12 PMID: 7945229
  3. Association of pRas and pRaf-1 in a complex correlates with activation of a signal transduction pathway.
    Curr Biol. 1993 Dec 1;3(12):805-12 PMID: 15335813
  4. Role of Raf-1 serine/threonine protein kinase in growth factor signal transduction.
    Oncogene. 1991 Apr;6(4):495-500 PMID: 2030909
  5. 14-3-3: modulators of signaling proteins?
    Science. 1994 Oct 7;266(5182):56-7 PMID: 7939645
  6. Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane.
    Nature. 1994 Jun 2;369(6479):411-4 PMID: 8196769
  7. A protein kinase similar to MAP kinase activator acts downstream of the raf kinase in Drosophila.
    Cell. 1993 Feb 12;72(3):407-14 PMID: 8381718
  8. Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro.
    Nature. 1993 Jul 22;364(6435):352-5 PMID: 8332195
  9. Raf functions downstream of Ras1 in the Sevenless signal transduction pathway.
    Nature. 1992 Dec 10;360(6404):600-3 PMID: 1461284
  10. The eukaryotic host factor that activates exoenzyme S of Pseudomonas aeruginosa is a member of the 14-3-3 protein family.
    Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2320-4 PMID: 8460141
  11. Protein kinase C alpha activates RAF-1 by direct phosphorylation.
    Nature. 1993 Jul 15;364(6434):249-52 PMID: 8321321
  12. Demonstration of the phosphorylation-dependent interaction of tryptophan hydroxylase with the 14-3-3 protein.
    Biochem Biophys Res Commun. 1993 Jul 15;194(1):144-9 PMID: 8101440
  13. Stimulatory effects of yeast and mammalian 14-3-3 proteins on the Raf protein kinase.
    Science. 1994 Sep 16;265(5179):1716-9 PMID: 8085159
  14. Cell biology. A signal chain of events.
    Nature. 1992 Dec 10;360(6404):534-5 PMID: 1334231
  15. 14-3-3 protein homologs required for the DNA damage checkpoint in fission yeast.
    Science. 1994 Jul 22;265(5171):533-5 PMID: 8036497
  16. Activation of Raf as a result of recruitment to the plasma membrane.
    Science. 1994 Jun 3;264(5164):1463-7 PMID: 7811320
  17. Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase.
    Mol Cell Biol. 1993 Nov;13(11):7170-9 PMID: 7692235
  18. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor.
    Cell. 1978 Jul;14(3):725-31 PMID: 210957
  19. Association of polyomavirus middle tumor antigen with 14-3-3 proteins.
    Science. 1994 Jul 22;265(5171):535-7 PMID: 8036498
  20. 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):9544-8 PMID: 7937802
  21. The 3Rs of life: Ras, Raf and growth regulation.
    Trends Genet. 1994 Feb;10(2):44-8 PMID: 8191584
  22. Mutational activation of c-raf-1 and definition of the minimal transforming sequence.
    Mol Cell Biol. 1990 Jun;10(6):2503-12 PMID: 2188091
  23. Definition of the human raf amino-terminal regulatory region by deletion mutagenesis.
    Mol Cell Biol. 1989 Feb;9(2):639-47 PMID: 2710120
  24. Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1.
    Nature. 1993 Jul 22;364(6435):308-13 PMID: 8332187
  25. Interaction of Ras and Raf in intact mammalian cells upon extracellular stimulation.
    J Biol Chem. 1994 Feb 11;269(6):3913-6 PMID: 8307946
  26. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs.
    Nucleic Acids Res. 1984 Sep 25;12(18):7057-70 PMID: 6207484
  27. GTP-dependent association of Raf-1 with Ha-Ras: identification of Raf as a target downstream of Ras in mammalian cells.
    Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8683-6 PMID: 8378348
  28. Identification of the major phosphorylation sites of the Raf-1 kinase.
    J Biol Chem. 1993 Aug 15;268(23):17309-16 PMID: 8349614
  29. Requirement for Raf and MAP kinase function during the meiotic maturation of Xenopus oocytes.
    J Cell Biol. 1993 Aug;122(3):645-52 PMID: 8335690
  30. Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase.
    Science. 1993 Jun 11;260(5114):1658-61 PMID: 8503013
  31. Raf-1 interacts with Fyn and Src in a non-phosphotyrosine-dependent manner.
    J Biol Chem. 1994 Jul 1;269(26):17749-55 PMID: 7517401
  32. The role of Raf-1 phosphorylation in signal transduction.
    Adv Cancer Res. 1992;58:53-73 PMID: 1312290
  33. Activation of Raf-1 by 14-3-3 proteins.
    Nature. 1994 Oct 13;371(6498):612-4 PMID: 7935795
  34. Interaction of the protein kinase Raf-1 with 14-3-3 proteins.
    Science. 1994 Oct 7;266(5182):126-9 PMID: 7939632
  35. Complex formation between RAS and RAF and other protein kinases.
    Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6213-7 PMID: 8327501
  36. 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):2922-6 PMID: 1372995
  37. Ras controls coupling of growth factor receptors and protein kinase C in the membrane to Raf-1 and B-Raf protein serine kinases in the cytosol.
    Oncogene. 1992 Sep;7(9):1867-73 PMID: 1386920
  38. raf family serine/threonine protein kinases in mitogen signal transduction.
    Cold Spring Harb Symp Quant Biol. 1988;53 Pt 1:173-84 PMID: 2978288
  39. C. elegans lin-45 raf gene participates in let-60 ras-stimulated vulval differentiation.
    Nature. 1993 May 13;363(6425):133-40 PMID: 8483497
  40. Mammalian Ras interacts directly with the serine/threonine kinase Raf.
    Cell. 1993 Jul 16;74(1):205-14 PMID: 8334704
  41. 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):1041-50 PMID: 1312393
  42. Association of the protein kinases c-Bcr and Bcr-Abl with proteins of the 14-3-3 family.
    Science. 1994 Oct 7;266(5182):129-33 PMID: 7939633
  43. Requirement for c-ras proteins during viral oncogene transformation.
    Nature. 1986 Apr 10-16;320(6062):540-3 PMID: 2938016
  44. Raf-1 N-terminal sequences necessary for Ras-Raf interaction and signal transduction.
    Mol Cell Biol. 1995 Jan;15(1):398-406 PMID: 7799948
  45. The MAP kinase cascade is essential for diverse signal transduction pathways.
    Trends Biochem Sci. 1993 Apr;18(4):128-31 PMID: 8388132
  46. 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):5982-6 PMID: 8016101
  47. Binding of 14-3-3 proteins to the protein kinase Raf and effects on its activation.
    Science. 1994 Sep 16;265(5179):1713-6 PMID: 8085158
  48. Mitogen regulation of c-Raf-1 protein kinase activity toward mitogen-activated protein kinase-kinase.
    J Biol Chem. 1993 Jul 25;268(21):16009-19 PMID: 8340422
  49. 14-3-3 proteins: a highly conserved, widespread family of eukaryotic proteins.
    Trends Biochem Sci. 1992 Dec;17(12):498-501 PMID: 1471260
Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.15.6.3390
Published
1995-06-00
Pages
3390-7
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC230573
Subset
IM
Grants
NCI NIH HHS · N01-CO-46000 · United States
External Links
PubMed source DOI: 10.1128/MCB.15.6.3390 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2