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PMID: 10022854 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

p50(cdc37) acting in concert with Hsp90 is required for Raf-1 function.

Molecular and cellular biology ·Vol. 19 ·No. 3 ·1999-03-00 ·Pages 1661-72

Grammatikakis N, Lin JH, Grammatikakis A, Tsichlis PN, Cochran BH

Abstract

Genetic screens in Drosophila have identified p50(cdc37) to be an essential component of the sevenless receptor/mitogen-activated kinase protein (MAPK) signaling pathway, but neither the function nor the target of p50(cdc37) in this pathway has been defined. In this study, we examined the role of p50(cdc37) and its Hsp90 chaperone partner in Raf/Mek/MAPK signaling biochemically. We found that coexpression of wild-type p50(cdc37) with Raf-1 resulted in robust and dose-dependent activation of Raf-1 in Sf9 cells. In addition, p50(cdc37) greatly potentiated v-Src-mediated Raf-1 activation. Moreover, we found that p50(cdc37) is the primary determinant of Hsp90 recruitment to Raf-1. Overexpression of a p50(cdc37) mutant which is unable to recruit Hsp90 into the Raf-1 complex inhibited Raf-1 and MAPK activation by growth factors. Similarly, pretreatment with geldanamycin (GA), an Hsp90-specific inhibitor, prevented both the association of Raf-1 with the p50(cdc37)-Hsp90 heterodimer and Raf-1 kinase activation by serum. Activation of Raf-1 via baculovirus coexpression with oncogenic Src or Ras in Sf9 cells was also strongly inhibited by dominant negative p50(cdc37) or by GA. Thus, formation of a ternary Raf-1-p50(cdc37)-Hsp90 complex is crucial for Raf-1 activity and MAPK pathway signaling. These results provide the first biochemical evidence for the requirement of the p50(cdc37)-Hsp90 complex in protein kinase regulation and for Raf-1 function in particular.

MeSH Terms
Animals Benzoquinones COS Cells Calcium-Calmodulin-Dependent Protein Kinases/metabolism Cell Cycle Proteins/genetics,metabolism Cell Line Chaperonins Chickens Dimerization Drosophila Proteins Enzyme Activation Enzyme Inhibitors/pharmacology Epidermal Growth Factor/metabolism,pharmacology HSP90 Heat-Shock Proteins/genetics,metabolism Humans Lactams, Macrocyclic Molecular Chaperones Proto-Oncogene Proteins c-raf/genetics,metabolism Quinones/pharmacology Rabbits Recombinant Fusion Proteins/genetics,metabolism Signal Transduction Spodoptera
Chemicals
Benzoquinones CDC37 protein, human Cell Cycle Proteins Drosophila Proteins Enzyme Inhibitors HSP90 Heat-Shock Proteins Lactams, Macrocyclic Molecular Chaperones Quinones Recombinant Fusion Proteins Epidermal Growth Factor Proto-Oncogene Proteins c-raf Calcium-Calmodulin-Dependent Protein Kinases Chaperonins geldanamycin
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Grammatikakis N
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA. ngrammat@earthlink.net
Lin J H
Grammatikakis A
Tsichlis P N
Cochran B H
References (80)
80 references, click to expand
  1. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase.
    Gene. 1988 Jul 15;67(1):31-40 PMID: 3047011
  2. Serum-, TPA-, and Ras-induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase.
    Genes Dev. 1992 Apr;6(4):545-56 PMID: 1313769
  3. Mammalian p50Cdc37 is a protein kinase-targeting subunit of Hsp90 that binds and stabilizes Cdk4.
    Genes Dev. 1996 Jun 15;10(12):1491-502 PMID: 8666233
  4. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation.
    Cell. 1995 Jan 27;80(2):179-85 PMID: 7834738
  5. Reversal of Raf-1 activation by purified and membrane-associated protein phosphatases.
    Science. 1995 Jun 30;268(5219):1902-6 PMID: 7604263
  6. A novel glycosaminoglycan-binding protein is the vertebrate homologue of the cell cycle control protein, Cdc37.
    J Biol Chem. 1995 Jul 7;270(27):16198-205 PMID: 7608185
  7. Molecular cloning and cell cycle-dependent expression of a novel gene that is homologous to cdc37.
    DNA Cell Biol. 1995 Dec;14(12):1017-23 PMID: 8534368
  8. 14-3-3: modulators of signaling proteins?
    Science. 1994 Oct 7;266(5182):56-7 PMID: 7939645
  9. The native structure of the activated Raf protein kinase is a membrane-bound multi-subunit complex.
    J Biol Chem. 1994 Mar 4;269(9):6695-701 PMID: 8120027
  10. Cdc37 is required for association of the protein kinase Cdc28 with G1 and mitotic cyclins.
    Proc Natl Acad Sci U S A. 1995 May 9;92(10):4651-5 PMID: 7753858
  11. The heat shock protein 83 (Hsp83) is required for Raf-mediated signalling in Drosophila.
    EMBO J. 1997 Apr 15;16(8):1961-9 PMID: 9155022
  12. Mechanisms regulating Raf-1 activity in signal transduction pathways.
    Mol Reprod Dev. 1995 Dec;42(4):507-14 PMID: 8607983
  13. Mechanism of dimer formation of the 90-kDa heat-shock protein.
    Eur J Biochem. 1995 Oct 1;233(1):1-8 PMID: 7588731
  14. The Hsp90 complex--a super-chaperone machine as a novel drug target.
    Biochem Pharmacol. 1998 Sep 15;56(6):675-82 PMID: 9751071
  15. Reconstitution of the multiprotein complex of pp60src, hsp90, and p50 in a cell-free system.
    J Biol Chem. 1992 Feb 15;267(5):2902-8 PMID: 1310678
  16. Physical interaction of mammalian CDC37 with CDK4.
    J Biol Chem. 1996 Sep 6;271(36):22030-4 PMID: 8703009
  17. Activation of the MAP kinase pathway by the protein kinase raf.
    Cell. 1992 Oct 16;71(2):335-42 PMID: 1330321
  18. Cell cycle-dependent association of Gag-Mil and hsp90.
    FEBS Lett. 1994 Apr 18;343(1):15-21 PMID: 8163010
  19. Nucleotides and two functional states of hsp90.
    J Biol Chem. 1997 Mar 21;272(12):8007-12 PMID: 9065472
  20. The native v-Raf.hsp90.p50 heterocomplex contains a novel immunophilin of the FK506 binding class.
    J Biol Chem. 1994 Sep 2;269(35):22157-61 PMID: 7520912
  21. Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase.
    Mol Cell Biol. 1993 Nov;13(11):7170-9 PMID: 7692235
  22. In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone.
    Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12949-56 PMID: 9371781
  23. A 50 kilodalton protein associated with raf and pp60(v-src) protein kinases is a mammalian homolog of the cell cycle control protein cdc37.
    Biochemistry. 1997 Mar 25;36(12):3600-7 PMID: 9132011
  24. Disruption of the Raf-1-Hsp90 molecular complex results in destabilization of Raf-1 and loss of Raf-1-Ras association.
    J Biol Chem. 1995 Oct 13;270(41):24585-8 PMID: 7592678
  25. The phosphorylation state of the reticulocyte 90-kDa heat shock protein affects its ability to increase phosphorylation of peptide initiation factor 2 alpha subunit by the heme-sensitive kinase.
    Biochemistry. 1989 Feb 21;28(4):1435-8 PMID: 2719907
  26. hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures.
    Mol Cell Biol. 1989 Sep;9(9):3919-30 PMID: 2674684
  27. Cdc37 is a molecular chaperone with specific functions in signal transduction.
    Genes Dev. 1997 Jul 15;11(14):1775-85 PMID: 9242486
  28. Expression of raf family proto-oncogenes in normal mouse tissues.
    Oncogene. 1990 Mar;5(3):345-51 PMID: 1690378
  29. Steroid receptor interactions with heat shock protein and immunophilin chaperones.
    Endocr Rev. 1997 Jun;18(3):306-60 PMID: 9183567
  30. Effects of herbimycin derivatives on src oncogene function in relation to antitumor activity.
    J Antibiot (Tokyo). 1988 Jun;41(6):831-4 PMID: 3403381
  31. Binding of p23 and hsp90 during assembly with the progesterone receptor.
    Mol Endocrinol. 1995 Jun;9(6):670-8 PMID: 8592513
  32. Sequential modification of serines 621 and 624 in the Raf-1 carboxyl terminus produces alterations in its electrophoretic mobility.
    J Biol Chem. 1997 Jan 24;272(4):2136-42 PMID: 8999914
  33. Cdc37: a protein kinase chaperone?
    Trends Cell Biol. 1997 Apr;7(4):157-61 PMID: 17708934
  34. Functional mapping of the N-terminal regulatory domain in the human Raf-1 protein kinase.
    J Biol Chem. 1995 Jun 9;270(23):14100-6 PMID: 7539798
  35. The 90-kDa heat shock protein (hsp-90) possesses an ATP binding site and autophosphorylating activity.
    J Biol Chem. 1991 Mar 15;266(8):4943-50 PMID: 2002041
  36. Oligomerization activates c-Raf-1 through a Ras-dependent mechanism.
    Nature. 1996 Sep 12;383(6596):181-5 PMID: 8774885
  37. Progesterone receptor structure and function altered by geldanamycin, an hsp90-binding agent.
    Mol Cell Biol. 1995 Dec;15(12):6804-12 PMID: 8524246
  38. Mutational activation of c-raf-1 and definition of the minimal transforming sequence.
    Mol Cell Biol. 1990 Jun;10(6):2503-12 PMID: 2188091
  39. Modular folding and evidence for phosphorylation-induced stabilization of an hsp90-dependent kinase.
    J Biol Chem. 1998 Apr 3;273(14):8475-82 PMID: 9525961
  40. Definition of the human raf amino-terminal regulatory region by deletion mutagenesis.
    Mol Cell Biol. 1989 Feb;9(2):639-47 PMID: 2710120
  41. Possible role for serine/threonine phosphorylation in the regulation of the heteroprotein complex between the hsp90 stress protein and the pp60v-src tyrosine kinase.
    J Biol Chem. 1995 Dec 1;270(48):28654-9 PMID: 7499384
  42. Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90.
    Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14536-41 PMID: 8962087
  43. Interaction of the Rous sarcoma virus protein pp60src with the cellular proteins pp50 and pp90.
    Curr Top Microbiol Immunol. 1986;123:1-22 PMID: 2419040
  44. Interaction of Ras and Raf in intact mammalian cells upon extracellular stimulation.
    J Biol Chem. 1994 Feb 11;269(6):3913-6 PMID: 8307946
  45. 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):21711-6 PMID: 8408024
  46. Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro.
    Mol Cell Biol. 1995 Aug;15(8):4125-35 PMID: 7623807
  47. Developmental and cell lineage specificity of raf family gene expression in mouse testis.
    Oncogene. 1993 Apr;8(4):1055-62 PMID: 8455932
  48. Differential effects on cAMP on the MAP kinase cascade: evidence for a cAMP-insensitive step that can bypass Raf-1.
    Mol Biol Cell. 1995 Aug;6(8):1025-35 PMID: 7579705
  49. Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone.
    Cell. 1997 Jul 11;90(1):65-75 PMID: 9230303
  50. Identification of the major phosphorylation sites of the Raf-1 kinase.
    J Biol Chem. 1993 Aug 15;268(23):17309-16 PMID: 8349614
  51. Control of the ERK MAP kinase cascade by Ras and Raf.
    Cancer Surv. 1996;27:101-25 PMID: 8909797
  52. Interaction between Cdc37 and Cdk4 in human cells.
    Oncogene. 1997 Apr 24;14(16):1999-2004 PMID: 9150368
  53. The role of heat shock proteins in regulating the function, folding, and trafficking of the glucocorticoid receptor.
    J Biol Chem. 1993 Oct 15;268(29):21455-8 PMID: 8407992
  54. Benzoquinonoid ansamycins possess selective tumoricidal activity unrelated to src kinase inhibition.
    Cancer Res. 1992 Apr 1;52(7):1721-8 PMID: 1551101
  55. The amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an ATP/ADP switch domain that regulates hsp90 conformation.
    J Biol Chem. 1997 Sep 19;272(38):23843-50 PMID: 9295332
  56. Role of SAPK/ERK kinase-1 in the stress-activated pathway regulating transcription factor c-Jun.
    Nature. 1994 Dec 22-29;372(6508):794-8 PMID: 7997269
  57. Mutations in Hsp83 and cdc37 impair signaling by the sevenless receptor tyrosine kinase in Drosophila.
    Cell. 1994 Jul 1;77(7):1027-36 PMID: 8020093
  58. Hsp90 is obligatory for the heme-regulated eIF-2alpha kinase to acquire and maintain an activable conformation.
    J Biol Chem. 1997 Apr 25;272(17):11648-56 PMID: 9111082
  59. The carboxy-terminal region of mammalian HSP90 is required for its dimerization and function in vivo.
    Mol Cell Biol. 1994 Feb;14(2):1459-64 PMID: 8289821
  60. A 50-kDa cytosolic protein complexed with the 90-kDa heat shock protein (hsp90) is the same protein complexed with pp60v-src hsp90 in cells transformed by the Rous sarcoma virus.
    J Biol Chem. 1991 Sep 5;266(25):16436-40 PMID: 1653236
  61. Activation of Raf-1 by Ras in intact cells.
    Methods Enzymol. 1995;255:301-10 PMID: 8524115
  62. Differential regulation of Raf-1, A-Raf, and B-Raf by oncogenic ras and tyrosine kinases.
    J Biol Chem. 1997 Feb 14;272(7):4378-83 PMID: 9020159
  63. Assisting spontaneity: the role of Hsp90 and small Hsps as molecular chaperones.
    Trends Biochem Sci. 1994 May;19(5):205-11 PMID: 7914036
  64. p50(cdc37) binds directly to the catalytic domain of Raf as well as to a site on hsp90 that is topologically adjacent to the tetratricopeptide repeat binding site.
    J Biol Chem. 1998 Aug 7;273(32):20090-5 PMID: 9685350
  65. Oligomeric forms of the 90-kDa heat shock protein.
    Biochem J. 1998 Mar 1;330 ( Pt 2):989-95 PMID: 9480920
  66. Transit of pp60v-src to the plasma membrane.
    Proc Natl Acad Sci U S A. 1982 Dec;79(23):7117-21 PMID: 6296817
  67. Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation.
    Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8324-8 PMID: 8078881
  68. Activation of c-Raf-1 by Ras and Src through different mechanisms: activation in vivo and in vitro.
    EMBO J. 1997 May 1;16(9):2384-96 PMID: 9171352
  69. The function of steroid hormone receptors is inhibited by the hsp90-specific compound geldanamycin.
    J Biol Chem. 1997 Jul 25;272(30):18694-701 PMID: 9228040
  70. The hsp90-based chaperone system: involvement in signal transduction from a variety of hormone and growth factor receptors.
    Proc Soc Exp Biol Med. 1998 Apr;217(4):420-34 PMID: 9521088
  71. CDC37 is required for p60v-src activity in yeast.
    Mol Biol Cell. 1996 Sep;7(9):1405-17 PMID: 8885235
  72. Raf meets Ras: completing the framework of a signal transduction pathway.
    Trends Biochem Sci. 1994 Jul;19(7):279-83 PMID: 8048167
  73. The selection of S. cerevisiae mutants defective in the start event of cell division.
    Genetics. 1980 Jul;95(3):561-77 PMID: 7002718
  74. Destabilization of Raf-1 by geldanamycin leads to disruption of the Raf-1-MEK-mitogen-activated protein kinase signalling pathway.
    Mol Cell Biol. 1996 Oct;16(10):5839-45 PMID: 8816498
  75. The complexity of Raf-1 regulation.
    Curr Opin Cell Biol. 1997 Apr;9(2):174-9 PMID: 9069260
  76. Organization of the chick CDC37 gene.
    J Biol Chem. 1998 Feb 6;273(6):3598-603 PMID: 9452487
  77. Analysis of native forms and isoform compositions of the mouse 90-kDa heat shock protein, HSP90.
    J Biol Chem. 1991 Jun 5;266(16):10099-103 PMID: 2037568
  78. Geldanamycin, a new antibiotic.
    J Antibiot (Tokyo). 1970 Sep;23(9):442-7 PMID: 5459626
  79. Activation of the Raf-1 kinase cascade by coumermycin-induced dimerization.
    Nature. 1996 Sep 12;383(6596):178-81 PMID: 8774884
  80. Dynamic activation of endothelial nitric oxide synthase by Hsp90.
    Nature. 1998 Apr 23;392(6678):821-4 PMID: 9580552
Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.19.3.1661
Email
ngrammat@earthlink.net
Published
1999-03-00
Pages
1661-72
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC83960
Subset
IM
Grants
NIGMS NIH HHS · R01 GM051551 · United States
NIGMS NIH HHS · GM51551 · United States
External Links
PubMed source DOI: 10.1128/MCB.19.3.1661 PMC Full Text View journal details
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