The hsp90-binding Antibiotic Geldanamycin Decreases Raf Levels and Epidermal Growth Factor Signaling without Disrupting Formation of Signaling Complexes or Reducing the Specific Enzymatic Activity of Raf Kinase

Abstract

We have expressed the mitogenic signaling proteins Src, Ras, Raf-1, Mek (MAP kinase kinase), and Erk (MAP kinase) in baculovirus-infected Sf9 insect cells in order to study a potential role for the chaperone hsp90 in formation of multiprotein complexes. One such complex obtained by immunoadsorption with anti-Ras antibody of cytosol prepared from cells simultaneously expressing Ras, Raf, Mek, and Erk contained Ras, Raf, and Erk. To detect directly the protein-protein interactions involved in forming multiprotein complexes, we combined cytosols from single infections in vitro in all possible combinations of protein pairs. We detected complexes between Ras⅐Raf, Ras⅐Src, Raf⅐Mek, and Raf⅐Src, but no complex containing Erk was obtained by mixing cytosols. Thus, cellular factors appear to be required for assembly of the Erk-containing multiprotein complex. One cellular factor thought to be involved in signaling protein complex formation is the chaperone hsp90, and we show that Src, Raf, and Mek are each complexed with insect hsp90. Treatment of Sf9 cells with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, did not decrease coadsorption of either Raf or Erk with Ras, although it did decrease the level of cytosolic Raf. To study geldanamycin action, we treated rat 3Y1 fibroblasts expressing v-Raf and showed that the antibiotic blocked assembly of Raf⅐hsp90 complexes at an intermediate stage of assembly where Raf is still bound to the p60 and hsp70 components of the assembly mechanism. As in Sf9 cells, Raf levels decline with geldanamycin treatment of 3Y1 cells. To determine if geldanamycin affects mitogenic response, we treated HeLa cells with epidermal growth factor (EGF) and showed that geldanamycin treatment decreased EGF signaling and decreased the level of Raf protein without affecting the EGF-mediated increase in Raf kinase activity. We conclude that hsp90 is not required for forming complexes between the mitogenic signaling proteins or for Raf kinase activity and that EGF signaling is decreased indirectly by geldanamycin because the antibiotic increases degradation of Raf and perhaps other components of the signaling pathway.Several receptors for polypeptide ligands, including those for insulin, epidermal growth factor, platelet-derived growth factor, and nerve growth factor, transduce signals by activating the mitogen-activated protein (MAP) 1 family of serine/threonine kinases (also called Erks for extracellular signal-regulated kinases) (see Refs. 1 and 2, for review). The receptors themselves are tyrosine kinases that undergo ligand-induced autophosphorylation leading to the recruitment of the Grb2 adaptor and its associated Ras activator protein Sos. Subsequent Ras binding to the Raf-1 serine/threonine kinase leads to phosphorylation by Raf-1 of another kinase called Mek (also called MAP kinase kinase), which in turn, phosphorylates and activates Erk. Erk is a terminal effector of this signal transduction pathway in that it can directly phosphorylate transcription factor...

Hsp90 inhibitors deplete key anti‐apoptotic proteins in pediatric solid tumor cells and demonstrate synergistic anticancer activity with cisplatin

“…44 Hsp90 activity can be assessed in tumor samples indirectly by evaluating changes in levels of client proteins that are regulated by Hsp90. The functions of numerous cancer-associated proteins are regulated by Hsp90 and are altered by Hsp90 inhibitors, [45][46][47][48] but it is not yet clear which of the affected proteins will be most predictive of activity against a particular tumor type in vivo. In an attempt to identify useful markers for monitoring the activity of Hsp90 inhibitors in pediatric cancer patients enrolled on a clinical trial, we evaluated effects of Hsp90 inhibitors on 2 client proteins that are important in childhood solid tumors.…”

Targeting of the protein chaperone, HSP90, by the transformation suppressing agent, radicicol

“…The position of HSP90 is indicated by arrows in lanes 5 and 10. (b) Identical experiment to the one in Figure 5a, except that the lysates were from SRC-3T3 cells active degradation of Raf and reduces signaling through the Raf-MAP kinase pathway Schulte et al, 1995Schulte et al, , 1996Stancato et al, 1997). In addition to Raf, geldanamycin appears capable of e ecting the depletion of other growth-regulatory signaling proteins, as well as nuclear hormone receptors, a property that is generally believed to be responsible for its anti-neoplastic activity (Stebbins et al, 1997, and references therein).…”

“…The ability of radicicol to bind to HSP90 and cause the destabilization of Raf (Soga et al, 1998) is reminiscent of geldanamycin Schulte et al, 1995Schulte et al, , 1996Stancato et al, 1997). To compare the HSP90 binding ability of radicicol and geldanamycin, the ability of geldanamycin to block the speci®c binding of radicicol to HSP90 was examined ( Figure 5).…”

“…Besides Ras, other proteins that interact with Raf, and perhaps regulate it, include Mitogen-or extracellular-regulated kinase (MEK1/2); proteins of the 14-3-3 family; molecular chaperones such as HSP90 and Cdc37/p50; and the protein Kinase Suppressor of Ras (KSR) (reviewed in Morrison and Cutler, 1997). Interaction of HSP90 and Cdc37/p50 appears to be important for the proper intracellular localization and stability of Raf (Schulte et al, 1995(Schulte et al, , 1996Stancato et al, 1997;reviewed in Hunter and Poon, 1997).…”

Hsp90 Is Required for Pheromone Signaling in Yeast

“…Hsp90 may also be required for growth factor signaling. 1) Raf-1, a serine/threonine kinase involved in mitogenic signal transduction in vertebrates, exists in a geldanamycin-sensitive heterocomplex with Hsp90 (Stancato et al, 1993(Stancato et al, , 1994Lovric et al, 1994;Wartmann and Davis, 1994;Schulte et al, 1995Schulte et al, , 1996Stancato et al, 1997). 2) Mutations in Drosophila HSP83 reduce signaling by the torso (Doyle and Bishop, 1993) and sevenless receptors (Cutforth and Rubin, 1994;van der Straten et al, 1997), which may be due, at least in part, to a requirement for Hsp90 for Raf function (van der Straten et al, 1997). 3) The insulin receptor binds Hsp90, and antibodies to Hsp90 interfere with insulin signaling (Takata et al, 1997).…”