©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
p185 Binds to GRP94 in Vivo
DISSOCIATION OF THE p185/GRP94 HETEROCOMPLEX BY BENZOQUINONE ANSAMYCINS PRECEDES DEPLETION OF p185(*)

(Received for publication, November 7, 1995)

Christine Chavany (1) Edward Mimnaugh (1) Penny Miller (3) Roberto Bitton (1) Phongmai Nguyen (2) Jane Trepel (2) Luke Whitesell (4) Rodney Schnur (3) James D. Moyer (3) Len Neckers (1)(§)

From the  (1)Clinical Pharmacology Branch and (2)Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, (3)Pfizer Central Research, Groton, Connecticut 06340, and the (4)Department of Pediatrics, University of Arizona Health Sciences Center, Steele Memorial Research Center, Tucson, Arizona 85724

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Treatment of SKBr3 cells with benzoquinone ansamycins, such as geldanamycin (GA), depletes p185, the receptor tyrosine kinase encoded by the erbB2 gene. In the same cells, a biologically active benzoquinone photoaffinity label specifically binds a protein of about 100 kDa, and the ability of various GA derivatives to reduce the intracellular level of p185 correlates with their ability to compete with the photoaffinity label for binding to this protein. In this report, we present evidence that the 100-kDa ansamycin-binding protein is GRP94. Membrane-associated p185 exists in a stable complex with GRP94. GA binding to GRP94 disrupts this complex, leading to degradation of pre-existing p185 protein, and resulting in an altered subcellular distribution of newly synthesized p185.


INTRODUCTION

Herbimycin A (HA) (^1)and geldanamycin (GA) are benzoquinone ansamycins with potent antiproliferative activity that specifically bind to the heat shock protein hsp90(1) , with which several tyrosine kinases, as well as other intracellular signal transduction molecules, are complexed(2, 3, 4, 5, 6, 7, 8, 9) . Geldanamycin dissociates certain multi-molecular complexes containing HSP90(1, 10) , leading to target protein (i.e. v-Src, c-Raf-1) instability(1, 9) .

The erbB2 gene (also known as her-2/neu) encodes a 185-kDa receptor-like protein (p185) with tyrosine kinase activity. This protein is overexpressed in many breast, ovarian, and prostate carcinomas and is associated with poor prognosis. Miller et al.(11) have reported that p185 is rapidly depleted in human breast cancer cells (SKBr3) following exposure to HA or GA. Although the p185 protein level is markedly reduced, p185 mRNA and protein synthesis are only slightly affected, and it appears that one of the primary effects of these drugs is to significantly reduce the half-life of p185(11, 12) . However, unlike v-Src and c-Raf-1 (see above), p185 cannot be demonstrated to form a complex with HSP90(12) . Intriguingly, when they used a biologically active, I-labeled photoaffinity derivative of GA (CP202509), Miller et al.(13) demonstrated specific binding to a 100-kDa protein in SKBr3 cells, but not to p185 itself. The ability of various GA derivatives to reduce the intracellular level of p185 correlated with their ability to specifically compete with the I-photoaffinity label for binding to this protein.

In this report, we present evidence that this ansamycin-binding protein is the glucose-regulated protein GRP94, an endoplasmic/sarcoplasmic reticulum protein with homology to the molecular chaperone HSP90 (14, 15, 16) . An abundant cellular glycoprotein, GRP94 is induced in response to glucose deprivation, hypoxia, calcium ionophores, glycosylation inhibitors, and low pH, and its expression is up-regulated in pathological states during which these conditions naturally occur, such as ischemia and tumor growth (reviewed in (17) ). While the function of GRP94 has not been well defined, it has been implicated in Ca regulation(18) , protein folding, and antigen presentation(19, 20) . In addition, we demonstrate here the existence of a stable heterocomplex between p185 and GRP94. Finally, we show that GA rapidly dissociates GRP94 from p185 and that this occurs prior to degradation of p185 protein.


MATERIALS AND METHODS

Cell Culture Conditions

SKBr3 human breast carcinoma cells were purchased from the American Type Culture Collection (Rockville, MD) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biologicals), 1 mM glutamine, and 10 mM Hepes (pH 7.3), at 37 °C in an atmosphere of 6% carbon dioxide, or as described previously(13) .

Preparation of Cytosol and Crude Membrane Fractions from SKBr3 Cells

10^7 SKBr3 cells were plated in 100-mm^2 plastic dishes. Following treatment, cells were washed twice with ice-cold PBS and scraped with a cell scraper into 1 ml of ice-cold TESV buffer (50 mM Tris, pH 7.5, 2 mM EDTA, 100 mM NaCl, 1 mM vanadate) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 20 µg/ml each aprotinin and leupeptin). Cells were homogenized using a Dounce tissue grinder and then lysed by sonication on ice for 10 s three times, letting samples chill in ice water between sonications. The cell lysate was ultracentrifuged at high speed (100,000 times g, 60 min, 4 °C) for preparation of membrane and cytosol fractions, or at low speed (12,000 times g, 15 min, 4 °C) to remove the insoluble fraction. The supernatant fraction from high speed centrifugation (cytosol) was saved, while the crude membrane pellet was resuspended by sonication in 0.5 ml of ice-cold TESV buffer containing protease inhibitors. The membrane suspension was then clarified by centrifugation at 12,000 times g for 15 min (4 °C), and the supernatant (soluble membrane fraction) was saved. The protein content of each fraction was determined using the BCA protein assay (Pierce).

Co-immunoprecipitation of GRP94 and p185 and Western Immunoblot

GRP94 protein was immunoprecipitated from both membrane and cytosol fractions (500-1000 µg of protein, prepared fresh) using 2 µg of rat monoclonal anti-human GRP94 antibody (StressGen) with an incubation period of 1 h at 4 °C. Rabbit anti-rat protein A-Sepharose beads, preswollen and equilibrated in TESV, were added, and samples were rotated in a tumbler for 40 min at 4 °C. The beads were washed three times with TESV buffer, resuspended in 1 times sample loading buffer (0.0627 M Tris-HCl, pH 6.8, 1% SDS, 1% 2-mercaptoethanol, 10% glycerol, 0.0005% bromphenol blue), and heated for 5 min at 100 °C. Samples were then electrophoresed through a 7% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. p185 was detected with a monoclonal antibody (Ab3, 1:2,000 dilution, Oncogene Science) using a chemiluminescence-based Western blotting kit (DuPont) according to supplier's instructions. Membranes were exposed for various times to Kodak X-Omat AR film. Films were scanned by densitometry to obtain semi-quantitative analysis of p185 level. Alternatively, the whole cell lysate, cleared of insoluble particles after sonication, was immunoprecipitated with 2 µg of a monoclonal antibody targeted against human p185 (Ab 5, Oncogene Science). Detection of GRP94 was performed using a 1:1,000 dilution of the StressGen monoclonal antibody, as described above. The content of GRP94 and p185 in cytosol and crude membrane fractions was monitored by immunoblotting 100 µg of cytosol and 15 µg of membrane protein with the proper monoclonal antibody, as described above.

I-CP202509 Labeling of SKBr3 Cells

Two million cells were seeded in 60-mm plates. After overnight culture, cell monolayers were incubated with 11 nMI-CP202509 (15 µCi) for 1 h at 37 °C in the dark in the presence or absence of 5 µM unlabeled CP127374, a geldanamycin analog. After aspiration of the medium containing compound and addition of 1 ml of PBS, the cells were irradiated for 10 min with a UV light ( = 254 nm) at a distance of 8-10 cm. The monolayers were washed twice with PBS and lysed with hot 2% SDS, 50 mM Tris, pH 7.4. Lysates were boiled for 10 min and pulse-sonicated for 1 min in a bath sonicator (Heat Systems Inc., Farmingdale, NY).

Immunoprecipitation of I-CP202509-labeled Ansamycin-binding Protein

Denatured lysates of SKBr3 cells were first diluted 20-fold in PBS containing 0.25% bovine serum albumin to reduce the SDS concentration to a level suitable for the formation of antigen-antibody complexes. Diluted lysates were incubated on ice for 10 min and centrifuged at 100,000 times g for 30 min. Lysate supernatants were incubated with goat anti-rat conjugated agarose beads (Sigma), which had been coated with rat monoclonal antibody to either GRP94 or HSP90 (StressGen, Vancouver, British Columbia, Canada). The bead-lysate mixtures were incubated for 2 h at 4 °C. on a rocker. Finally, beads were washed six times with wash buffer (50 mM Tris, pH 7.4, 0.1% SDS) and boiled for 5 min with 2 times Laemmli sample buffer (120 mM Tris, pH 6.8, 4% SDS, 20% glycerol, 100 mM dithiothreitol, 0.016% bromphenol blue). Eluted proteins were analyzed by PAGE and autoradiography.

To verify the specificity of the immunoprecipitation, lysates of unlabeled SKBr3 cells were prepared in parallel with the above samples. Immunoprecipitation and immunoblotting with both antibodies revealed that neither cross-reacted with the other's target (native or denatured), demonstrating that both antibodies, whether used for immunoprecipitation or immunoblotting, are specific for their cognate antigens.

Subcellular Localization of Synthesized p185 in the Presence or Absence of GA

Subcellular localization of p185 protein in untreated SKBr3 cells, and in cells exposed for 18 h to 2 µM GA, was visualized by immunofluorescence. Briefly, cells were grown on coverslips, rinsed in PBS, fixed with 3.7% formaldehyde for 10 min at room temperature, and rinsed again with PBS. Coverslips were overlaid with p185 antibody (Ab 5, 10 µg/ml in PBS, Oncogene Science) and kept at 4 °C for 1 h. Following rinsing in PBS, coverslips were overlaid with Cy3TM-conjugated goat anti-mouse immunoglobulin (1:500 in PBS; Jackson ImmunoResearch Laboratories, Inc.) and kept at 4 °C for an additional hour. After rinsing in PBS and water, coverslips were air-dried and mounted with SlowFade(TM) mounting medium (Molecular Probes). Fluorescence was visualized using a Zeiss Axioskop microscope and an Optronics CCD camera.

Analysis of Newly Synthesized p185 in the Presence or Absence of GA

A total of 10^7 SKBr3 cells were plated in 100-mm^2 plastic dishes and allowed to grow for 24 h. Cells were exposed to medium alone or containing GA for 16 h, then washed twice with ice-cold PBS and incubated for an additional 6 h in 4 ml of Dulbecco's modified Eagle's medium lacking methionine and cysteine, supplemented with 10% dialyzed fetal calf serum and 100 µCi/ml [S]methionine/cysteine (TranS-label, ICN). GA (3 µM) was added back to cells previously exposed to the drug. At the end of the labeling period, cells were washed twice in cold PBS and lysed as described above. Protein content was determined, as well as the amount of radioactivity in 5 µl of cell lysate that was precipitable with trichloroacetic acid. p185 was immunoprecipitated from lysates containing equal acid-precipitable radioactivity (Ab 5, 2 µg/sample, Oncogene Science) as described above. Immunoprecipitates were solubilized by heating at 95 °C for 5 min in 1% SDS. After centrifugation, supernatants were transferred to tubes containing an equal volume of digestion buffer (0.1 M sodium acetate, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, pH 5.5) and incubated with endoglycosidase H (Boehringer Mannheim, 10 milliunits/tube) for 20 h at 37 °C. Samples were then mixed with 5 times reducing loading buffer and electrophoresed through a 7% SDS-polyacrylamide gel, which was then fixed in 50% methanol, 10% acetic acid, and the [S] signal was amplified using an enhancing solution (DuPont). Dried gels were autoradiographed using Kodak X-Omat AR films.


RESULTS

p185 and GRP94 Form an Intracellular Complex in SKBr3 Cells Which Is Dissociated by Brief Exposure to GA

After overnight incubation of SKBr3 cells in the presence or absence of GA, membrane and cytosol fractions were prepared. Detection of a native complex between p185 and GRP94 was demonstrated by co-precipitation of p185 with a monoclonal antibody targeted against human GRP94 (Fig. 1B). The native complex was easily seen in the membrane fraction, which contained the majority of p185, but a minority of GRP94. In most experiments, a p185bulletGRP94 complex could also be demonstrated in cytosol, but at a much lower level (for example, see Fig. 1B). The presence of a GRP94bulletp185 complex was confirmed by the converse experiment, i.e. immunoprecipitation of total lysate with antibody to p185 followed by Western blot detection of GRP94 (Fig. 1C). After overnight treatment with GA, the complex disappeared from both cellular fractions (see Fig. 1B). However, such treatment caused the level of p185, but not GRP94, to be dramatically reduced (Fig. 1, compare panels A and D).


Figure 1: p185 and GRP94 exist as a complex in SKBr3 cells. SKBr3 cells were incubated 18 h in the presence (+) or absence(-) of GA (3 µM). Prior to SDS-PAGE analysis, proteins contained in membrane (M) or cytosol (C) fractions were (panel B) or were not (panels A and D) immunoprecipitated with rat monoclonal anti-human GRP94. In panel C, proteins in total lysate (L) were (+) or were not(-) subjected to immunoprecipitation with monoclonal anti-human p185. The proteins were separated by electrophoresis and immunoblotted with monoclonal antibodies to p185 (panels A and B) or to GRP94 (panels C and D) as described under ``Materials and Methods.''



In order to determine whether disappearance of the p185bulletGRP94 complex following overnight exposure to GA was the result of loss of p185, or the result of complex dissociation prior to loss of p185, we performed the same experiment following a brief exposure of SKBr3 cells to GA. As seen in Fig. 2, after a 75-min exposure of SKBr3 cells to GA, total p185, as measured by Western blotting of cell lysates, was only minimally reduced (see Fig. 2A). However, co-precipitation of a p185bulletGRP94 complex was dramatically reduced (see Fig. 2B).


Figure 2: The native complex between p185and GRP94 is dissociated by GA prior to p185depletion. SKBr3 cells were incubated for 75 min in the presence (+) or absence(-) of GA (3 µM). Prior to SDS-PAGE analysis, proteins contained in membrane (M) or cytosol (C) fractions were (panel B) or were not (panel A) immunoprecipitated with rat anti-human GRP94. Immunoblotting of p185 was performed using a murine anti-human p185 antibody.



Benzoquinone Ansamycins Bind Directly to GRP94

When intact SKBr3 cells were labeled with I-CP202509, a GA analog incorporating a photoactivatable linker, a 100-kDa protein in the total lysate was specifically labeled in a photolysis-dependent manner (Fig. 3, compare lanes 1 and 2), as reported previously(13) . After GRP94 immunoprecipitation, the labeled ansamycin-binding protein was reduced in the cleared lysate (Fig. 3, compare lane 3 to lane 2), and concentrated in the GRP94 immunoprecipitate (Fig. 3, lane 4). Labeling of GRP94 by I-CP202509 was specific and saturable, because co-incubation of the cells with 5 µM unlabeled CP127374 markedly reduced labeling of the 100-kDa protein in both total lysate (Fig. 3, compare lane 5 to lane 2) and in the GRP94 immunoprecipitate (Fig. 3, compare lane 6 to lane 4). In contrast, the I-CP202509-labeled protein was not immunoprecipitated with anti-HSP90 (Fig. 3, lanes 7 and 8). Photoaffinity labeling of 10% Triton X-100 extracts of SKBr3 cells with I-CP202509 also resulted in specific labeling of a 100-kDa protein, which was immunoprecipitated by anti-GRP94 antibody, confirming the results obtained with intact cells (data not shown). Superimposition of an autoradiogram and GRP94 immunoblot from a single transfer membrane indicated that the I-CP202509-labeled band and GRP94 had identical electrophoretic mobility. Thus, these data identify the target of the GA photoaffinity analog, previously described as p100, as GRP94, on the basis of both molecular weight and immunoprecipitation with an anti-GRP94 monoclonal antibody.


Figure 3: GA binds to cellular GRP94. SKBr3 cells were labeled with I-CP202509 and lysed as described under ``Materials and Methods.'' Lysates were subjected to immunoprecipitation with monoclonal antibodies to GRP94 or HSP90 as described. The positions of protein molecular size standards are indicated (in kDa). Lane 1, lysate of cells incubated with I-CP202509 but not exposed to UV light. Lane 2, lysate of cells incubated with I-CP202509 followed by photolysis. Lane 3, lysate in lane 2 after clearing by immunoprecipitation with anti-GRP94. Lane 4, anti-GRP94 immunoprecipitate from lysate in lane 2. Lane 5, lysate of cells incubated with I-CP202509 and 5 µM CP127374 followed by photolysis. Lane 6, anti-GRP94 immunoprecipitate from lysate in lane 5. Lane 7, lysate in lane 2 after clearing with anti-HSP90. Lane 8, anti-HSP90 immunoprecipitate of lysate in lane 2.



Prolonged Exposure to GA Modifies the Subcellular Distribution of p185

Approximately 85% of p185 is associated with the membrane fraction of SKBr3 cells (based on densitometric analysis of data in Fig. 1A and similar experiments). Since GA treatment greatly depletes the steady-state level of p185 without affecting its synthesis(11, 12) , we wished to determine whether prolonged exposure to GA altered the subcellular distribution of newly synthesized protein. To do this, we analyzed p185 immunofluorescence in intact cells, both untreated or exposed to GA for 22 h. As can be seen in Fig. 4, in untreated SKBr3 cells p185 is primarily localized to the plasma membrane. This corresponds to the expected strong signal seen by PAGE analysis of the membrane fraction (see Fig. 1A).


Figure 4: GA alters the subcellular distribution of newly synthesized p185. A, cells, grown on coverslips, were treated with GA (2 uM for 22 h), and then p185 immunofluorescence was detected as described under ``Materials and Methods.'' Results are compared to untreated cells. B, cells treated as in A were pulse-labeled with [S]methionine/cysteine for the final 6 h. p185 was immunoprecipitated from total cell lysate and subjected to overnight digestion with endoglycosidase H as described under ``Materials and Methods.'' Lanes 1-3 represent p185 immunoprecipitations from control cells, and lanes 4-6 represent drug-treated cells. Lanes 1 and 4, no endoglycosidase H; lanes 2 and 5, 10 milliunits of endoglycosidase H; lanes 3 and 6, 20 milliunits of endoglycosidase H.



After 22 h in the presence of GA, however, immunofluorescence analysis revealed that p185 was no longer localized to the plasma membrane, but instead appeared restricted to cytoplasmic inclusions, as visualized by a punctate pattern of perinuclear fluorescence (Fig. 4A). Since GA depletes pre-existing p185 protein within several hours(11) , and reduces the half-life of newly synthesized protein from greater than 9 h to approximately 2 h(12) , the immunofluorescent signal observed in Fig. 4A most likely represents p185 newly synthesized in the presence of GA. Immunoprecipitation and PAGE analysis of newly synthesized p185 (using a 6-h pulse with [S]methionine) from cells treated with GA for 24 h revealed that the ratio of distribution of the protein between membrane and cytosol remained the same as in untreated cells (83% of total signal in membrane fraction; data not shown). However, drug treatment rendered all of the newly synthesized material sensitive to endoglycosidase H, while p185 synthesized in the absence of GA was only slightly sensitive to endoglycosdiase H (Fig. 4B). Endoglycosidase H sensitivity is characteristic of incomplete glycosylation and is consistent with the lower apparent molecular weight of p185 synthesized in the presence of GA (Fig. 4B, compare lanes 1 and 4). Since resistance to endoglycosidase H is acquired in the trans-Golgi (21) , these data are further consistent with the trapping of p185 synthesized in the presence of GA in the endoplasmic reticulum or cis-Golgi. Thus, although overnight exposure to GA does not change the fraction of protein that is membrane associated, it causes dramatic subcellular redistribution of newly synthesized p185.


DISCUSSION

In this report, we demonstrate that the p185 receptor tyrosine kinase forms a stable complex with the glucose-regulated chaperone protein GRP94. The benzoquinone ansamycin GA destabilizes this complex within 75 min, prior to significant loss of p185, which occurs over the next several hours. Exposure of SKBr3 cells to GA and other benzoquinoid ansamycins has been shown previously to result in rapid loss of p185(11) . This effect appears to be mediated primarily at the level of protein stability, since p185 mRNA level and rate of synthesis remain essentially unaltered, while the half-life of the protein is reduced from 9.5 to 2 h(11, 12) . Effects of benzoquinone ansamycins on other receptor tyrosine kinases have been noted. Thus, Murakami et al.(22, 23) reported destabilizing effects of HA on the epidermal growth factor receptor, while Sepp-Lorenzino et al.(24) described similar effects of HA on both the insulin receptor and the insulin-like growth factor receptor. Sepp-Lorenzino et al.(24) suggest that the protein instability caused by HA is mediated by the 20 S proteasome in a ubiquitin-dependent manner.

Previously, using an iodinated photoaffinity analog of GA, Miller et al.(13) demonstrated that this compound did not label p185, but instead bound an unknown protein of approximately 100 kDa. Furthermore, the ability of various GA analogs to deplete cellular p185 correlated with their ability to interact with p100(13) . The data presented here identify p100 as GRP94, which can be co-precipitated in a complex with p185. By binding to GRP94, GA either induces rapid dissociation of this complex or interferes with the dynamic equilibrium of complex association-dissociation. This occurs prior to significant loss of p185, suggesting a role for GRP94 in maintaining p185 stability. Whether ansamycin disruption of GRP94bulletp185 complexes leads to ubiquitination of p185 and its subsequent proteolysis by the 20 S proteasome is currently under investigation.

Other receptors may be regulated in a similar fashion. For example, the type I tumor necrosis factor receptor, which has no tyrosine kinase activity, has recently been shown to form a native complex, whose function is as yet undetermined, with a protein showing strong homology to members of the HSP90 family(25) .

Our current data further suggest that failure of newly synthesized p185 to associate with GRP94 prevents the translocation of the newly synthesized protein to the plasma membrane, instead trapping it in an intracellular vesicular compartment consistent with the endoplasmic reticulum/cis-Golgi. The data in Fig. 4demonstrate that, following overnight exposure to GA, p185 is only detectable in this vesicular compartment and not on the plasma membrane. Perhaps an association with GRP94 is required for both the proper intracellular trafficking and stability of a family of receptor proteins.

GA has previously been shown to bind to the heat shock protein HSP90, with which GRP94 shares 50% homology(26) . The drug causes dissociation of heterocomplexes composed of this protein and various signal transduction proteins, including v-Src, c-Raf-1, and the progesterone receptor(1, 9, 10) . In the case of c-Raf-1 and v-Src, heterocomplex dissociation results in protein instability and altered subcellular localization(1, 9, 27) . These findings are thus quite similar to those reported here, except p185 apparently associates with GRP94 and not HSP90. It is not clear why the GA photoaffinity label only recognizes GRP94 and not HSP90 in either intact cells or cell lysates. We have documented that SKBr3 cells contain normal amounts of HSP90, and that this HSP90 efficiently binds to solid phase-immobilized GA in a competable manner (1) . (^2)In addition, the photoaffinity label binds effectively to purified HSP90 in a photolysis-dependent manner in vitro.^2 Conversely, GRP94 binds to solid phase-immobilized GA, corroborating the photoaffinity label results, but GRP94 binding is less efficient than that of HSP90.^2 A possible explanation for these apparent discrepancies is that GA and its photoaffinity label derivative, although both effective in depleting p185(13) , possess different affinities for GRP94 and HSP90. At the same time, substitution with a bulky group at the 17-position of GA (necessary for solid phase immobilization of GA; see (1) ) may decrease the drug's binding affinity to GRP94 without affecting binding to HSP90. In fact, certain 17-substituted GA derivatives are poor depletors of p185(28) ,^2 presumably because of their failure to interact with GRP94.

The current results, together with previous reports of GA effects on HSP90(1, 10) , identify the molecular site of action of the benzoquinone ansamycins as the chaperonins, specifically HSP90 and GRP94. Binding of these inhibitors to the chaperonins appears to destabilize the complex of an array of signaling proteins, such as p185, v-Src, or c-Raf-1, with the chaperone protein, thereby directing the signaling protein to a degradative pathway. Further studies should better define the role of these chaperones in regulating signal transduction as well as the potential of the benzoquinone ansamycins in the pharmacologic manipulation of this process.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Clinical Pharmacology Branch, NCI, NIH, Bldg. 10, Rm. 12N226, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-402-3308; Fax: 301-402-1608; :len{at}helix.nih.gov.

(^1)
The abbreviations used are: HA, herbimycin A; GA, geldanamycin; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.

(^2)
C. Chavany, E. Mimnaugh, P. Miller, R. Bitton, P. Nguyen, J. Trepel, L. Whitesell, R. Schnur, J. D. Moyer, and L. Neckers, unpublished observations.


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