©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Herbimycin A Induces the 20 S Proteasome- and Ubiquitindependent Degradation of Receptor Tyrosine Kinases (*)

Laura Sepp-Lorenzino (1) (2), Zhengping Ma (1) (2), David E. Lebwohl (2), Alexander Vinitsky (3), Neal Rosen (1) (2)(§)

From the (1)Cell Biology and Genetics Program and (2)Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 and the (3)Department of Pharmacology, Mount Sinai School of Medicine, City University of New York, New York, New York 10029

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Herbimycin A is an ansamycin antibiotic isolated as an agent that reverses morphological transformation induced by v-src. Although herbimycin A is widely used as a tool for inhibiting multiple tyrosine protein kinases and tyrosine kinase-activated signal transduction, its mechanism of action is not well defined and includes a decrease in both tyrosine kinase protein levels and activity (Uehara, Y., Murakami, Y., Sugimoto, Y., and Mizuno, S.(1989) Cancer Res. 49, 780-785). We now show that herbimycin A induces a profound decrease in the total cellular activity of transmembrane tyrosine kinase receptors, such as insulin-like growth factor, insulin, and epidermal growth factor receptors. A substantial proportion of the in vivo inhibition could be explained by an increase in the rate of degradation. The enhanced degradation of insulin-like growth factor-insulin receptor was prevented by inhibitors of the 20S proteasome, whereas neither lysosomotropic agents nor general serine- and cysteine-protease inhibitors were active in preventing receptor degradation induced by herbimycin A. Moreover, in a temperature-sensitive mutant cell line defective in the E1-catalyzed activation of ubiquitin, herbimycin A treatment at the restrictive temperature did not result in the degradation of insulin receptor. These results suggest that herbimycin A represents a novel class of drug that targets the degradation of tyrosine kinases by the 20S proteasome. The ubiquitin dependence of this process indicates that this degradation of tyrosine kinases might involve the 20S proteasome as the proteolytic core of the ubiquitin-dependent 26S protease.


INTRODUCTION

Tyrosine kinases play key roles in the regulation of a variety of cellular processes, ranging from differentiation to malignant transformation(1, 2, 3, 4, 5, 6, 7, 8, 9) . The availability of specific tyrosine kinase inhibitors is a useful tool for dissecting molecular pathways regulated by these enzymes. They also represent a potential class of chemotherapeutic agents that functions by inhibiting proto-oncogene products. One such inhibitor, herbimycin A (HA),()is a benzoquinonoid ansamycin antibiotic originally isolated as an agent capable of reverting the transformed phenotype of Rous sarcoma virus-infected normal rat kidney cells(1) . Cellular and biochemical analysis of HA-treated cultures indicated that the morphological reversion was accompanied by a decrease in the kinase activity of the oncoprotein, together with increased turnover of pp60(4, 10) . HA also effectively reversed the morphologic transformation induced by other tyrosine kinase oncogenes such as yes, fps, abl, erbB, and ros but not by ras, raf, and myc(2) . Moreover, HA inhibited the tyrosine kinase activities of several src-family members and of p210in vitro(3, 5, 6, 11) , whereas no effect was observed on the in vitro or intracellular catalytic activities of serine/threonine kinases such as protein kinase A, protein kinase C, or Raf-1(3, 5, 6, 11) .

The establishment of autocrine and paracrine growth factor loops may play an important role in the pathogenesis of breast cancer. Insulin-like growth factors and transforming growth factor- are potent mitogens for breast cancer cells, and tyrosine kinase receptors such as IGF-IR, EGFR, and erbB-2 are overexpressed in a significant percentage of tumors(12, 13) . Moreover, several breast cancer cell lines are dependent on the activity of growth factor receptors for the maintenance of their transformed state(14, 15, 16) . Therefore, these tyrosine kinase receptors are potential targets for inhibitors such as HA. We have found that the breast cancer cell lines MCF-7 and MDA MB-468 (which express high levels of IGF-IR and EGFR, respectively) are profoundly inhibited by HA with respect to anchorage-dependent and -independent growth.

In the present study we investigated the mechanism of HA-mediated inhibition of tyrosine kinase receptor action in these cell lines. HA treatment effectively inhibited ligand-induced receptor activation and tyrosine phosphorylation of downstream targets. In vitro kinase activity of the activated receptors was inhibited by HA only slightly and required higher concentrations than those required for the intracellular effect. We have found that in the cell, HA induces a rapid decline in the level of tyrosine kinases without affecting the steady-state levels of other cellular proteins. The decrease in intracellular tyrosine kinase protein content is secondary to induction by the drug of an increased rate of degradation. The enhanced degradation of tyrosine kinases was found to be dependent on the 20S proteasome and the ubiquitin-conjugating pathway. Inhibitors of 20S proteasome proteolytic activities prevented the HA effect. Thus, HA leads to inhibition of transmembrane tyrosine kinases by causing their selective degradation in a process requiring the 20S proteasome, presumably as the catalytic core of the ATP- and ubiquitin-dependent 26S protease.


EXPERIMENTAL PROCEDURES

Materials

Herbimycin A (HA) was purchased from Life Technologies, Inc., dissolved in MeSO to 1 mg/ml, aliquoted, and kept frozen at -20 °C. [-P]ATP (3000 Ci/mmol) and S-Protein labeling mix (L]S]methionine, 1200 Ci/mmol) were purchased from Dupont NEN. Electrophoresis and electrotransfer reagents were from Fisher, Sigma, and Life Technologies, Inc. All other reagents were analytical grade and obtained from standard suppliers.

Antibodies

Monoclonal antibodies, anti-human IGF-IR antibody (IR-3), and anti-human IR (Ab-1) were from Oncogene Science, anti-phosphotyrosine antibody PT-66 was from Sigma, and anti-phospholipase C was from UBI. Polyclonal antibodies against EGFR, GAP, p85-PI3 kinase, and SHC were purchased from UBI, and anti-ubiquitin antibody was from Sigma. A polyclonal antibody that recognizes the subunit of IGF-IR in immunoblots was kindly provided by Dr. L.-H. Wang (Mt. Sinai Medical Center, New York), whereas a monoclonal antibody against human EGFR (mAb 225) was from Drs. J. Mendelsohn and H. Masui (Memorial Sloan-Kettering Cancer Center). A polyclonal antisera for baculovirus-produced human IR -subunit was raised by one of us (D. E. L., Ref. 17).

Protease Inhibitors

The peptidyl aldehyde inhibitors for the 20S proteasome were synthesized by the oxidation of corresponding alcohols by the procedure of Pfitzner and Moffat (18) as modified by Vinitsky et al.(19) . Their synthesis and characterization of their effect in vitro and in vivo is described in Ref. 19. The following 20S proteasome inhibitors were employed in this study (): Bz-Gly-Pro-Ala-phenylalaninal (GPAF-al), Bz-Pro-Gly-Ala-leucinal (PGAL-al), Bz-Gly-Pro-Phe-valinal (GPFV-al), Bz-Gly-Pro-Ala-leucinal (GPAL-al or 2al), Bz-Gly-Pro-Phe-leucinal (GPFL-al or 1al), Bz-Gly-Ala-Phe-leucinal (GAFL-al or 4al), and Bz-Gly-Leu-Ala-leucinal (GLAL-al or 5al). All inhibitors were dissolved in MeSO at 2 mM and stored at -20 °C. In order to extend the half-life of these inhibitors in vivo, cultures were simultaneously treated with Bz-Pro-prolinal (PP-al), a peptidyl aldehyde inhibitor of prolyl endopeptidase(20) . Phenylmethylsulfonyl fluoride (PMSF), leupeptin, E64d, and calpain inhibitor II were from Sigma and Calbiochem.

Cell Culture

The human breast cancer cell lines MCF-7 and MDA MB-468 cells were from the American Type Culture Collection, Rockville, MD. Cultures were maintained in DME/F12 (1:1) supplemented with 5% heat-inactivated fetal bovine serum (Gemini Bioproducts), 2 mM glutamine, and 50 units/ml each of penicillin and streptomycin, in a humidified 5% CO/air atmosphere at 37 °C. The temperature-sensitive E1-ts20 mutant cell line was provided by Dr. R. Kulka (The Hebrew University of Jerusalem). Cultures were maintained at 32 °C in RPMI 1640 supplemented with 10% fetal bovine serum, glutamine, and antibiotics.

Transfections

ts20 cells were co-transfected with a vector for human insulin receptor under the control of the cytomegalovirus promoter (pCMV-hIR(21) ) and a vector that confers geneticin resistance (pSV-neo). Transfection was performed with Lipofectin (Life Technologies, Inc.) according to the manufacturers' instructions. Pooled populations of stable transfectants were selected after 2 weeks of exposure to geneticin (0.5 mg/ml) and maintained under selective pressure.

Immunoprecipitation and Immunoblotting

Following experimental treatments, monolayers were washed twice with phosphate-buffered saline, and then either dissolved directly into SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer or solubilized in lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 1 mM NaV0, 40 mM NaF, and 10 µg/ml each of leupeptin, aprotinin, and soybean trypsin inhibitor) (750 µl/100 mm-dish)(22) . Cell lysates were centrifuged at 15,000 g for 10 min at 4 °C in a microfuge (Eppendorf), and the detergent solubilized proteins were immuno-precipitated with appropriate antibodies. Immunocomplexes were collected on protein A-Sepharose beads (Pharmacia), washed three times with lysis buffer, and subjected to SDS-PAGE. Gels were transferred onto nitrocellulose membranes and subjected to immunoblotting and detection via chemoluminescence (ECL, Amersham Corp.).

In Vitro Kinase Assays

IGF-IR immunoprecipitates from insulin-treated cells were washed twice in lysis buffer followed by a wash in lysis buffer lacking phosphatase inhibitors (NaF and NaVO). Washed immunoprecipitates were then incubated for 1 h at room temperature with either herbimycin A or MeSO in 40 µl of kinase buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl, 10 mM MnCl, 100 mM NaCl, 0.1% Triton X-100) in the presence or absence of 1 mM DTT. In vitro kinase reactions were initiated by the addition of 10 µCi of [-P]ATP/reaction plus unlabeled ATP to 90 µM. Reactions were carried out for various times (1, 5, 10, 15, and 30 min) at room temperature and stopped by the addition of 10 µl of 5 SDS-PAGE sample buffer. Phosphorylated products were separated by SDS-PAGE; gels were Coomassie-stained, dried, and autoradiographed. Band quantitation was performed using a FUJIX PhosphorImager with MacBAS-1000 software.

Pulse and Pulse-chase Experiments

In order to study the effect of HA on protein synthesis, MCF-7 cells were pulse-labeled with [S]methionine (100 µCi/ml, 1, 200 Ci/mmol) in methionine-free media for increasing time periods in the presence or absence of HA or its carrier (MeSO). Conversely, to assess the effect of HA on protein degradation, MCF-7 cells were prelabeled to isotopic equilibrium with [S]methionine (100 µCi/ml, 1, 200 Ci/mmol) in methionine-free media for 24 h and chased with unlabeled methionine (150 µg/ml) for a subsequent 24-h period. One h into the chase, cultures received either herbimycin A (5 µg/ml) or MeSO to 0.5%, and these conditions were maintained until the completion of the chase. Lysates were made at different time points in both types of experiments, and samples containing equal protein content were immunoprecipitated for IGF-IR as described above. Gels containing radiolabeled samples were Coomassie Blue-stained, fluorographed by impregnation with ENHANCE (DuPont NEN), dried, and exposed to x-ray film. Band quantitation was performed using a FUJIX PhosphorImager with MacBAS-1000 software.


RESULTS

Signaling via Receptor Tyrosine Kinases Is Antagonized in Vivo by Herbimycin A

IGFs and high concentrations of insulin activate IGF-IR and are mitogenic for MCF-7 cells under serum-free conditions (22). Treatment of MCF-7 cells with HA not only blocked insulin- and IGF-I-induced mitogenesis induced by activation of IGF-IR, but also inhibited proliferation induced by 10% fetal bovine serum (data not shown). This inhibitory effect was dose dependent and observed with HA concentrations as low as 10 ng/ml (17.4 nM) (data not shown). In order to understand the antiproliferative effect of HA in these cells, we first examined whether it impaired IGF-IR function.

As shown in Fig. 1A, insulin stimulation of serum-starved MCF-7 cells at concentrations that stimulate IGF-IR (5 min, 1 µM) led to two major changes on phosphotyrosine blots of total cellular protein: phosphorylation of the -subunit of the receptor (100 kDa) and of a family of insulin receptor substrates (IRS) with 185 kDa (Fig. 1). Preincubation of the cells with increasing concentrations of HA caused a dose-dependent decrease in the phosphotyrosine content of those insulin-responsive substrates (Fig. 1A). Immunoprecipitation of the IGF-IR followed by immunoblotting with an anti-phosphotyrosine antibody confirmed the identity of the 100 kDa band as the -subunit of the receptor (Fig. 1B). We have previously observed that in MCF-7 cells, the p120-rasGTPase-activating protein (GAP) becomes tyrosine phosphorylated following insulin treatment.()Tyrosine phosphorylation of GAP by insulin was also reduced in a dose-dependent manner following treatment with herbimycin A (Fig. 1C). Therefore, HA led to a dose-dependent inhibition of insulin-activated IGF-IR kinase activity and biological response as measured by phosphorylation of substrates and mitogenesis.


Figure 1: Herbimycin A inhibits tyrosine phosphorylation in response to insulin in MCF-7 cells in a dose-dependent manner. MCF-7 cells were serum-deprived for 24 h in the presence of increasing concentrations of herbimycin A in MeSO (0.1, 0.5, 1, 5, and 10 µg/ml). The final MeSO concentration in all cultures was 0.5%. Cells were subsequently stimulated or not (Control) with 1 µM insulin for 5 min and detergent-solubilized proteins were immunoprecipitated for IGF-IR (B) or for GAP (C). Immunoprecipitates and total lysate protein (A) were analyzed via SDS-PAGE, transferred onto nitrocellulose, and immunoblotted for phosphotyrosine content followed by detection via chemioluminescence. The positions of the IGF-IR receptor -subunit, pp185-IRS-1, GAP, and the GAP-associated proteins p190 and p62 are indicated with arrows on the right whereas the mobility of molecular weight markers are indicated on the left by their corresponding M.



Herbimycin A Inhibits IGF-IR Kinase Activity in Vitro

We investigated whether the ligand-activated tyrosine kinase activity of the IGF-IR was affected by the drug in in vitro kinase assays. Immobilized IGF-IR, immunoprecipitated from insulin-stimulated MCF-7 cells, was preincubated with 5 µg/ml of HA for 60 min prior to kinase assays. In the absence of DTT, 5 µg/ml (8.7 µM) of HA decreased the rate of IGF-IR autophosphorylation to approximately 50% of that obtained for vehicle alone (MeSO) (Fig. 2, A and BversusC and D). No inhibition was observed in the presence of reducing agents (Fig. 2, C and D), which supports the previous finding that sulfhydryl groups inactivate HA(3) . Although we were able to observe a direct effect of HA on the IGF-IR kinase in vitro, the concentrations required to observe this effect were at least 100-fold higher than those required to inhibit receptor function in the intact cell (Fig. 1).


Figure 2: IGF-IR kinase activity is inhibited in vitro by herbimycin A in the absence of reducing agents. IGF-IR was immunoprecipitated from lysates of insulin-stimulated MCF-7 cells with IR3, a monoclonal antibody for the -subunit of the human receptor. Immunocomplexes bound to protein A-sepharose beads were preincubated for 1 h at room temperature in the presence of either MeSO or 5 µg/ml herbimycin A in a standard in vitro kinase buffer supplemented or not with 1 mM DTT. The final MeSO concentration in both conditions was 2.5%. In vitro kinase reactions were carried out at room temperature for different times, terminated by addition of SDS-PAGE sample buffer and phosphorylated proteins were analyzed by SDS-PAGE followed by autoradiography. IGF-IR autophosphorylation was quantitated with a FUJIX PhosphorImager, by measuring the extent of P incorporation into the -subunit of the receptor in the absence and presence of DTT.



Herbimycin A Induces a Dose- and Time-dependent Decrease in the Steady-state Levels of Receptor Tyrosine Kinases in Breast Cancer Cells

The difference in concentrations required for inhibition of receptor tyrosine kinases in vitro and in the cell suggests either that intracellular HA is converted into a more potent inhibitor, or that other mechanisms of inhibition predominate. As shown in Fig. 3, HA led to a dose- and time-dependent reduction of the cellular content of IGF-IR (precursor as well as mature receptor) in MCF-7 cells, as detected on immunoblots of total cellular protein or of immunoprecipitated receptor. This effect was pronounced at 0.1 µg/ml of HA (Fig. 3A), and relatively rapid, noticeable after 4 h, and complete by 24 h (Fig. 3B).


Figure 3: Herbimycin A treatment leads to a decrease in the steady-state levels of tyrosine kinases in a time- and dose-dependent fashion, without affecting the levels of other (non-tyrosine kinase) cellular proteins. MCF-7 or MDA MB-468 cells were treated for 24 h with increasing concentrations of HA (A and D) as described for Fig. 1, or for different time periods with 5 µg/ml of the drug (B and E). Total cellular lysates and immunoprecipitates for IGF-IR (A and B) or immunoprecipitates for IR and EGFR (D) were immunoblotted with a specific antibodies for each receptor. Lysates of MCF-7 cells treated for 24 h with increasing HA concentrations were immunoblotted for phospholipase C-, GAP, the p85 subunit of PI3K, and SHC with specific antisera, as described for Fig. 1C.



To test whether this reduction in the receptor levels was due to a generalized effect on protein metabolism, we analyzed the effect of HA on the steady-state levels of other cellular proteins. The profile of [S]methionine-labeled total proteins revealed that there was no overall decrease in protein levels induced by HA treatment (not shown). Western blots for p85-phosphatidylinositol 3`-kinase (PI3K), phospholipase C-, SHC, and GAP (Fig. 3C) demonstrated that the cellular abundance of this group of proteins was not affected by the presence of HA in MCF-7 cells. The cellular abundance of other proteins such as - and -catenin, E-cadherin, and of serine/threonine protein kinases such as cdk2, erk-1, and erk-2 was also not affected by HA (not shown).

The same response to HA was observed in a different breast cancer cell line, MDA MB-468, which expresses abundant amounts of IR and overexpresses EGFR(22, 23) . In these cells, a similar dose- and time-dependent reduction in the steady-state levels of tyrosine kinase receptors by HA was observed (Fig. 4). Moreover, p56lck levels were decreased in response to HA in Colo 205 colon carcinoma cells, as was c-kit in mast cells (data not shown). It was recently shown that the steady-state levels of erbB-2 in MDA MB-453 cells (24) and of EGFR in A431 cells (41) were also reduced in response to HA treatment. In summary, HA does not cause a generalized effect on protein turnover, but rather it seems to affect protein tyrosine kinases primarily.


Figure 4: Herbimycin A induced an increase in the rate of receptor tyrosine kinase degradation. In order to assess the effect of HA on protein degradation, MCF-7 cells were prelabeled to isotopic equilibrium with [S]methionine (100 µCi/ml, 1, 200 Ci/mmol) in methionine-free media for 24 h and chased with unlabeled methionine (150 µg/ml) for a subsequent 24-h period (t = 0 h). One h into the chase (t = 1 h), cultures received either herbimycin A (5 µg/ml) or MeSO to 0.5%, and these conditions were maintained until the completion of the chase. Lysates made at different time points (t = 2, 3, 6, 12, and 24 h) were immunoprecipitated for IGF-IR. The IGF-IR precursor, as well as the mature - and -subunits, were identified by SDS-PAGE followed by autoradiography (A) and quantitation of the levels of [S]methionine incorporated into each species were measured with a FUJIX PhosphorImager (B and C). To analyze the effect of HA on protein synthesis (D), MCF-7 cells were pulse-labeled with [S]methionine (100 µCi/ml, 1, 200 Ci/mmol) in methionine-free media for increasing time periods, and the specific incorporation into the - precursor (arrow) for the IGF-IR was quantitated as described above.



Herbimycin A Treatment Induced an Accelerated Rate of Receptor Tyrosine Kinase Degradation

Changes in steady-state levels can be accomplished by altered rates of synthesis or degradation. Equilibrium labeling of MCF-7 cells with [S]methionine followed by chase with cold methionine in the presence or absence of HA was used to measure the effect of the drug on IGF-IR degradation. As shown in Fig. 4, A-C, HA induced an accelerated rate of IGF-IR degradation in MCF-7 cells. The apparent half-life of both receptor subunits (the extracellular, ligand-binding -subunit and the intracellular, tyrosine kinase-containing -subunit) was shortened from more than 24 h in control cultures to approximately 6-7 h in the presence of the drug. Similar results were observed for the EGFR in MDA MB-468 cells (data not shown). On the other hand, no significant differences in the rate of [S]methionine incorporation into IGF-IR were observed in pulse-labeling experiments, whether or not HA was present (Fig. 4D). Thus, tyrosine kinase synthesis is not appreciably affected by HA. Therefore, the rapid decrease in receptor tyrosine kinase steady-state levels observed upon HA treatment is due to an increased rate of receptor degradation.

Herbimycin A-induced Receptor Tyrosine Kinase Degradation Involves the 20S Proteasome

Most intracellular protein degradation is catalyzed by lysosomal proteases (25, 26) or by the ubiquitin- and ATP-dependent 26S protease complex(27, 28, 29, 30) . We employed a series of inhibitors of the lysosomal and ubiquitin-dependent proteolytic pathways to determine the mechanism by which HA induces receptor degradation. These experiments were performed in the absence of receptor ligand in order to exclude ligand-induced, lysosome-dependent degradation(25, 26) , a process involved in the down-regulation of activated receptors.

Lysosomal function was blocked by the lysosomotropic bases ammonium chloride and chloroquine. In order to inhibit the proteolytic activity of the 20S proteasome (the proteolytic core of the 26S protease), a series of peptidyl aldehydes(19, 31) ()was used (). The sequences of these inhibitors are based on known substrates of the 20S proteasome; the presence of a C-terminal aldehyde is required for inhibition of peptide bond hydrolysis(19) . Peptidyl aldehyde inhibitors were shown to block protein degradation catalyzed by the 20S proteasome in vitro and the 26S protease-mediated intracellular degradation of ubiquitinated proteins, whereas the peptidyl alcohols from which they were derived did not(19) . The most effective inhibitors of the 20S proteasome are GAFL-al (4al) and GLAL-al (5al) which strongly inhibit all of its five proteolytic components. When these inhibitors were added to cells together with HA they completely prevented the enhanced degradation of IGF-IR (Fig. 5, A and B). We next employed a series of peptidyl aldehyde inhibitors of the branched-chain amino acid preferring (BrAAP) component of the 20S proteasome, which is thought to be the major factor in protein degradation by this protease(19) . As shown in Fig. 5B, the potencies of the peptidyl aldehyde inhibitors of the BrAAP component in reverting the HA-induced IGF-IR degradation are directly correlated with their ability to inhibit that component in vitro(19) . That correlation also held for the ability of the inhibitors of BrAAP to affect accumulation of ubiquitin-protein conjugates in treated cells (not shown and 19). The most potent inhibitor from the series, GPFL-al, was able to revert IGF-IR levels to 75% of the levels from untreated controls (Fig. 5B). Bz-Pro-prolinal (PP-al), a peptidyl aldehyde inhibitor of prolyl endopeptidase (20) used to prolong the half-life of the peptidyl aldehyde inhibitors, had no effect on the levels of IGF-IR. Lysosomotropic agents or the inactive peptidyl alcohol analogs were also without effect in preventing the HA-induced IGF-IR degradation (Fig. 5, A and B). Moreover, the nonspecific serine- and cysteine-protease inhibitors PMSF, leupeptin, and E64d, and calpain inhibitor II (a potent inhibitor of cytosolic calpains and of lysosomal cathepsin B(25, 26, 30) ) were also ineffective in restoring IGF-IR levels in HA-treated cultures (Fig. 5, A and B). None of these general protease inhibitors affects the rate of protein degradation by the 20S proteasome at the range of concentrations employed here. Identical results were obtained for IR in MDA MD-468 cells (data not shown). These results imply that HA-induced degradation of IGF-IR and IR is catalyzed by the 20S proteasome.


Figure 5: IGF-IR degradation induced by herbimycin A is dependent on the 20S proteasome. A, MCF-7 cells were treated with 1 µg/ml HA for 9 h in the presence or absence of lysosomotropic agents, general serine- and cysteine-protease inhibitors (left panel) or with a series of peptidyl aldehyde inhibitors for the 20S proteasome (right panel). Lanes 1 and 7, untreated controls; lanes 2 and 8, HA alone; all others HA plus: NHCl (10 mM, lane 3), chloroquine (0.5 mM, lane 4), leupeptin (175 µg/ml, lane 5), PMSF (2 mM, lane 6), 20S proteasome inhibitors 4-al (GAFL-al), 5-al (GLAL-al), 1-al (GPFL-al) and 2-al (GPAL-al) (50 µM, lanes 9-13). Lanes 14 and 15 are samples from HA-treated cultures simultaneously treated with the inactive peptidyl-alcohols 5-ol (GLAL-ol) and 1-ol (GPFL-ol) (50 µM, lanes 14 and 15). All cultures were treated with benzyloxycarbonyl-prolyl-prolinal (PP-al), a prolyl endopeptidase inhibitor, included to prolong the half-life of the 20S proteasome inhibitors in vivo. PP-al by itself had no effect on the steady-state levels of tyrosine kinases. B, densitometric analysis of IGF-IR receptor levels from at least three independent experiments were combined in a graph and expressed as percentage of IGF-IR recovered ± S.E. The K values for the BrAPP component for five of these 20S proteasome inhibitors were from Vinitsky et al. (19). Inhibitors which were employed but not shown in A are GPFV-al, PGAL-al, GPAF-al, E64d, and calpain inhibitor II, all at 50 µM. Treatment of MCF-7 cells with any of these compounds in the absence of HA did not affect the basal steady-state levels of IGF-IR.



Degradation of Tyrosine Kinases Induced by Herbimycin A Is Dependent on the Ubiquitin-conjugating System

The 20S proteasome is proposed to be involved in ubiquitin-dependent and -independent proteolysis(27, 28, 33, 34) . In order to discriminate between these two possibilities, we decided to investigate whether ubiquitination of tyrosine kinases was necessary for their sensitivity to HA. Experiments to detect ubiquitinated receptors in immunoprecipitates failed to provide clear results (see ``Discussion''). We used a temperature-sensitive mutant cell line defective in E1-catalyzed ubiquitin activation (35) to determine the ubiquitin requirement of the HA effect. Activation of ubiquitin by E1 is an essential step for all ubiquitin-dependent processes(27) , and at the restrictive temperature, cells defective in this step are unable to carry on protein ubiquitination(35) . ts20 cells were stably transfected with human IR (pCMV-hIR) (21) and selected for geneticin resistance. Pooled populations were treated with or without HA for 5 h at either 25 or 37 °C. At the permissive temperature, HA induced the degradation of IR; however at 37 °C, the degradation of IR was greatly prevented (Fig. 6). Densitometric analysis indicated that at 25 °C IR levels were reduced by 77% in the presence of HA, whereas at 37 °C IR levels were decreased by only 13%. The steady-state levels of total cellular ubiquitin-protein conjugates were markedly decreased at 37 °C, indicative of an impaired E1 function (Fig. 6). The dependence of HA-induced degradation on the ubiquitin-conjugating system together with its sensitivity toward 20S proteasome inhibitors suggest that exposure of protein tyrosine kinases to HA leads to their selective degradation by the ubiquitin- and ATP-dependent 26S protease.


Figure 6: Receptor tyrosine kinase degradation induced by herbimycin A is dependent on the presence of an active ubiquitin pathway. Temperature-sensitive E1 ts20 mutants transfected with the human IR were pretreated for 4 h at either 25 or 37 °C and subsequently treated with HA (1 µg/ml) or MeSO (0.01%) in serum-free media for 5 h. The levels or IR were measured by immunoprecipitation and immunoblotting (left panel), and the levels of total ubiquitin-protein conjugates were analyzed by Western blots of lysate protein with anti-ubiquitin antibody (1:500, Sigma) (right panel).




DISCUSSION

In this report we show that HA leads to the functional inhibition of cellular tyrosine kinase receptors by inducing their degradation. We found that HA caused a time- and dose-dependent reduction in the steady-state levels of IGF-IR, IR, and EGFR receptors (Fig. 3). Analysis of the steady-state levels of other cellular proteins (including serine and threonine protein kinases) after HA treatment revealed that the HA effect seems to be specific for tyrosine kinases. Measurements of the kinetics of protein turnover showed that HA enhanced the degradation of tyrosine kinase receptors (Fig. 4, A-C, and data not shown) with no significant changes in the rate of protein synthesis (Fig. 4D). Analysis of the molecular pathways involved in this effect suggested that the 20S proteasome and the ubiquitin pathway of protein degradation were involved ( Fig. 5and Fig. 6). We employed a set of inhibitors of a variety of cellular proteases and of lysosomal function to study this phenomenon (Fig. 5). We found that neither inhibition with the nonspecific serine- and cysteine-protease inhibitors PMSF, leupeptin, and E64d, nor inhibition of the Ca-dependent protease calpain with calpain inhibitor II (which is also a potent inhibitor of the lysosomal protease cathepsin B) (25, 26, 30) were effective in preventing the HA-induced degradation of tyrosine kinases. Moreover, blockade of lysosomal function with ammonium chloride or chloroquine at concentrations capable of preventing ligand-activated EGFR down-regulation via the endocytic pathway were also incapable of preventing the HA effect. Therefore, we conclude that the observed degradation of tyrosine kinases induced by HA does not occur via the lysosomal pathway or by cytosolic proteases such as calpains. It is important to note that although activated receptors are degraded by the lysosome(26) , the experiments presented here were conducted in the absence of activating ligands in order to prevent activation of this pathway, which would complicate the analysis of the HA effect.

We also employed a set of peptidyl aldehyde inhibitors designed to inhibit the catalytic activities of the 20S proteasome ()(19) . The 20S proteasome or multicatalytic proteinase complex is a large cytosolic and nuclear proteinase which is thought to constitute the proteolytic core of the ubiquitin-dependent proteolytic machinery(27, 28, 31, 33) . It is distinguished by the wide specificity of the proteolytic reactions it catalyzes. At least five distinct proteolytic components of the 20S proteasome have been described(31) . These are referred to as tryspin-like, chymotrypsin-like, peptidyl-glutamyl peptide hydrolyzing, small neutral amino acid-preferring, and BrAAP. Each of these activities is catalyzed by a physically distinct subunit or a set of subunits of 20S proteasome, not by a single site with a broad range of substrate specificities(31, 33) . A series of peptidyl aldehyde inhibitors for the 20S proteasome were designed based on sequences derived from known 20S proteasome substrates of the BrAAP component. These inhibitors possess a C-terminal aldehyde, which by reacting with a nucleophilic center in the active site of a protease forms a hemiacetal or thiohemiacetal, which is thought to mimic the putative transition state of peptide bond hydrolysis(19) . These peptidyl aldehyde inhibitors effectively blocked protein degradation catalyzed by the 20S proteasome in vitro and led to the accumulation of ubiquitin-protein conjugates in cells (19). Conversely, the peptidyl alcohol precursors of these inhibitors had no effect in either setting(19) , thus confirming the necessity of the aldehyde group for inhibition. We observed that there was a dose-dependent accumulation of ubiquitin-protein conjugates in cells treated with the aldehyde inhibitors, with no change in response to the inactive alcohols (Ref. 19 and data not shown). When a combination of HA and each of these compounds were added to cells, we observed that the enhanced degradation of tyrosine kinases was prevented (Fig. 5). Moreover, when a series of peptidyl aldehyde inhibitors were employed, we observed a direct relationship between the efficacy of these compounds in preventing the HA effect and their potencies as inhibitors of the BrAAP activity of the 20S proteasome (Fig. 5, Ref. 19, and data not shown). It is important to note that, in the absence of HA, the BrAAP inhibitors had no effect per se on the levels of tyrosine kinases. Also, none of the lysosomotropic agents or general serine-and cysteine-protease inhibitors tested affected protein degradation by the 20S proteasome at the range of concentrations employed. Therefore, our data point to a model in which HA leads to the degradation of tyrosine kinases in a process that involves their selective targeting to the 20S proteasome.

The best studied function of the 20S proteasome is that of the proteolytic core of the ATP-dependent 26S protease which primarily catalyzes ubiquitin-dependent degradation of proteins(27, 28, 31) . Therefore, we studied whether ubiquitination was required for the HA effect. Immunoprecipitated IGF-IR from HA-treated cells is associated with ubiquitin as assessed by Western blotting with an anti-ubiquitin antibody (data not shown). However, it was difficult to produce a blot of high quality, due to the poor reactivity of the anti-ubiquitin antibodies employed and the rapid loss in IGF-IR in HA-treated cells. Therefore, we used a conditional mutant cell line to test whether ubiquitination played a role in the HA-induced degradation of these receptors. As shown in Fig. 6, in a temperature-sensitive cell line (35) deficient in the ubiquitin-activating enzyme E1 (the first enzyme in the ubiquitin conjugation pathway)(27, 29) , HA was unable to induce the degradation of transfected IR under restrictive conditions, while effectively reducing IR levels at the permissive temperature. Although this result does not unequivocally prove that ubiquitination of the tyrosine kinase occurs, it indicates that a functional ubiquitin pathway is necessary for HA to induce degradation. We are currently studying the molecular mechanism of this effect in a cell-free system.

A proposed mechanism for the in vitro inhibition of tyrosine kinase activity by HA involves the formation of an adduct between a reactive cysteine residue in the kinase with the benzoquinone group of the drug, thereby leading to the inactivation of the enzyme(3, 6) . This model was based on the observations that sulfhydryl compounds such as DTT, 2-mercaptoethanol, glutathione (reduced form), or cysteine inactivated HA, whereas methionine, cystine, or oxidized glutathione had no effect (3, 6, and Fig. 2). Comparative sequence analysis of protein kinases revealed the presence of 2 conserved Cys in the kinase domain of receptor and cytosolic tyrosine kinases, which are conspicuously absent from most serine/threonine protein kinases(36, 37) . The primary role of these Cys residues in catalysis was illustrated in mutants generated for p56(38) . Cys to Ala p56 mutants in the catalytic domain, at positions 464 and 475, abolished catalytic activity in vitro as well as in the cell. Furthermore, C475A was highly unstable, exhibiting a half-life six times shorter than the wild type protein(38) . Whether this accelerated turnover of the mutant proteins was due to a profound change in conformation or whether it involved a mechanism similar to that induced by HA (see below) is currently under investigation.

A model for the cellular effect of HA could be proposed wherein the ubiquitin conjugating system might recognize the HAprotein complex itself or a motif in the protein exposed by HA binding. In this regard, it has been recently demonstrated that geldanamycin, a HA analog, inhibited the formation of the hsp90v-src complex(39) . Whether this event is sufficient to target tyrosine kinases for conjugation with ubiquitin and subsequent proteolysis is unknown. Nevertheless, the inducibility of tyrosine kinase degradation by the 20S proteasome and its dependence on the ubiquitination pathway will allow the study of the elements of the proteolytic system involved in the cells response to HA. The results presented here suggest that induction of receptor tyrosine kinase degradation is an important component of the mechanism by which HA inhibits the activity of transmembrane and cytosolic tyrosine kinases in the cell. During the preparation of this article, Murakami et al.(32) reported the enhanced degradation of EGFR induced by HA in A431 cells. They showed that degradation occurred by a lysosomal-independent pathway, as lysosomotropic agents had no effect of preventing EGFR loss. In this report, we show that degradation is dependent upon the 20S proteasome and on the ubiquitin-conjugating system. Induction of degradation of a specific class of proteins represents a novel mechanism for drug action and identifies an agent, HA, which may represent a prototype of this new class of drugs. It also affords a strategy for causing cytotoxicity in tumors whose transformed phenotype is dependent on the presence of activated tyrosine kinases.

  
Table: 20S proteasome inhibitors

Standard one or three letters abbreviations for amino acids are used.



FOOTNOTES

*
This work has been supported by the National Cancer Institute of the NIH Grant CA 58706-01 (to N. R.) and by an Aaron Diamond Foundation postdoctoral fellowship (to A. V.). 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: Memorial Sloan-Kettering Cancer Center, 1275 York Ave., Box 271, New York, NY 10021. Tel.: 212-639-2369; Fax: 212-717-3627.

The abbreviations used are: HA, herbimycin A; IR, insulin receptor; IGF-IR, insulin-like growth factor receptor; IGF, insulin-like growth factor; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GAP, p120-rasGTPase-activating protein; PI3 kinase, phosphatidylinositol-3` kinase; Bz, benzyloxycarbonyl; PP-al, Bz-Pro-prolinal; PMSF, phenylmethylsulfonyl fluoride; BrAAP, branched-chain amino acid preferring; PAGE, polyacrylamide gel electrophoresis; MeSO, dimethyl sulfoxide.

L. Sepp-Lorenzino, Z. Ma, D. E. Lebwolhl, A. Vinitsky, and N. Rosen, unpublished observations.

A. Vinitsky, C. Cardozo, and M. Orlowski, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. L-H. Wang for providing us with anti-IGF-IR antibody, Drs. J. Mendelsohn and K. Masui for the anti-EGFR monoclonal antibody 225, and Dr. R. Kulka for the ts20 cell line. We gratefully acknowledge Drs. M. Orlowski and C. Cardozo for support and Dr. J. Rothman for a critical review of the manuscript.


REFERENCES
  1. Uehara, Y., Hori, M., Takeuchi, T., and Umezawa, H. (1985) Jpn. J. Cancer Res. (Gann)76, 672-675 [Medline] [Order article via Infotrieve]
  2. Uehara, Y., Murakami, Y., Mizuno, S., and Kawai, S. (1988) Virology164, 294-298 [Medline] [Order article via Infotrieve]
  3. Uehara, Y., Fukazawa, H., Murakami, Y., and Mizuno, S. (1989) Biochem. Biophys. Res. Commun.163, 803-809 [Medline] [Order article via Infotrieve]
  4. Uehara, Y., Murakami, Y., Sugimoto, Y., and Mizuno, S. (1989) Cancer Res.49, 780-785 [Abstract]
  5. June, C. H., Fletcher, M. C., Ledbetter, J. A., Schieven, G. L., Siegel, J. N., Phillips, A. F., and Samelson, L. E. (1990) Proc. Natl. Acad. Sci. U. S. A.87, 7722-7726 [Abstract]
  6. Fukazawa, H., Mizuno, S., and Uehara, Y. (1990) Biochem. Biophys. Res. Commun.173, 276-282 [Medline] [Order article via Infotrieve]
  7. Wilks, A. F. (1993) Adv. Cancer Res.60, 43-73 [Medline] [Order article via Infotrieve]
  8. Bolen, J. B. (1993) Oncogene8, 2025-2031 [Medline] [Order article via Infotrieve]
  9. Kazlauskas, A. (1994) Curr. Op. Genet. & Develop.4, 5-14
  10. Uehara, Y., Hori, M., Takeuchi, T., and Umezawa, H. (1986) Mol. Cell. Biol.6, 2198-2206 [Medline] [Order article via Infotrieve]
  11. Fukazawa, H., Li, P.-M., Yamamoto, C., Murakami, Y., Mizuno, S., and Uehara, Y. (1991) Biochem. Pharmacol.42, 1661-1671 [CrossRef][Medline] [Order article via Infotrieve]
  12. Papa, V., Gliozzo, B., Clark, G. M., McGuire, W. L., Moore, D., Fujita-Yamaguchi, Y., Vigneri, R., Goldfine, I. D., and Pezzino, V. (1993) Cancer Res.53, 3736-3740 [Abstract]
  13. Van de Vijver, M. J., and Nusse, R. (1991) Biochim. Biophys. Acta1072, 33-50 [Medline] [Order article via Infotrieve]
  14. Arteaga, C. L., Coronado, E., and Osborne, C. K. (1988) Mol. Endocrinol.2, 1064-1069 [Abstract]
  15. Arteaga, C. L., Kitten, L. J., Coronado, E. B., Jacobs, S., Kull, J. F. C., Alfred, D. C., and Osborne, C. K. (1989) J. Clin. Invest.84, 1418-1423 [Medline] [Order article via Infotrieve]
  16. Ennis, B. W., Valverius, E. M., Lippman, M. E., Bellot, F., Kris, R., Schlessinger, J., Masui, H., Goldenberg, A., Mendelsohn, J., and Dickson, R. B. (1989) Mol. Endocrinol.3, 1830-1838 [Abstract]
  17. Herrera, R., Lebwohl, D., Garcia de Herreros, A., Kallen, R. G., and Rosen, O. M. (1988) J. Biol. Chem.263, 5560-5568 [Abstract/Free Full Text]
  18. Pfitzner, K. E., and Moffat, J. G. (1965) J. Am. Chem. Soc.87, 5661-5670
  19. Vinitsky, A., Cardozo, C., Sepp-Lorenzino, L., Michaud, C., and Orlowski, M. (1994) J. Biol. Chem.269, 29860-29866 [Abstract/Free Full Text]
  20. Wilk, S., and Orlowski, M. (1983) J. Neurochem.41, 69-75 [Medline] [Order article via Infotrieve]
  21. Russell, D. S., Gherzi, R., Johnson, E. L., Chou, C. K., and Rosen, O. M. (1987) J. Biol. Chem. 262, 11833-11840
  22. Sepp-Lorenzino, L., Rosen, N., and Lebwohl, D. E. (1994) Cell Growth & Diff.5, 1077-1083
  23. Dickson, R. B., Bates, S. E., McManaway, M. E., and Lippman, M. E. (1986) Cancer Res.46, 1707-1713 [Abstract]
  24. Miller, P., DiOrio, C., Moyer, M., Schnur, R. C., Bruskin, A., Cullen, W., and Moyer, J. D. (1994) Cancer Res.54, 2724-2730 [Abstract]
  25. Knop, M., Schiffer, H. H., Rupp, S., and Wolf, D. H. (1993) Curr. Opinion Cell Biol.5, 990-996 [Medline] [Order article via Infotrieve]
  26. Gruenberg, J., and Howell, K. E. (1989) Annu. Rev. Cell Biol.5, 453-481 [CrossRef]
  27. Ciechanover, A. (1994) Cell79, 13-21 [Medline] [Order article via Infotrieve]
  28. Rechsteiner, M., Hoffman, L., and Dubiel, W. (1993) J. Biol. Chem.268, 6065-6068 [Free Full Text]
  29. Hershko, A., and Ciechanover, A. (1992) Annu. Rev. Biochem.61, 761-807 [CrossRef][Medline] [Order article via Infotrieve]
  30. Rock, K. L., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick, L., Hwang, D., and Goldberg, A. L. (1994) Cell78, 761-771 [Medline] [Order article via Infotrieve]
  31. Orlowski, M., Cardozo, C., and Michaud, C. (1993) Biochemistry32, 1563-1572 [Medline] [Order article via Infotrieve]
  32. Murakami, Y., Mizuno, S., and Uehara, Y. (1994) Biochem. J.301, 63-68 [Medline] [Order article via Infotrieve]
  33. Orlowski, M. (1990) Biochemistry29, 10289-10297 [Medline] [Order article via Infotrieve]
  34. Rivett, A. J. (1993) Biochem. J.291, 1-10 [Medline] [Order article via Infotrieve]
  35. Kulka, R. G., Raboy, B., Schuster, R., Parag, H. A., Diamond, G., Ciechanover, A., and Marcus, M. (1988) J. Biol. Chem.263, 15726-15731 [Abstract/Free Full Text]
  36. Hanks, S. K., Quinn, A. M., and Hunter, T. (1988) Science241, 42-52 [Medline] [Order article via Infotrieve]
  37. Hanks, S. K., and Quinn, A. M. (1991) Methods Enzymol.200, 38-62 [Medline] [Order article via Infotrieve]
  38. Veillette, A., Dumont, S., and Fournel, M. (1993) J. Biol. Chem.268, 17547-17553 [Abstract/Free Full Text]
  39. Whitesell, L., Mimnaugh, E. G., De Costa, B., Myers, C. E., and Neckers, L. M. (1994) Proc. Natl. Acad. Sci. U. S. A.91, 8324-8328 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.