©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Autophagy and Multiple Steps in Asialoglycoprotein Endocytosis by Inhibitors of Tyrosine Protein Kinases (Tyrphostins) (*)

Ingunn Holen , Per E. Str , Paul B. Gordon , Monica Fengsrud , Trond O. Berg , Per O. Seglen (§)

From the (1) Department of Tissue Culture, Institute for Cancer Research, The Norwegian Radium Hospital, 0310 Oslo, Norway

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In isolated rat hepatocytes, several tyrosine protein kinase inhibitors (tyrphostins) reduced the autophagic sequestration of electroinjected [H]raffinose by 40-75% at doses that did not significantly affect cellular ATP levels or plasma membrane integrity. Tyrphostin 46 specifically inhibited autophagy, whereas tyrphostins 1, 25 and 51 also suppressed the receptor-mediated endocytic uptake of I-tyramine-cellobiose-asialoorosomucoid, I-TC-AOM, by 20-30% and its degradation by 70-90%. Tyrphostins 1 and 51, and the microtubule inhibitor vinblastine, inhibited an early endocytic step (endosome maturation/multivesiculation?), causing accumulation of endocytosed I-TC-AOM in a recycling compartment that corresponded to light endosomes (1.10-1.11 g/ml) in sucrose density gradients. In the electron microscope, these endosomes could be recognized as small, peripheral endocytic vesicles and tubules accumulating endocytosed AOM-gold. The serine/threonine protein phosphatase inhibitor okadaic acid inhibited an intermediate endocytic step (detachment of multivesicular endosomes from the tubulovesicular network?), causing accumulation of I-TC-AOM in a recycling compartment corresponding to light endosomes (1.10-1.11 g/ml), but with a multivesicular rather than a tubulovesicular morphology. Tyrphostin 25 inhibited endocytosis at a late step (endosome-lysosome fusion?), causing accumulation of I-TC-AOM in a non-recycling compartment corresponding to dense, multivesicular endosomes (1.14 g/ml) that had probably detached from the light endosomal network.


INTRODUCTION

The pathways of autophagy and endocytosis are known to be subject to regulation by protein phosphorylation. In isolated rat hepatocytes, autophagy can be inhibited by cyclic AMP, an activator of the cyclic AMP-dependent protein kinase (1) , as well as by okadaic acid (2) , a protein phosphatase inhibitor that causes a general hyperphosphorylation of liver cell proteins (3) . The effect of okadaic acid on autophagy can be antagonized by a specific inhibitor of Ca/calmodulin-dependent protein kinase II (2), implicating this, or a closely related enzyme, in the negative regulation of autophagy. On the other hand, several inhibitors of tyrosine protein kinases suppress autophagy, suggesting that this class of enzymes may be involved in positive control of the process (2) .

The endocytic pathway is apparently regulated by protein phosphorylation at several steps, e.g. receptor internalization (4, 5) , receptor recycling (4) , receptor routing (6) , endosome-endosome fusion (7, 8) , endosome binding to microtubules (9) , and transport of endosomes along microtubules (10) . In addition, several endosomal proteins have been shown to be phosphorylated (11) . The present study was undertaken to investigate the role of tyrosine protein phosphorylation in regulation of autophagy and endocytosis in freshly isolated hepatocytes. For this purpose we tested the effects of various tyrosine protein kinase-inhibitory benzenemalononitrile derivatives, known as tyrphostins. The tyrphostins were developed as substrate-competitive inhibitors of the EGF() receptor tyrosine kinase (EGFR) (12) , and shown to be effective blockers of EGF-dependent processes in intact cells, e.g. cell proliferation (13, 14) . Some of the tyrphostins can inhibit EGF-independent tyrosine protein kinases as well, such as the receptors for insulin, platelet-derived growth factor, or nerve growth factor (12, 15, 16) . We have measured autophagy as the sequestration of electroinjected [H]raffinose (17) and receptor-mediated endocytosis as the uptake, recycling, and degradation of I-TC-AOM. This probe has the advantage that acid-soluble degradation products remain inside the vacuoles where they are generated and can therefore be used to distinguish between proteolytic and non-proteolytic endocytic vacuoles, separated on sucrose density gradients (18) . In addition, endocytic vacuoles have been characterized ultrastructurally after uptake of AOM-coated gold particles. The results indicate that tyrphostins can inhibit the initial sequestration step of autophagy as well as several steps in the endocytic pathway.


EXPERIMENTAL PROCEDURES

Cell Preparation and Incubation

Hepatocytes were isolated from 18-h starved male Wistar rats (250-300 g) by two-step collagenase perfusion (19) . The cells were washed and suspended in suspension buffer containing 15 mM pyruvate and extra Mg (to 2 mM). Two ml of cell suspension (15-20 mg cellular wet weight) were incubated at 37 °C in 5-cm albumin-coated plastic Petri dishes. For measurement of endocytosis, the cells were preincubated for 30 min at 37 °C in the presence of the drugs before addition of I-TC-AOM, and the cells were then incubated for a further 2 h at 37 °C. For isopycnic centrifugation the cells were incubated with drugs for 30 min at 37 °C, and then for a further 30 or 120 min in the presence of I-TC-AOM. After the incubation the cells were cooled to 0 °C, transferred to 15-ml plastic centrifugation tubes, and washed once with perfusion buffer containing 10 mM EGTA, pH 7.5, to remove receptor-bound I-TC-AOM from the cell surface. For uptake and degradation measurements, the cells were washed twice with 10% sucrose (w/v) or once with perfusion buffer.

Measurement of Autophagy

Autophagy was measured as the sequestration of electroinjected [H]raffinose into sedimentable vacuoles as described previously (17) .

Measurement of ATP

For measurement of intracellular ATP, the incubated cells (2 ml) were precipitated with 0.5 ml of 10% perchloric acid and centrifuged for 15 min at 3700 g. A 1-ml aliquot of the supernatant was transferred to a separate tube and neutralized with freshly made 2 M KOH. The amount of ATP in the sample was measured luminometrically by use of a luciferin/luciferase assay (Pharmacia LKB Biotechnology, Sweden).

Measurement of Protein Synthesis

Protein synthesis was measured as the incorporation of [C]valine into the acid-precipitable material of cells incubated in the presence of amino acids (20) .

Measurement of Uptake and Degradation of I-TC-AOM

After washing, the cell pellets were counted in an LKB-gamma counter to measure the cellular uptake of I-TC-AOM, i.e. the amount of cell-associated radioactivity, expressed as the percent of the total acid-insoluble radioactivity initially added. To each pellet, 1 ml of 10% trichloroacetic acid was added, the samples were kept on ice for a minimum of 15 min, and thereafter centrifuged for 15 min at 3700 g. The supernatant, containing the acid-soluble degradation products of I-TC-AOM, was transferred to a separate tube and counted. The amount of I-TC-AOM degraded was calculated as a percentage of the total acid-insoluble radioactivity added to each sample initially.

Measurement of I-TC-AOM Recycling

To measure the release of endocytosed I-TC-AOM from ligand-recycling compartments, hepatocytes were incubated for 30 min at 37 °C in the presence of drugs. I-TC-AOM was then added, and the cells incubated for another 60 min. The cells were then rapidly cooled by addition of ice-cold buffer and washed once at 0 °C with buffer containing 10 mM EGTA to remove surface-bound ligand. The cells were subsequently incubated at 37 °C in the presence of 2.5 mM EGTA for up to 60 min. In the presence of the chelator, any AOM that recycles to the cell surface and is released into the medium will be prevented from rebinding to the receptor. The amount of recycled I-TC-AOM was measured and expressed as percent of the total cell-associated radioactivity.

Cell Fractionation and Isopycnic Sucrose Density Gradient Centrifugation

All fractionation and gradient work was performed at 0 °C. Cell samples were resuspended in 0.5 ml of unbuffered 10% sucrose and pooled before electrodisruption. An equal volume of buffered sucrose (0.25 M sucrose, 10 mM HEPES, 1 mM EDTA, pH 7.3) was then added to each sample, followed by 10-20 strokes in a Dounce homogenizer with a tight-fitting pestle. The homogenate was centrifuged at 3000 g min to obtain a nuclear pellet and an post-nuclear supernatant. The nuclear pellet was washed once in 4.5 ml of buffered sucrose, and the two post-nuclear supernatants were combined. A 1.5-ml sample of post-nuclear supernatant was layered on top of a linear sucrose density gradient. The gradient was made by layering 5.5 ml of 63% buffered sucrose underneath 5.5 ml of 15% buffered sucrose and mixing with a Biocomp Gradient Master (Nycomed Pharma, Oslo, Norway) to obtain densities ranging from 1.04 g/ml in the top fraction to 1.23 g/ml in the bottom fraction. The gradients were centrifuged at 4 °C in a Beckman L8-70 M ultracentrifuge with an SW40/9E 2800 rotor for 1 h 45 min at 40,000 revolutions/min (3.110 gmin). Following centrifugation the gradients were split into approximately 20 fractions by upward displacement using Maxidenz as a displacement fluid. The densities of the fractions were calculated from the refractive indices. Acid phosphatase activity was determined spectrophotometrically in a Technicon RA-1000 autoanalyzer and expressed as percentage of the total acid phosphatase in the cell. Total radioactivity, acid-soluble, and acid-insoluble radioactivity in each fraction were measured in a gamma counter as described above, except that albumin was added to a final concentration of 0.1% (w/v) before acid precipitation (0.4 ml gradient fraction, 0.1 ml of albumin, and 0.5 ml of 20% trichloroacetic acid). Radioactivities were expressed as percentage of the total acid-insoluble radioactivity initially added to the sample.

Electron Microscopy

Colloidal gold particles (3- and 10-nm diameter) were prepared according to Slot and Geuze (21) and coated with AOM by adsorption according to established procedures. Gold sol (250 ml) was complexed with AOM (1.5 mg for 10-nm gold; 0.75 mg for 3-nm gold) by mixing at room temperature, and the gold-protein complexes recovered by centrifugation at 47,000 g for 120 min (3-nm) or 45,000 g for 45 min (10-nm). The gold-AOM probes were dialyzed against 500 ml of 0.9% NaCl (w/v) overnight. To prelabel hepatocytic lysosomes, 1 ml of 3-nm AOM-gold in collodial suspension (with an absorbance value of 1.70 at 520 nm after 100-fold dilution) was injected intravenously into rats 24 h before cell isolation. To label endosomes and lysosomes in vitro, 2 ml of hepatocytes in suspension (15 mg cells/ml, wet weight) were incubated at 37 °C with 15 µl of 10-nm AOM-gold (final absorbance of 2.0 at 520 nm) for 120 min. The cells were washed once in perfusion buffer containing 10 mM EGTA to remove dead cells and surplus gold-AOM, and twice in ice-cold 0.1% glutaraldehyde, 0.1 M cacodylate buffer, pH 7.4. Pellets were fixed in 2% glutaraldehyde, 0.1 M cacodylate buffer overnight and postfixed for 60 min with 2% OsO containing 1.5% potassium ferrocyanide, followed by en bloc staining with 1.5% uranyl acetate. After serial dehydration in ethanol and propylene oxide, specimens were embedded in Epon, sectioned, and post-stained with 0.2% lead citrate. Sections were examined in a Phillips CM10 electron microscope at 80 kV.

Reagents

I-TC-AOM was kindly provided by dr. Tor Gjand Prof. Trond Berg, University of Oslo. [H]Raffinose (5 Ci/mmol, 1 Ci/liter) was from NEN Du Pont and [C]valine (260 Ci/mol, 50 mCi/l) from Amersham International, Amersham, Bucks, United Kingdom. Okadaic acid was from Moana Bioproducts Inc., Hawaii. Metrizamide and Maxidenz were from Nycomed A/S, Oslo, Norway. Texas red sulfonyl chloride (T-1905) and rhodamine green succinimidyl ester (R-6112) were purchased from Molecular Probes Inc., Eugene, OR. Tyrphostins and all other biochemicals were from Sigma.


RESULTS

Effects of Tyrphostins on Autophagic Sequestration, Protein Synthesis, Intracellular ATP Levels, and Viability

The structural formulas of the tyrphostins investigated in this study, and their potencies against the EGF receptor tyrosine protein kinase (EGFR), are listed in Fig. 1. All of the tyrphostins tested inhibited the autophagic sequestration of [H]raffinose (). Tyrphostins 25, 46, and 51 inhibited autophagy by 70-76%, tyrphostin 1 by 54%, without significantly affecting hepatocytic integrity (measured as the loss of total cellular radioactivity). Tyrphostin 25 was somewhat more potent than the other two, with maximal effect at 100 µM. At higher concentrations, cytotoxicity (reduced cellular integrity) became more prominent. With tyrphostins 23 and 63, cytotoxicity was evident already at 30 and 150 µM, respectively.


Figure 1: Molecular structure of the tyrphostins used in the present study. The data, including the potency of tyrphostins against EGF-receptor tyrosine kinase activity, have been adapted from Ref. 12.



Protein synthesis was inhibited by 15-40% at nontoxic tyrphostin concentrations. Intracellular ATP levels were not reduced by any of the tyrphostins tested, even at high concentrations, showing that the drugs did not interfere with the mitochondrial respiratory chain as has been reported for some tyrphostins (22) . The inhibition of autophagy by tyrphostins would, therefore, be unlikely to be the result of nonspecific cytotoxicity.

Effects of Tyrphostins on Uptake and Degradation of an Endocytic Probe

Isolated hepatocytes rapidly endocytose I-TC-AOM by a receptor-mediated mechanism (18) : within 2 h, 80% of the added probe had been taken up by control cells (Fig. 2A), and approximately 50% of this had become degraded (Fig. 2B). Two of the tyrphostins (1 and 25) had moderate effects on I-TC-AOM uptake (Fig. 2A) but strong inhibitory effects on I-TC-AOM degradation (Fig. 2B). Similar effects were exerted by the microtubule inhibitor, vinblastine, the serine/threonine protein phosphatase inhibitor, okadaic acid, and, somewhat paradoxically, by the tyrosine protein phosphatase inhibitor, vanadate. Fig. 3shows dose-response curves for the effects of various tyrphostins on uptake and degradation of I-TC-AOM during a 3-h incubation period. Tyrphostins 23 (panel A), and 46 (panel B) caused only small alterations in the uptake and degradation of I-TC-AOM, even at the highest doses employed. Tyrphostins 1, 25, 51, and 63 (panels C-F) had moderate inhibitory effects (30% or less) on I-TC-AOM uptake, but inhibited the degradation of endocytosed I-TC-AOM strongly (70-90%) in the concentration range 100-300 µM. These tyrphostins would thus seem to be effective inhibitors of I-TC-AOM endocytosis/degradation at some step(s) beyond ligand uptake.


Figure 2: Effects of tyrphostins, okadaic acid, vinblastine, and vanadate on accumulation and degradation of endocytosed I-TC-AOM. Hepatocytes were incubated for 30 min at 37 °C without additions (), with 50 µM vinblastine (), 30 nM okadaic acid (), 100 µM tyrphostin 1 (), 100 µM tyrphostin 25 (), or 10 mM vanadate (). I-TC-AOM was then added, and the cells incubated further at 37 °C for the length of time indicated. The cells were then cooled to 0 °C, washed, precipitated with ice-cold 10% trichloroacetic acid, and the total cell-associated radioactivity (A) as well as the acid-soluble radioactivity (B) was measured and expressed as percent of the acid-insoluble radioactivity initially added. The data are from a single experiment.




Figure 3: Effects of tyrphostins on endocytic uptake and degradation of I-TC-AOM. Hepatocytes were incubated for 30 min at 37 °C, in the presence of tyrphostin at the doses indicated. I-TC-AOM was then added, and the incubation continued for another 2 h at 37 °C. The cells were then cooled to 0 °C, washed, and precipitated with 10% ice-cold trichloroacetic acid. The total cell-associated radioactivity (uptake, ), as well as the acid-soluble radioactivity (degradation, ), was measured and expressed as percent of the acid-insoluble radioactivity initially added. Each value is the mean ± S.E. of three to six independent experiments.



Effects of Tyrphostins on Density Distribution of I-TC-AOM-containing Endocytic Vacuoles

After 30 min of continuous I-TC-AOM uptake, radiolabeled endocytic vacuoles from control hepatocytes banded in a sucrose density gradient as a major peak at 1.10 g/ml, with a shoulder at 1.13-1.14 g/ml (Fig. 4). This distribution was not much changed by any of the inhibitors applied, although the light peak was somewhat sharper and the heavy shoulder somewhat less developed after treatment with okadaic acid, vinblastine, and some of the tyrphostins (1, 51, and 63). At this time, no radioactive material coincided with the lysosomes, i.e. the major peak of acid phosphatase at 1.18 g/ml. Vinblastine shifted this peak to a lighter position (1.14 g/ml), whereas the other treatments had no effect on lysosome distribution.


Figure 4: Effects of tyrphostins on density distribution of endocytic vacuoles after 30 min of I-TC-AOM endocytosis. Hepatocytes were incubated for 30 min with no additions (control), or in the presence of 30 nM okadaic acid, 50 µM vinblastine, 30 µM tyrphostin 23, 100 µM tyrphostins 46, 25, or 1, 300 µM tyrphostin 51 or 150 µM tyrphostin 63. I-TC-AOM was then added and the cells incubated for another 30 min at 37 °C, then rapidly cooled to 0 °C, and washed twice with 10% sucrose, electrodisrupted, and homogenized. Post-nuclear supernatants were prepared and fractionated by isopycnic sucrose density gradient centrifugation. Radioactivity was measured and acid phosphatase (ACP) assayed in each fraction, and the values per fraction are expressed as percent of the total cell-associated radioactivity () or acid phosphatase (). The data are from a single experiment.



After 2 h of I-TC-AOM endocytosis, some of the radioactivity in control cells was still present in the light gradient peak at 1.10 g/ml, but most of it had been shifted to peaks at 1.14 and 1.18 g/ml, the latter colocalizing with the lysosomal marker enzyme (Fig. 5). The radioactivity at 1.18 g/ml was largely acid-soluble, indicating that I-TC-AOM had been degraded by lysosomal enzymes (results not shown). Cells treated with tyrphostins 23 or 46 (which had negligible effects on I-TC-AOM endocytosis, cf.Fig. 3 ) displayed the same distribution as control cells.


Figure 5: Effects of tyrphostins on density distribution of endocytic vacuoles after 120 min of I-TC-AOM endocytosis. The experiment and the analyses were performed as described in the legend to Fig. 4, except that the hepatocytes were incubated for 120 min after the addition of I-TC-AOM. The data are from a single experiment.



In cells treated with okadaic acid, a major fraction of the endocytosed I-TC-AOM was still retained at 1.10 g/ml after 120 min, although some had also reached the lysosomal peak at 1.18 g/ml (Fig. 5). With tyrphostin 25, most of the radioactivity accumulated at 1.14 g/ml, less at 1.10 g/ml, and very little at 1.18 g/ml. Tyrphostins 1, 51, and 63 produced yet another pattern, essentially the same as vinblastine, with radioactivity retained in a major peak at 1.10 g/ml and a minor peak at 1.14 g/ml, and little or none at 1.18 g/ml. Unlike the tyrphostins, vinblastine shifted the lysosome peak to 1.14 g/ml, but no acid-soluble radioactivity was found in this region in either vinblastine- or tyrphostin-treated cells (results not shown).

Effects of Tyrphostins on Ligand Recycling

Endocytosed I-TC-AOM undergoes extensive recycling from early endosomes, but when the probe reaches late endosomes it dissociates from the receptor and recycling gradually decreases (18) . Fig. 6shows the degree of I-TC-AOM recycling, measured as the release of radioactivity from the cells in the presence of EGTA (to cause ligand detachment at the cell surface), during a 1-h incubation in the presence of okadaic acid, vinblastine, or various tyrphostins. Vinblastine increased the extent of recycling 4-fold, and okadaic acid 3-fold, indicating increased retention of ligand in a recycling compartment (Fig. 6A). Tyrphostins 1, 51, and 63 similarly increased I-TC-AOM recycling severalfold (Fig. 6B). Tyrphostins 23 and 46, which had little or no effect on I-TC-AOM endocytosis, respectively (Fig. 3), also had little or no effect on recycling (Fig. 6A). Tyrphostin 25 caused much less recycling than the other active tyrphostins (Fig. 6B), suggesting ligand retention primarily in a non-recycling compartment.


Figure 6: Effects of tyrphostins on ligand recycling. Hepatocytes were incubated for 30 min in the presence of various drugs as indicated. A, , no addition (control); , 50 µM vinblastine; , 30 nM okadaic acid; , 100 µM tyrphostin 46; , 30 µM tyrphostin 23. B, , no addition (control); , 100 µM tyrphostin 25; , 300 µM tyrphostin 51; , 100 µM tyrphostin 1; , 150 µM tyrphostin 63. I-TC-AOM was then added, and the incubation continued for 1 h at 37 °C. The cells were then rapidly cooled to 0 °C and washed once in ice-cold perfusion buffer containing 10 mM EGTA to remove surface-bound ligand. Finally, the cells were incubated in suspension buffer containing 2.5 mM EGTA (to prevent rebinding of released ligand) at 37 °C for the length of time indicated. I-TC-AOM released to the medium was measured and expressed as percent of the total radioactivity in the system. Each value is the mean ± S.E. or range of two to eight experiments.



Effects of Tyrphostins on the Distribution of Endocytic Vacuoles Identified by Electron Microscopy

To obtain a structural identification of the endocytic vacuoles affected by tyrphostins, hepatocytes were prelabeled with 3 nm of AOM-gold in vivo overnight (to label lysosomes) and allowed to endocytose 10 nm of AOM-gold during the period of drug treatment (to identify endosomes). In control cells, three major vacuole types could be identified: 1) small endocytic vesicles and tubules (50-100 nm diameter). These were the predominant structures labeled with 10 nm of AOM-gold after 5 min of endocytosis (Fig. 7A). 2) Multivesicular endosomes (200-600 nm diameter), the predominant type of endosome at later times of endocytosis (Fig. 7B). 3) Lysosomes (300-1500 nm diameter), recognized as vacuoles containing 3 nm of AOM-gold or visibly degraded material of cellular origin (Fig. 7C). Both small, dense lysosomes (Fig. 7C, upper right-hand corner) and large, electron-lucent lysosomes (Fig. 7C, lower left-hand corner) could be seen; in addition, the lysosome class as defined here may include some of the ``prelysosomes'' and ``late endosomes'' characterized by other workers on the basis of different criteria (23) .


Figure 7: Morphology of endocytic vacuoles. Hepatocytes, isolated from a rat injected with 3-nm AOM-gold 24 h before sacrifice, were preincubated for 30 min without additions (control, A-C), 50 µM vinblastine (D), 100 µM tyrphostin 1 (E), 100 µM tyrphostin 25 (F), or with 30 nM okadaic acid (G). 10-nm AOM-gold was then added, and the cells incubated for another 2 h at 37 °C before harvesting and processing for conventional electron microscopy. 3-nm gold was found in both light and dense lysosomes (C). Endocytosed 10-nm gold could be found in early endocytic vesicles/tubules (A, D, and E), in multivesicular endosomes (B, F, and G) and in light and dense lysosomes (C). In cells treated with vinblastine (D) or tyrphostin 1 (E), 10-nm gold was present mainly in early endocytic vesicles/tubules. In cells treated with tyrphostin 25 (F) or okadaic acid (G), 10-nm gold was primarily found in multivesicular endosomes. Bar, 100 nm.



A quantitative morphometric analysis revealed that after 2 h of endocytosis in control cells, the bulk of the endocytosed 10-nm AOM-gold was approximately equally distributed between multivesicular endosomes and lysosomes, only a small fraction (4%) remaining in endocytic vesicles/tubules (). A similar distribution was seen in cells treated with tyrphostin 46, which does not affect endocytosis detectably. However, vinblastine treatment altered the distribution radically, causing two-thirds of the endocytosed 10-nm AOM-gold to be retained in endocytic vesicles/tubules, while hardly anything (2%) reached the lysosomes (Fig. 7D, ). A similar distribution was seen in cells treated with tyrphostin 1 (Fig. 7E, ).

In cells treated with okadaic acid (Fig. 7G) or tyrphostin 25 (Fig. 7F), endocytosed 10-nm AOM-gold accumulated predominantly in multivesicular endosomes (). It thus seems clear that different tyrphostins can affect different steps in the endocytic pathway, in accordance with the results obtained by the sucrose gradient analysis (Fig. 5) and the ligand recycling studies (Fig. 6).


DISCUSSION

The effects of the tyrphostins indicate that autophagic sequestration as well as receptor-mediated endocytosis may be dependent on tyrosine protein phosphorylation. In contrast, increased serine/threonine protein phosphorylation, induced by okadaic acid or other protein phosphatase inhibitors, has been shown to inhibit autophagy (2, 24) . It is, therefore, possible that members of the two classes of protein kinase may regulate autophagy antagonistically.

All the active tyrphostins had to be given at relatively high concentrations (100-300 µM) in order to be effective on isolated hepatocytes. At these levels, some cytotoxicity was observed with tyrphostins 23 and 63, but not with tyrphostins 1, 25, 46, or 51. Among the latter, tyrphostin 46 was uniquely autophagy-specific, whereas the other three inhibited both autophagy and endocytosis. There was no correlation between the effects of the individual tyrphostins on I-TC-AOM degradation and their reported potencies as inhibitors of the EGFR (12) : two of the most effective inhibitors in our studies, tyrphostins 1 and 63, are in fact essentially inactive as EGFR inhibitors (cf. Fig. 1 ). Wijetunge et al. (25) concluded that the cellular effects of tyrphostin 1 could not be due to EGFR inhibition, and other observations likewise indicate that tyrphostin effects are not necessarily the result of inhibition of tyrosine protein kinases (22) . Any implication of these enzymes to explain the effects of tyrphostins on autophagy or endocytosis should, therefore, be regarded as tentative. As an additional paradox, a tyrosine protein phosphatase inhibitor, vanadate, has been found to inhibit asialoglycoprotein (ASGP) endocytosis (26) as effectively as do the tyrosine protein kinase inhibitors.

Our results indicate that tyrphostins have an early effect on I-TC-AOM uptake as well as later effects on intracellular endocytic flux. The ASGPR has a single cytoplasmic tyrosine residue that might be a substrate for a receptor-associated tyrosine protein kinase (27) , although the functional role of phosphorylation at this tyrosine has been questioned (28) . Fallon et al. showed that several tyrosine kinase inhibitors, among them genistein and a tyrphostin, inhibited tyrosine phosphorylation of the ASGPR in vitro(5) and internalization of the ASGPR in intact HepG2 cells, suggesting a causal relationship. In our studies, we found no effect of genistein at concentrations that strongly affected other hepatocellular processes (2) and at most a 30% inhibition of I-TC-AOM uptake by the tyrphostins. In normal hepatocytes, at least, tyrosine phosphorylation of the ASGPR would, therefore, seem to have a modulating rather than an obligatory function in ASGP uptake.

In contrast to their moderate effect on I-TC-AOM uptake, several tyrphostins strongly reduced the transfer of I-TC-AOM to lysosomes and its degradation to acid-soluble material. Tyrphostins 1 and 51, as well as the microtubule-disrupting drug vinblastine, exerted their effect at a relatively early endocytic step, causing accumulation of endocytosed ASGP in a recycling compartment identified as small (50-100 nm), light (1.10-1.11 g/ml) endocytic vesicles and tubules. These tyrphostins may thus inhibit the microtubule-dependent (29) transfer of ligand from tubulovesicular to multivesicular endosomes within a continuous endocytic network (30) , possibly by interfering with endosome maturation/multivesiculation (31, 32) . However, the tyrphostins did not share the ability of vinblastine to disrupt the hepatocytic microtubule organization,() suggesting a different mechanism of action, e.g. interference with the binding of endosomes to microtubules (33) .

Like tyrphostins 1 and 51, okadaic acid caused retention of endocytosed ASGP in a recycling compartment corresponding to light endosomes (1.10-1.11 g/ml). However, in contrast to the tyrphostin-treated cells, these endosomes were identified morphologically as multivesicular rather than as tubulovesicular. Okadaic acid would thus seem to inhibit endocytosis at a later step than tyrphostins 1 and 51, possibly by preventing the detachment of multivesicular endosomes from a continuous endosomal network (30) containing tubulovesicular as well as multivesicular elements of similar low density (1.10-1.11 g/ml).

Tyrphostin 25 would appear to inhibit endocytosis at an even later step. In the presence of this drug, ASGP accumulated predominantly in dense (1.14 g/ml), still multivesicular endosomes which had the properties of a non-recycling compartment, indicating their detachment from the recycling endosomal network to become ``carrier vesicles'' (34). Tyrphostin 25 apparently inhibited fusion between these dense multivesicular endosomes and lysosomes, thereby preventing the formation of active, light (1.14-1.16 g/ml) lysosomes (35, 36) , or ``prelysosomes'' (37) , recognized by their contents of both acid-soluble and acid-insoluble I-TC-AOM-derived material. This precluded the subsequent degradation of I-TC-AOM and the concomitant formation of dense (1.18-1.19 g/ml) lysosomes containing acid-soluble degradation products only. A tyrosine protein kinase involvement at a late step in ASGP endocytosis has previously been suggested (38) , but on the basis of inhibition by staurosporine, a broadly nonspecific protein kinase inhibitor. Hopefully, the use of tyrphostins with different specificities may help to elucidate the complexity of the endocytic pathway in greater detail.

  
Table: Effects of tyrphostins on autophagic sequestration, protein synthesis, intracellular ATP levels, and cellular integrity

Hepatocytes preloaded with [H]raffinose were incubated for 3 h at 37 °C in the presence of tyrphostins at the concentrations indicated. Autophagy was measured as the net sequestration of [H]raffinose, and expressed as percent of the autophagic rate measured in control cells (3.7%/h). Cellular (plasma membrane) integrity was measured as the loss of total cellular radioactivity between 1 and 3 h of incubation, and expressed as percent of the loss from control cells (3.0%/h). Protein synthesis was measured as the net incorporation of [C]valine during a 3-h incubation in the presence of a complete, balanced amino acid mixture, and expressed as percent of the protein synthesis rate measured in control cells (0.5%/h). ATP was measured luminometrically in acid-precipitated cells after 3 h of incubation at 37 °C, and expressed as percent of the ATP level measured in control cells (2.1 µg/g cellular wet mass). Each value is the mean ± S.E. or range of the number of experiments given in parentheses.


  
Table: Effects of okadaic acid, vinblastine, and various tyrphostins on endocytosis of 10 nm gold-AOM

Lysosomes were labeled by injecting rats intravenously with 3 nm of AOM-gold 24 h before cell isolation. The freshly isolated hepatocytes were incubated for 30 min at 37 °C with no additions (control) or in the presence of 50 µM vinblastine, 30 nM okadaic acid, or 100 µM tyrphostin 1, 25, or 63. 10 nm of AOM-gold was then added and the cells incubated for another 2 h at 37 °C. After the incubation the cells were rapidly cooled to 0 °C and washed once in ice-cold perfusion buffer containing 10 mM EGTA to remove surface-bound ligand. The cells were processed for conventional electron microscopy as described under ``Experimental Procedures.'' Ten cell profiles from each treatment group were examined, and the total number of 10-nm gold particles in all vacuoles belonging to a certain morphologically defined compartment (endocytic vesicles/tubules, multivesicular endosomes or lysosomes) was counted and express as percent of the total number of vacuole-associated gold particles/cell profile. Each value is the mean ± S.E. of 10 cell profiles.



FOOTNOTES

*
This work was generously supported by the Norwegian Cancer Society and by the Research Council of Norway. 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: Dept. of Tissue Culture, Institute for Cancer Research, The Norwegian Radium Hospital, N-0310 Oslo, Norway. Tel.: 47-22-93-59-47; Fax: 47-22-73-29-44.

The abbreviations used are: EGF, epidermal growth factor; ASGP, asialoglycoprotein; ASGPR, asialoglycoprotein receptor; AOM, asialoorosomucoid; cAMP, cyclic 3`5`-adenosine monophosphate; EGFR, EGF receptor tyrosine protein kinase; TC-AOM, tyramine-cellobiose-asialoosomucoid.

H. Blankson, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Tor Gjand Prof. Trond Berg for providing the I-TC-AOM. The skillful technical assistance of Mona Birkeland is gratefully acknowledged.


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