Temperature-sensitive mutants of avian erythroblastosis virus: Surface expression of the erbB product correlates with transformation

Cell, Vol. 36, 963-972,

April 1964, Copyright

Q 1964 by MIT

0092.6674/64/040963-i

0 $02.00/O

Temperature-Sensitive Mutants of Avian Erythroblastosis Virus: Surface Expression of the erbl3 Product Correlates with Transformation Hartmut Beug* and Michael J. Hayman+ *European Molecular Biology Laboratories Postfach 10.2209 Meyerhofstrasse 1 6900 Heidelberg, Federal Republic of Germany ‘Imperial Cancer Research Fund Laboratories St. Bartholomews Hospital Dominion House London EC1 A 7BE, England

Summary The w-erbB gene of avian erythroblastosis virus (AEV) codes for an integral plasma membrane glycoprotein, gp74eW. Expression of gp74’* and its intracellular precursors, gp66and gp66*-, has been studied in cells transformed by two temperature-sensitive mutants of AEV. After shift to 42OC, the processing of gp66is blocked in tsAEV-transformed, but not in wtAEV-transformed, erythroblasts and fibroblasts. In addition, gp74’* disappears from the surface of tsAEV cells within 12 hr after shift. Thus tsAEV mutants probably bear a lesion in v-erb6 that affects the maturation and subcellular localization of gp74tiB. The tsAEV erythroblasts, when “committed” to differentiation by a pulse-shift to 42OC, reexpress gp7QM during terminal differentiation at 36OC. This suggests that tsAEV erythroblasts become insensitive to the transforming functions of gp74- at a certain stage of differentiation. Introduction Avian erythroblastosis virus (AEV) is a retrovirus that induces acute erythroid leukemias as well as sarcomas upon injection into young chicks. In vitro, AEV induces the outgrowth of transformed erythroblast-like cells from infected bone marrow and also transforms chicken embryo fibroblasts (for review see Graf and Beug, 1978). The genome of AEV contains two putative oncogenes, v-erbA and v-erb6, which are flanked by nonfunctional parts of the replicative genes gag and env (Roussel et al., 1979; Lai et al., 1979; Vennstrom and Bishop, 1982). Because of this lack of replicative genes, AEV is dependent on a helper virus for replication. The v-erbA oncogene codes for a 75 kilodalton (kd) gag-erbA fusion protein, which is located predominantly in the cytoplasm (Hayman et al., 1979, 1983). The product of the verbs oncogene was recently identified as a plasma membrane glycoprotein of 74 kd, gp74”- (Hayman et al., 1983; Hayman and Beug, manuscript submitted; Privalsky et al., 1983). Biosynthesis of gp74&’ starts from a 62.5 kd precursor, which is glycosylated (and phosphorylated) to form 66 kd and 68 kd intermediate products, gp66*and gp68etibs. These are sensitive to the enzyme endoglycosidase H (Tarentino and Maley, 1974) and are located

mainly on intracellular membranes (rough endoplasmic reticulum and/or Golgi membranes). gp68tiB is further glycosylated to form the mature gp74e*B, which is located almost exclusively in the plasma membrane and insensitive to endo H (Hayman and Beug, manuscript submitted). It is likely, therefore, that the protein detected on the cell surface of AEV-transformed cells by immunofluorescence using anti-erbB sera (Hayman et al., 1983) is identical with gp74e? Indications of how the two oncogenes of AEV contribute to generate the transformed phenotype in erythroblasts and fibroblasts have come from experiments with viral mutants that carry large deletions in either v-erbA or verbB (Frykberg et al., 1983). These studies showed that verb6 is the principal transforming gene of AEV. In the absence of v-erbA, v-erb6 causes fibroblast transformation and induces an aberrant type of leukemic erythroblast in infected bone marrow. These erythroblasts require complex growth conditions and partially differentiate into mature erythrocytes (Frykberg et al., 1983; Beug et al., unpublished). In contrast, v-erbA in combination with v-erbB is apparently capable of arresting the leukemic cells in an early stage of differentiation and of rendering them able to grow in simple tissue culture media unsuitable for normal erythroid precursors (Beug et al., 1982a). Insight into the mechanism by which AEV transforms hematopoietic cells came from the isolation of AEV mutants temperature-sensitive for leukemic transformation (tsAEV; Graf et al., 1978; Palmieri et al., 1982). Erythroblasts transformed by such tsAEV mutants and cultivated at the nonpermissive temperature, under conditions suitable for differentiation of normal erythroid precursors (CFUE), grew into typical, CFU-E-like erythrocyte colonies within 4 days (Samarut and Gazzolo, 1982; Beug et al., 1982a). This indicated that AEV-transformed leukemic erythroblasts retain the capacity to proceed along the normal pathway of erythroid differentiation, if the function of the erb oncogene(s) is turned off at the nonpermissive temperature. Pulse-shift experiments demonstrated that a period of 24 to 48 hr at 42°C was sufficient to “commit” the cells irreversibly to differentiate (Beug et al., 1982a). It was unclear, however, whether or not v-erb oncogene products would be reexpressed in the “committed” cells if allowed to complete their differentiation at 36°C. The observation that all five tsAEV mutants isolated so far are temperature-sensitive both for erythroblast transformation and for fibroblast transformation (Palmieri et al., 1982) pointed to v-erbB as the oncogene carrying the temperature-sensitive lesion (Frykberg et al., 1983). This prompted us to study the expression of the cytoplasmic and cell surface forms of the v-erb6 gene product(s) during temperature-induced differentiation and in “committed” tsAEV erythroblasts. We show that a shift to the nonpermissive temperature rapidly arrests the processing of the gp68 precursor into the mature gp74eti protein. At the same time, gp74erbB disappears from the cell surface. “Committed” tsAEV erythroblasts, however, when shifted

Cell 964

back to 36°C reexpress gp74”*’ at the cell surface even at later stages of differentiation. This indicates that the committed cells have lost the ability to respond to the transforming function of the v-erbB gene product.

Results Temperature-Sensitive Expression of gp74tsl67AEV Erythroblasts

ts 167 AEV

wt AEV

P75

A in

To study v-erb6 protein expression in differentiating tsAEV erythroblasts, two main approaches were chosen. First, cell surface expression of v-erb/3 protein was analyzed by immunofluorescence using anti-erbB sera (Hayman et al., 1983). Second, synthesis of the immature forms (gp66*, gp68MB) and mature form (gp74”*‘) of verb6 protein was assayed for by immunoprecipitation of extracts from cells labeled with either 3H-glucosamine or %-methionine using anti-erbB sera (Hayman et al., 1983; Hayman and Beug, manuscript submitted). To test for biosynthesis of verb products, cells of the clonal strain ts167E3 were kept at 42°C in differentiation medium for 2 days, and the cells were separated from dead and nondifferentiating cells by Percoll density gradient centrifugation (see Experimental Procedures; Adkins et al., submitted). Aliquots of these cells as well as of ts167E3 cells kept at 36°C for 2 days and cells from a wild-type AEV clone (B5) kept at 36°C and 42% under the same conditions were then labeled and analyzed by immunoprecipitation. Figure 1A shows that synthesis of ~75~“~~ and of the erbt3 precursors gp66e*B and gp68&’ was not drastically altered by shift to 42%. Processing of gp66*M into gp68&, however, oc curred more rapidly at 42’C, resulting in the disappearance of gp66’*’ at 42% under the labeling conditions used. In contrast, expression of gp74”M was completely blocked in ts167E3 cells shifted to 42”C, although it was present in the same cells at 37’C (Figure 1B). A similar result was obtained with the ts34AEVtrans formed, temperature-inducible erythroblast cell line HD3 (Beug et al., 1982b). In a pulse-chase experiment, processing of gp68&’ into gp74&’ proceeded normally in HD3 cells grown at 37’C, whereas no detectable gp74& was generated from gp68@” after 1, 2, or 4 hr of chase in HD3 cells shifted to 42°C for 3 days (Figure IC and data not shown). To study ceil surface expression of v-erbB protein at the nonpermissive temperature, tsl67E3 cells were tested by immunofluorescence using anti-erbB serum after shift to 42% for 12, 24, and 48 hr. Temperature-sensitive AEV cells kept at 36°C and wild-type AEV cells from both 36°C and 42% were treated similarly. To demonstrate the specificity of possible changes in the erbB fluorescence, the cells were also stained with antierythroblast serum (Beug et al., 1979) labeled with another fluorochrome. Figure 2 shows that all the erbB protein detectable by fluorescent anti-erbB antibody had already disappeared from the cell

w68 gp66

36”

42”

36”

42”

c, 9P74 gp68 -9P66

6 36”

42”

36”

42”

1

ts 34 AEV 36”

42”

/9P74 azpp68 gp66

,N,I,

I

P

4h

Figure 1. Processing tsAEV Erythroblasts

of gp66 -

-N

into gp74-

I P

I

I 4h

Is Temperature

Sensitive

in

(A) tsl67AEV erythroblast clone E3 (tsl67AEV) and wtAEV erythroblast clone 85 (wtAEV) were kept at 36°C or shifted to 42% for 2 days. Extracts of these cells labeled with %methionine were then immunoprecipftated with antisera detecting both p75 (~75) and the erbB gene products gp66. gp68, and gp74. The weak band of gp74obtained under these labeling conditions is obscured by ~75. Note the mere rapid processing of gp66 into gp66 at 42%. (B) tsl67AEV and wtAEV cells were treated as in (A), but were labeled with %-glucosamine and immunoprecipftated with antiserum detecting erb8 gene products only. (C) Cells of the ts34AEV-transformed erythroblast cell line HD3 (ts34AEV) were treated as in (A), but pulse-lab&d with 36S-methionine for only 30 min (P). An aliquot was chased in medium containing cold methionine for 4 hours (4h). Extracts were immunoprecipitated with anti-erbs serum (I) or normal rat serum (N). Asterisks indicate background bands precipitated by both normai and immune serum.

surface after only 12 hr at 42°C whereas no change in staining intensity was detectable with antierythroblast serum (Figure 2). In contrast, no decrease in erbB surface fluorescence could be seen in wild-type AEV erythroblasts

Temperature-Sensitive 965

Mutants

of AEV

unable to differentiate in response to temperature shift. These cells were fibroblasts transformed by tsl67AEV and cells of the noninducible ts34 erythroblast line HD2 (Beug et al., 1982b). Figure 38 shows that in both cell types grown at 42°C the synthesis of gp74* is greatly reduced, but not completely abolished, as compared to cells at 36°C and control wild-type cells (Figure 38 and data not shown). A similar result was obtained with v-e&3 protein detected by immunofluorescence (Figure 3A). After shift to 42°C surface erb6 expression, as judged both by the number of fluorescence-positive cells and by fluorescence intensity, was significantly reduced but not completely absent (from 70% to 34% in ts167 fibroblasts and from 74% to 43% in HD2 erythroblasts).

Differential Expression of v-erbA and v-erbB Proteins during Terminal Differentiation of tsAEV Erythroblasts

Figure 2. gp74Shifted to 42%

Disappears

from the Cell Surface of tsAEV Erythroblasts

ts167AEVE3 or wtAEV-65 erythroblasts were grown at 36°C or shifted to 42°C as described in the legend to Figure 1. The same tsAEV cells doublelabeled with anti-erb6 serum plus FITC-labeled goat antirat antibody (erb6) and antrerythroblast serum plus TRITC-labeled goat antirabbit antibody (EblAg) are shown in the top and mrddle panels, respectively. wtAEV cells (bottom panels) were stained in the same way, but only the erbB fluorescence is shown. White arrows show nonspecific fluorescence of dead cells.

kept at 42°C for periods of 12 hr to 3 days (Figure 2 and data not shown).

Expression of gp74-’ Is Also Reduced in tsAEV Fibroblasts and Nondifferentiating Erythroblast Variants at 42°C The observed disappearance of cell-surface-expressed verbi3 protein in tsAEV erythroblasts at 42°C could be a consequence of changes associated with the onset of terminal differentiation in these cells. Alternatively, it may reflect heat-sensitive properties of the putatively thermolabile v-erb6 protein itself. In an attempt to resolve this question, we used cells transformed with tsAEV that were

Terminal differentiation of tsAEV erythroblasts into erythrocyte-like cells is associated with a major reprogramming of the pattern of protein synthesis, especially at later stages of maturation (Beug et al., 1982a; Adkins et al., submitted). To study how these differentiation-induced changes in protein synthesis would affect expression of the v-erbA and v-erb/3 products, ts167E3 cells (50 X lo6 each) were shifted to 42°C for 1, 2, 3, or 4 days or left at 36°C. The resulting cell populations were purified on Percoll density gradients (see Experimental Procedures) and characterized for their state of differentiation by neutral benzidine staining (see Figure 5C; Beug et al., 1982a). They were then labeled and immunoprecipitated with anti-v-erb sera. In addition, they were subjected to immunofluorescent staining as described above, either as live cells or after fixation and permeabilization, to reveal intracellular erbB protein. By 3H-glucosamine labeling, it could be shown that gp74embs was already absent 24 hr after shift at 42°C as expected (Figure 4A). In contrast, gp68&’ was produced at roughly constant levels until day 2. Its synthesis was clearly decreased, however, in day 3 reticulocytes and was almost absent in the fairly mature erythrocytes obtained at day 4 (Figure 48) in which large amounts of adult-type hemoglobin (HbA) and spectrin were detected by monoclonal antibodies (data not shown). In contrast, P75 gag-erbAwas continuously synthesized throughout terminal differentiation, being present even in the mature day4 cells (Figure 46). The differential expression of p75QaQerbAand gp74e’be observed during the late stages of erythroid differentiation was reflected by similar behavior of the virus structural protein precursors pr76Qag and pr92”““. pr76Q”Q was produced throughout differentiation in a similar fashion to p75QWabA, whereas synthesis of pr92”“” was shut down at the same time as gp68e*bs (Figure 4C). These results were confirmed by immunofluorescence analysis. Cell surface v-erbB protein had completely disappeared 24 hr after shift to 42°C (Figure 5A). In contrast, cytoplasmic v-erb6 protein (detected in fixed, permeabil-

Cell 966

A 36°C

42°C 36°C

42°C

IN

IN

ts167AEV fibroblasts and cells of the nondifferentiating erythroblast cell line HD2 were kept at WC or shifted to 42% for 6 and 3 days, respectively. In (A), these cells are shown after immunofluorescent staining with antieM antibodies. To demonstrate the specificity of anti&S staining. the fibroblasts were double-labeled with ant&b6 antibodies and antibodies to the virus structural protein pi9 (p19) as described in the legend to Figure 2. (6) shows rmmunoprecipitation of the erbB gene products from these cells after labeling with 3H-glucosamine. Symbols as in legend to Figure 1.

ized cells) was abundant until day 3, but clearly reduced or absent in terminally differentiated day-4 erythrocytes (Figure 5B). Staining of the same cells with a variety of antibodies specific for erythroblasts (antierythroblast serum, Beug et al., 1979; MC 4.5.A.5, Hayman et al., 1982); for reticulocytes (MC 4.6.C.1, Hayman et al., 1982; Beug et al., 1982a); and for erythrocytes (antierythrocyte serum, Beug et al., 1982a; and a monoclonal antibody to spectrin, MC F.13.4.9; H. Weintraub, personal communication) showed that the major “switch” from an immature to a more mature phenotype takes place around day 2, i.e. at a time when synthesis of the v-erbB intermediate gp6ae*’ is not yet affected (Figure 5B and data not shown).

Cell Surface gp74Is Reexpressed in Committed tsAEV Erythroblasts Differentiating at 36% Previous studies have shown that tsAEV erythroblasts acquire the capacity to mature into apparently normal erythrocytes even at the permissive temperature (36°C) if they are “committed” to differentiate by a pulse shift to 42’C (Beug et al., 1982a, and unpublished results). With the ts167E3 clone used in these studies, the time at 42°C required to “commit” more than 80% of the cells was 42-

48 hr (data not shown). Since the synthesis of gp6adB is still essentially normal after this time we asked whether or not cell surface gp74e*B was reexpressed after shift of the committed cells to 36°C. Accordingly, tsl67E3 cells (75 x 106) were shifted to 42°C for 42 hr and purified by Percoll gradient centrifugation, and an aliquot was analyzed by immunofluorescence to verify that surface v-erb was absent in the essentially undifferentiated cells (Figure 6B). The cells were further incubated at 36°C for 1, 2, or 3 days. Resulting cell populations were purified on suitable Percoll gradients and analyzed by immunofluorescence for v-erb expression and state of differentiation. Cell surface v-erbB protein was first detected 6 hr after backshift to 36°C and was found to be strongly expressed on the partially differentiated cells obtained after 1 day (data not shown) or 2 days at 36°C (Figure 6C). The terminally differentiated ceils obtained 3 days after shift to 36°C showed greatly reduced surface v-erbB expression (data not shown), probably as a result of the suppression of verbB precursor synthesis occurring in very mature cells. To determine whether reexpression of surface v-erbB protein could also be detected biochemically, committed ts167E3 cells that had been allowed to differentiate at

Temperature-Sensitrve 967

Mutants

of AEV

A

37°C

c

42”C, Id

91074 w68 w66

-

pr 180

-pr92 +pr76 cp75

B p75+

w68+ w66” 1 I.

36°C

42°C

/I Id

Figure 4. Differential Expression

2d of verbA

and verb8

36°C ’ 3d

4d’

Gene Products

’ Id

I

during Terminal Differentiation

42°C 2d

3d

\ 4d

of tsAEV Erythroblasts

ts167E3 cells kept at 36°C or shifted to 42°C for 1, 2, 3, or 4 days (l-4d) were labeled with ‘H-glucosamine (A) or %-methionine (6, C). (A) Extracts immunoprecipitated wrth antr-erb8 antibody (I) or normal rat serum (N). In (6) and (C), extracts were immunoprecipitated with anti-erbA+B antibodies or antibodies to the vtrus structural proteins p76w (pr76), p19F (pr92), and prl6CP”-‘~ (pr166), respectively. Asterisks in (A) and (8) show background tmmunoprecipitatrons also seen with the respective prermmune sera.

36°C for 1 and 2 days were labeled and analyzed by immunoprecipitation. In both cases, a gp74”‘bE-like molecule with a slightly increased electrophoretic mobility (gp72) was detected in the committed cell population reexpressing cell surface v-erbB protein (Figure 6F and data not shown). The above results raise the question why the v-erbB protein is unable to function as a transforming protein in committed tsAEV erythroblasts. A possible explanation is that erythroid differentiation leads to changes in the plasma membrane, which in turn could affect possible functions of gp74=-. We had previously observed that differentiation-specific antigens on immature normal and tsAEVtransformed erythroid cells can undergo redistribution into “patches” and “caps” as a consequence of cross-linking by the antibodies used for immunofluorescent staining. “Patching” and “capping” of the same antigens did not occur, however, in more mature reticulocytes and erythrocytes (H. B., unpublished). To determine whether erythroid differentiation would also affect the ability of gp74e’bB to undergo redistribution into “caps,” the following experiment was performed. ts167E3 cells were either kept at 36°C or pulse-shifted to 42’C to induce commitment, then incubated at 36’C for at least 24 hr. After double-staining with anti-&B antibodies plus antierythroblast or antierythrocvte antibodies at 0°C both cell preparations were

divided into two aliquots. The first aliquot was immediately fixed and processed for fluorescence microscopy, while the second aliquot was incubated at 37°C for 15 to 60 min prior to fixation to induce “patching” and “capping.” Figure 7A shows that gp74as well as the erythroblast antigen(s) undergo extensive capping in undifferentiated tsAEV erythroblasts, when incubated at 37°C for 15 min. In the committed, differentiating tsAEV erythroblasts, however, gp74etiE either did not redistribute at all after 15 min at 37°C as indicated by a ring-like staining, or assembled into tiny patches (Figure 78). After 60 min at 37°C most cells showed patching of gp74eb’, but no caps were observed. In contrast, the erythrocyte antigen(s) detected by differentiation-specific antibodies did not undergo redistribution at all, even after prolonged incubation at 37% This suggests that the erythrocyte antigen(s) are immobilized by interaction with the rigid, submembrane cytoskeleton of erythrocytes, while gp74ti is not. Discussion tsAEV Mutants Probably Bear a Lesion in v-erbB That Affects the Processing and Localization of gp74The results have demonstrated a correlation between the formation of the mature erbB gene product, gp74-, and

Cdl 968

Figure 5. Cell-Surface

and Intracellular

Expression

of v-erb6 Gene Products

during Differentiation

of tsAEV Erythroblasts

Cells from the same preparation as in Figure 4 were stained as live cells with antierb6 antibodies (A) or fixed and permeabilized and then double-labeled as described in the legend to Figure 2 with antierb8 antibc$ies (erbB) and antiefythrocyte serum (Ery Ag; Beug et al., 1979) (B). (C) shows cytospin preparations of the cells used in Figures 4 and 5 after staining for hemoglobin (Hb) with neutral benzidine (Beug et al., 1982a) to demonstrate the homogeneity of the cell populations used. Bar represents 20 pm.

the presence of erbS protein on the cell surface, as detected by immunofluorescence in all cells tested. This strongly suggests that surface erbB protein corresponds to gp74--, an interpretation that is in agreement with the available data on biosynthesis and subcellular localization of gp74(Hayman and Beug, manuscript submitted). In tsAEV erythroblasts induced to differentiate at 42°C the formation of gp74eM from its precursor gp68eM is completely blocked within 24 hr after shift to 42°C. Furthermore, preexisting gp74”ti disappears from the cell surface after 12 hr at 42%. In contrast, expression of the intermediate forms, gp66em and gp68eM, does not change significantly during the first 48 hr after shift. No changes in cell surface gp74& expression were seen in wtAEV-transformed erythroblast clones or in a wtAEV cell line (LSCC HDl; Beug et al., 1982b). The finding that

expression of cell surface gp74”*’ was also reduced in tsl67-transformed fibroblasts and in a nondifferentiating variant of ts34 erythroblasts at 42% (LSCC HD2; Beug et al., 1982b) strongly suggests that the observed block in the processing of gp68to gp74*’ is due to a thermosensitivity of the mutant v-erbB protein rather than to events connected with terminal erythroid differentiation. According to this interpretation, incubation at the nonpermissive temperature would prevent correct completion of the carbohydrate side chains of gp74”hB and/or its transition from intracellular inembranes to the cell surface. Further work will be necessary to determine how and at which step the maturation of the mutant v-erbB gene product is affected by its thermolability. No changes in synthesis, turnover rate, or cytoplasmic location of p75ga@*A were seen in tsAEV erythroblasts at

Temperature-Sensitive 969

Mutants

of AEV

P

IErbB

IEry-

&I

I

N

1 N

I N

b-P76 9P72X w68+

-9P68 ‘9w66

Glu Frgure 6. gp74ti

Is Reexpressed

Met in Committed

Glu tsAEV Erythroblast

As outlined by the temperature-shift programs at top of shifted to 4TC and then kept at 36°C (C, F); numerals antibodies (erbB) and antierythrocyte serum (Ery-Ag) products from the same cell preparations after labeling (I) and normal rat serum (N) or anti-erbA+B antibodies,

Differentiation

Met

Glu

Met

at 36’C

(A), (B), and (C), tsl67AEV E3 erythroblasts were kept at 36’C (A, D), shifted to 42°C (B. E), or pulsel-4 indicate duration of shift in days. In (A-C), the cells are shown after double-labeling with anti-erbB as described in the legend to Figure 2. (D-F) show immunoprecipitations of erbA and erb8 gene with 3H-gIucosamine (Glu) or %-methionine (Met) and immunoprecipitation with anti-erb6 antibodies respectively.

42’C (Figure 4B and data not shown), suggesting that there is no mutation in v-erbA. Thus the temperaturesensitive lesion of ts34 and tsl67AEV appears to be located in the v-erbB gene, a conclusion that is in accord with the proposed central role of v-erbB in transformation (Frykberg et al., 1983) and with the results of a comparative analysis of tsAEV mutants in erythroblasts and fibroblasts (Palmieri et al., 1982). Although it is surprising at first that apparently normal amounts of ~75~~~ do not detectably interfere with terminal differentiation of tsAEV erythroblasts at 42°C this finding agrees with the observation that AEV mutants in which only the v-erbA gene is expressed (AEV

A%; Frykberg et al., 1983) are nontransforming fibroblasts and erythroblasts.

in both

Why Are v-erbA and v-er6B Expressed Differentially in Mature Erythroid Cells? Terminal differentiation of erythroid cells is characterized by the shutdown of synthesis and/or degradation of most cellular proteins and by the selective expression of a rather small number of polypeptides such as hemoglobin and a few specialized membrane proteins (for review see Branton et al., 1981; Marchesi, 1983). In differentiating tsAEV erythroblasts, p75*-etiA and pr76gW were found to be

Cell 970

cells. It is also interesting to note that the products of the v-erbB and env genes are translated from spliced, subgenomic viral messages, whereas pr76m and p75QaQerbAare products of genomic mRNAs. Further experiments are required to understand why certain viral gene products can be expressed in mature erythroid cells while others cannot.

What Is the Mechanism of Cell Transformation Caused by gp74eM?

ISmin 37°C

Figure 7. Redistribution tsAEV Erythroblasts

of gp74 amB into Caps

Is Inhibited

in Committed

tsl67AEV E3 cells were kept at 36°C (A) or committed to differentiation by pulse-shift (B) as outlined by the temperature-shift programs at top of (A) and (6). Cells were double-labeled with anti-erbB antibodies (e&S) plus antierythroblast serum (EblAg) or antierythrocyte serum (EtyAg) as described in the legend to Figure 2. Before fixation and mounting, cells were either kept at 4°C or warmed to 37% as described in the text. White arrows: pronounced caps seen in the uncommitted cells with both antierbB and antierythroblast antibodies.

expressed at all stages of differentiation. In contrast, synthesis of the v-erbB gene products gp66tiB, gp68”*‘, and gp74e’be and of pr92”“” was suppressed in mature erythroid cells. The block of v-erbB expression late in differentiation was not due to the thermolability of the mutant gp74e*bs protein, since v-erbB products were also absent in the mature cells that spontaneously differentiated from several types of leukemic erythroblasts transformed by the wildtype v-erbB gene alone (Frykberg et al., 1983; Fung et al., 1983; Yamamoto et al., 1983; Graf, Hayman, and Beug, unpublished results). Since v-erbA and v-erbB are expressed under the control of the same viral promoter, it is difficult to understand why only the production of v-erbB and env proteins is shut down in mature erythroid cells. One possibility is that the v-erbB and env gene products are actually synthesized at similar rates to p75QwerbA or pr76QaQ, but that the former glycoproteins are rapidly degraded in mature erythroid

We have demonstraed that cell surface expression of verbB is abolished in tsAEV-transformed cells that are released from the transformed phenotype by shift to 42°C. This could mean that the presence of gp74&’ on the cell surface is required for the maintenance of transformation, a hypothesis we refer to as the “surface hypothesis” of AEV transformation. A second idea is that an as yet undetected protein kinase associated with gp74e’be is responsible for the transforming activity of gp74e*B and is thermolabile in tsAEV mutants as well. In this “kinase hypothesis” cell surface expression of gp74”*’ would not be required for transformation. A third possibility, combining both models is that gp74hB represents a receptor-like molecule with an associated kinase activity. Some circumstantial evidence exists in favor of the surface hypothesis. First, gp74”ti does not completely disappear from the surface of variant tsAEV erythroblasts that fail to differentiate at 42°C. Second, the gene product of the c-erbB gene that is overexpressed in RAV-1 -induced erythroleukemias as a consequence of a promoter-insertion mechanism (Fung et al., 1983) is also expressed at the cell surface as a membrane glycoprotein of up to 84 kd (Hayman and Beug, unpublished). Third, cell surface glycoproteins seem to be involved in transformation of erythroblasts by other retroviruses such as the Friend murine erythroleukemia virus (Ruta et al., 1983). Two lines of evidence are in favor of the kinase hypothesis. First, gp74e’bs contains a (probably intracellular) domain, which shows extensive amino acid sequence homology to the tyrosine kinase pp60”“, the transforming protein of RSV (Yamamoto et al., 1983, and personal communication). Second, it was recently found, that RSV can also induce the outgrowth of transformed erythroid cells from infected chicken bone marrow (P. Kahn and T. Graf, personal communication). The “receptor hypothesis” combines both of the above possibilities. It postulates that gp74**’ represents a hormone-receptor-like molecule with associated kinase activity, as exemplified by the epidermal growth factor receptor (Cohen et al., 1980), the insulin receptor (Kasuga et al., 1983), or the platelet-derived growth factor receptor (Ek et al., 1982). The transmembrane glycoprotein nature of gp74 e*E, its apparently high turnover rate, its phosphorylation, and its homology to pp60s’” (Hayman et al., 1983; Hayman and Beug, manuscript submitted; Privalsky et al., 1983; Toyoshima, personal communication) as in accord with this postulate. In addition, our observation that redis-

Temperature-Sensrtive 971

Mutants

of AEV

tribution of surface gp74 erbB into caps is greatly impaired in committed tsAEV erythroblasts that differentiate at 36°C offers a possible explanation why these cells cannot respond to the transforming function of gp74”*‘. One could speculate that the hypothetical “hormone receptor” gp74erbB requires “clustering” for proper function, as proposed for the epidermal growth factor receptor (Schreiber et al., 1981). Although it is clear that patching and capping are processes distinct from the “microclustering” thought to be required for receptor function, it is still possible that the membrane and “red cell skeleton” (Marchesi, 1983) changes that impair patching and capping of gp74Wba could also affect “microclustering” and thus the transforming function of this molecule. In addition to the obvious usefulness of tsAEV mutants for learning more about the transformation mechanism of AEV, the study of gp74 erb8 in tsAEV-transformed cells might also constitute a new model system in which to study glycoprotein synthesis and processing, especially since a continuous, tsAEV-transformed chicken erythroblast line expressing mutant gp74e*B is available (LSCC HD3; Beug et al., 1982b). Temperature-sensitive mutations affecting the processing of glycoproteins have been developed so far only for virus structural genes, such as the genes for envelope glycoproteins of vesicular stomatitis, influenza, or Semliki Forest viruses (for review see Tartakoff, 1983). The v-erbB gene product is unique, however, in that it is homologous to a normal cellular protein and that it affects the growth and differentiation properties of erythroid cells. Experimental

Procedures

Cells and Viruses Clonal strains of erythroblasts transformed by tsl67AEV (RAV-2) (Pafmieri et al., 1982) ts34AEV (Graf et al., 1978) and wtAEV strain ES4 (Graf et al., 1976) as well as continuous erythroblast cell lines infected with these viruses (LSCC HDl, LSCC HD3, LSCC HD4; Baug et al., 1982b). were used in the experiments described. For the production of large amounts of tsAEV erythroblasts homogeneous for their state of differentiation, it was necessary to isolate well differentiating tsl87AEV erythroblast clones with an in vitro life span of more than 50 population doublings. For this, 75 tsl67AEV clones obtained from single Methocel colonies were grown in CFU-E medium without anemic chicken serum (Radke et al., 1982; concentration of fetal calf serum and detoxified BSA raised to 10% and 10 mg/ml, respectively, and 15% sterile distilled water added to optimize osmolarity) and screened for differentiation into erythrocytes as described (Beug et al., 1982a). Fifteen clones yielding more than 95% erythrocytes plus late reticulocytes within 4 days were frozen in liquid nitrogen, and afiquots were assayed for their in vitro life span by serial passaging (1:5) in the above medium. Two suitable clones (E3 and El 1) could be isolated that grew for 40-45 population doublings without losing their potential to differentiate. Chicken embryo fibroblasts infected with tsl67AEV and wtAEV were obtained as described (Palmieri et at., 1982). These cells, as well as the tsAEV and wtAEV erythroblast cell lines, were grown in standard growth medium. Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 8% fetal calf serum, 2% chicken serum, IO mM Hepes (pH 7.3) and (for erythroblasts) 5 x 10s5 M thioglycerol. Production of Cell Populations Homogeneous for Their State of Differentiation Erythroblast clones and cell lines were seeded at l-1.5 X 1@ cells/ml in a modrfied differentiation medrum (Beug et al.. 1982a), consisting of DMEM

supplemented with 15% sterile water, 6% fetal calf serum, 6% high-titer anemic chicken serum, 2% normal chicken serum, IO-’ M mercaptoethanol, 30 pg/ml iron saturated chicken ovotransferrin (Sigma), 8 mg/ml detoxified BSA, and 1.6 mg/ml sodium bicarbonate, and incubated at 37’C or 42°C and 2% CO1, with great care to avoid any evaporation of medium. After appropriate times, the cells were harvested. and about 50 X ld cells were layered on top of discontinuous Percoll density gradients preformed In conical 13 ml plastic tubes using Percoll solutions of four different densities. These solutions were made up as v/v mixtures from undiluted Percoll (rendered isotonic with phosphate-buffered saline) and standard growth medium. The following gradients were used (top to bottom): tsAEV erythroblasts grown at 36OC, wild-type erythroblasts grown at 36°C and 42°C and temperature-sensitive erythroblasts shifted for 1 day to 42°C 37%. 42%, 47%, 49%; temperature-sensitive eiythroblasts shifted for 2 days, 37%, 42%, 49%. 51%; for 3 and 4 days, 47%, 49%. 51%, 62%. The latter gradient was also used to purify committed tsAEV erythroblasts completing their differentiation at 36°C. Dead cells were removed from undifferentiated and partially differentiated cell populations by centrifugation through Ficoll (Beug et al., 1982a) prior to Percoll gradient centrifugation. Gradrents were spun for 20 min at 1500 x g. after which the majority of cells usually banded at one characteristic density. These cells were aspirated from the gradient and immediately used for further experimentation, Antisera Rat antisera to either ~75~ plus gp74or to gp74*“8 alone were produced and characterized as described (Hayman et al., 1983; Hayman and Beug, manuscript submitted). Differentiation-specific rabbit antisera to chicken erythrocytes and erythroblasts as well as monoclonal antibodies to erythroblasts (MC4.5.A.5) and reticulocytes (MC4.6.C.l) and affinitypurified rabbit antibody to virus structural proteins have been described earlier (Beug et al., 1979, 1981, 1982a; Hayman et al., 1982). Detection of v-erb Proteins by lmmunofluorescence Stainrng of live cells for surface verbB was performed as described (Beug et al.. 1982a; Hayman et al., 1983) except that rat antiserum specific for verbB (Hayman and Beug. manuscript submitted) plus fluoresceine(FITC)labeled goat antirat antibody was used in all experiments in combination with differentiation-specific rabbit antibodies plus rhodamine(TRITC)-labeled goat antirabbit antibody. To detect verbB and differentiation-specific antigens in fixed and permeabilized cells, cells were washed once in Hanks balanced salt solution, fixed for 30 min at IO x IO6 cells/ml and 4°C in 3.7% paraformaldehyde plus 0.02% glutaraldehyde in PBS, washed once each with PBS and DMEM plus 10% fetal calf serum and 25 mM Hepes, pH 7.3 (Beug et al., 1979) permeabilrzed for 10 min at 4°C in the same medium plus 0.05% NP40, and washed twrce in the above medium. The frxed cells were then processed for immunofluorescence exactly as live cells except that the final ethanol fixation step was omitted. Radioactive Cell Labeling and lmmunoprecipitation Labeling of cells with “S-methionine and 3H-glucosamine was performed as described (Hayman et al., 1979, 1983; Hayman and Beug, manuscript submitted) except that clonal strains of tsl67AEV and wtAEV erythroblasts were labeled at 36°C or 42°C for 2 hr with ?-methionine (600 &i/ml) in differentration medrum (in which DMEM was replaced with methionine-free medium). Cells from the same clonal strains were labeled for 12 to 17 hr with w-glucosamine (200-500 &i/ml in differentiation medium; the final glucose concentration in the medium was lowered to 1.2 g/l). Lysates were prepared, and aliquots containing the same amounts of acid-precipitable radioactivity were subjected to immunoprecipitation analysis as described earlier (Beug et al., 1981; Hayman et al., 1983; Hayman and Beug, manuscript submitted). Induction of Surface Antigen Redistribution Loving tsAEV erythroblasts were stained at 0°C with ant&b6 sera in combination with differentiation-specific antibodies as above. Before ethanol fixation, the stained cells were divided into two aliquots. One aliquot was immedrately frxed and mounted as described (Beug et al., 1979). The remaining cells were resuspended at 1 x IO6 cells/ml in differentiation medrum and incubated for 15 to 60 min at 37°C and 2% CO,. Thereafter

Cell 972

they were quickly cooled to O°C, washed once in cold Hanks balanced solution, then fixed and mounted as described above.

salt

Acknowledgments The authors would like to thank Drs. T. Graf and 8. Vennstrom for helpful suggestions and discussions as well as for help with the manuscript; Drs. P. Kahn, T. Graf, and T. Yamamoto for communication of data prior to publication; G. Doedertein and G. Kitchener for excellent technical assistance; and B. Blanasch for typing. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. Received

December

6, 1983; revised

Tyrostne-specific protein kinasa activity is associated receptor. Proc. Nat. Acad. Sci. USA 80, 2137-2141,

with the purified insulin

Lai, M. M. C., Hu, S. S. F., and Vogt, P. K. (1979). Avian erythroblastosis virus: transformation specific sequences form a contiguous segment of 3.25 kb located in the middle of the 6kb genome. Virology 97, 366-377. Marches+ V. T. (1983). The red-cell membrane Blood67, l-11.

skeleton-recent

progress.

Palmieri, S., Beug, H., and Graf, T. (1982). Isolation and characterization of four new temperature-sensitive mutants of avian erythroblastosis virus (AEV). Virology 124, 296-31 I. Privalsky, M. L., Scaly, L., Bishop, J. M.. McGrath, J. P., and Levinson, A. D. (1983). The product of the avian erythroblastosis virus erbB locus is a glycoprotein. Cell 32, 1257-l 267. Radke, K.. Beug, H.. Komfeld, S., and Graf, T. (1982). Transformatron of both erythroid and myeloid cells by E26, an avian leukemra virus that contarns the myb gene. Cell 37, 643-653.

Jaunary 30, 1984

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