of Temperature-Sensitive Rous Sarcoma Virus Mutants - Europe PMC

MOLECULAR AND CELLULAR BIOLOGY, Aug. 1984. p. 1508-1514 0270-7306/84/081508-07$02.00/0 Copyright ©3 1984, American Society for Microbiology

Vol. 4. No. 8

Functional Domains of the pp60vs1( Protein as Revealed by Analysis of Temperature-Sensitive Rous Sarcoma Virus Mutants ANDREW W. STOKER,* PAULA J. ENRIETTO, AND JOHN A. WYKE Laboratories of Tunmour Virologycand Viral Leuikaemogeniesis, Imperial Cancer Research Fund Laboratories, St. Bartholomew's Hospital, London ECIA 7BE, Englanid Received 27 December 1983/Accepted 16 May 1984

Four temperature-sensitive (ts) Rous sarcoma virus src gene mutants with lesions in different parts of the gene represent three classes of alteration in pp6Osrc. These classes are composed of mutants with (i) heat-labile protein kinase activities both in vitro and in vivo (tsLA27 and tsLA29), (ii) heat-labile kinases in vivo but not in vitro (tsLA33), and (iii) neither in vivo nor in vitro heat-labile kinases (tsLA32). The latter class indicates the existence of structural or functional pp6Osrc domains that are required for transformation but do not grossly affect tyrosine kinase activity.

The transforming src gene of Rous sarcoma virus (RSV) encodes a phosphoprotein with a molecular weight of 60,000, pp6Osr , which is required for the transformation of chicken embryo fibroblasts (CEF) in vitro (30, 47). pp605'' is a cyclicAMP-independent tyrosine protein kinase (6, 10, 12, 24, 31, 36, 37), the presence of which leads to an increase in the to, al cellular phosphotyrosine levels in transformed cells (13, 14). The transformation mechanism of this protein is still unknown, but the bulk of pp6O0s appears to be localized at the inner surface of the plasma membrane (27, 29, 45, 50), suggesting that it exerts its effects in this cellular compartment. Moreover, several proteins showing increased phosphotyrosine levels have been identified in RSV-transformed cells (4, 15, 39, 49). These include vinculin (53), a 50kilodalton (Kd) protein (28), a 36-Kd protein (23, 48), and three non-rate-determining glycolytic enzymes (17). Although the presence of several of these phosphoproteins may not be a prerequisite for morphological transformation or other transformation parameters, individually they could be involved in the appearance of specific features of transformation, as certain parameters may be divorced from each other and thus can be controlled independently (1-3, 16, 40, 51, 56, 61). The mechanism by which pp6os5' transforms cells has been studied with mutants of src. Work with temperaturesensitive (ts) mutants helped to establish the importance of the src gene and its kinase activity in maintenance of the transformed state (27, 33, 55). Mutants generated by in vitro mutagenesis of the cloned src gene are being used to define important regions of the molecule with respect to kinase activity and transforming ability (7, 20, 58). Such studies, taken together, suggest that pp605r( has perhaps more than one domain (of which the kinase site is one) that are implicated in full expression of the transformed phenotype (26, 34, 38, 44, 59). We have previously isolated and mapped a number of temperature-sensitive src mutations of the Prague strain of RSV, finding that the lesions were distributed along the length of the gene (25, 62). This distribution suggests that the lesions may reflect the underlying structural and functional pattern of the protein. To characterize these domains more carefully we analyzed four temperature-sensitive mutants of

*

src, two with lesions which map in the carboxy-terminal region of the protein and two with lesions in the aminoterminal half of the protein. MATERIALS AND METHODS

Cells and viruses. CEF were prepared from fertile eggs from a brown leghorn and white leghorn cross (Wickham Laboratories, Wickham, Hampshire, United Kingdom). Methods of culture and infection were as previously described (60). The temperature-sensitive mutations (permissive temperature, 35°C; restrictive temperature, 41°C) (62) were mapped by Fincham et al. (25). Soft agar growth assays with either freshly infected cells or transformed cultures were carried out essentially as described by Wyke and Linial (62), but without additional amino acids and folic acid in the medium. Immunoprecipitation and gel electrophoresis. Cells were washed three times in phosphate-buffered saline and lysed in buffer A (0.1 M NaCl, 1 mM EDTA; 10 mM Tris-hydrochloride [pH 7], 1% Nonidet P-40, 1 mg of bovine serum albumin per ml, 40 mM NaF, 50 mM ZnCl,) or buffer B (0.15 M NaCl, 20 mM Tris-EDTA [pH 7], 1% Nonidet P-40, 1 mg of bovine serum albumin per ml). The lysates were clarified in an Anderman microfuge for 10 min at 4°C. The antisera used were (1) preimmune rabbit serum, (2) tumor-bearing rabbit serum raised against B77 strain avian sarcoma virus injected into newborn rabbits (21), and (3) rabbit anti-pp60src serum raised against pp6Osl' made in Escherichia coli cells (kindly provided by R. L. Erikson [22]). Immunoprecipitations and sodium dodecyl sulfatepolyacrylamide slab gel electrophoresis were performed as described previously (21). Kinase assays. Transformed CEF were seeded onto 60-mm plates and incubated at 35 and 41°C for 18 to 24 h before kinase assays were performed. Cells were lysed in buffer B on ice and clarified. Kinase assays were performed as described by Collett and Erikson (10), using tumor-bearing rabbit antiserum, with the following modification. Before the reactions were started, the immune complexes were incubated at 35 or 41°C (i.e., the growth temperatures of the cells from which the pp60r'U had been immunoprecipitated) for 10 min in kinase reaction buffer (10) without [y-32PIATP. The ATP was then added, and the reaction was allowed to run for 10 min at the respective temperatures. V8 protease digestion. V8 protease digestion of the labeled

Corresponding author. 1508

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ts MUTATIONS IN FUNCTIONAL DOMAINS OF pp6O'--'

pp60src proteins was carried out as described by Cleveland et al. (9). Cells were labeled and immunoprecipitated as before, and the products of digestion were visualized by autoradiography. Phosphoamino acid analysis. Plates (35 mm) of RSVinfected cells were labeled with 1.5 to 2.0 mCi of 32p; for 18 h (in phosphate-free medium supplemented with 5% dialyzed calf serum), then lysed (on ice) in 200 ,ul of buffer A, and immediately clarified. The proteins were extracted from 40,ul samples of lysate and hydrolyzed, and phosphoamino acid separation was performed as described by Hunter and Sefton (31). Internal phosphoserine. phosphothreonine, and phosphotyrosine standards were visualized with ninhydrin, and the 32P-labeled phosphoamino acids were detected by autoradiography of the plates. The areas containing internal

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amino acid standards and background areas were scraped off the plates and counted by scintillation in Aquasol. Immunoprecipitation and alkali treatment of 36K. The anti36,000-molecular weight (36K) protein serum was kindly provided by S. A. Courtneidge (19). Plates (35-mm) of cells were labeled with 1 mCi of 3-P, per ml for 4 h (in phosphatefree medium plus 5% dialyzed calf serum after preincubation in this medium for 30 min). After immunoprecipitation and polyacrylamide gel electrophoresis, the gels were fixed, dried, and reswollen in 1 M KOH. The gels were then maintained at 55°C for 2 h (with gentle shaking). The gels were refixed and dried, and autoradiographs were made. The autoradiographs were scanned (with a Joyce Loebel Chromoscan densitometer), and absorbances were normalized for trichloroacetic acid counts in each sample.

FIG. 1. Foci of transformed CEF at the permissive temperature (35°C). Infecting RSV mutant viruses were A. tsLA27; B, tsLA29; C, tsLA32; D, tsLA33.

MOL. CELL. BIOL.

STOKER. ENRIETTO, AND WYKE

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TABLE 1. Soft agar growth of RSV mutant-infected CEF 5% Colony %c Colony formation' formation" Infecting virus remaining at wtPrA tsLA27 tsLA29 tsLA32 tsLA33

at 35°C

410C

0.5 0.4 0.8 1.0

145 0.2 1.6 17.2'

1.0

8.5'

aMean of two representative assays, seeding fully transformed cells at 2 x O0s and 2 x 104 per dish. b Calculated as 100 x (number of colonies at 41°C/number of colonies at 35°C). Colonies at 41'C were, in general, smaller than those at 35°C.

RESULTS Characterization of CEF transformed by temperature-sensitive mutants. The four mutants studied were tsLA27 and tsLA29, whose mutations are in the 3'-terminal 100 nucleotides of the coding region of src, and tsLA32 and tsLA33, that have lesions in the 5' half of the gene (25). All four mutants are fully temperature sensitive for morphological transformation (62), but the transformed phenotypes of LA32 and LA33 differ from LA27 and LA29 in that the former are less rounded and more fusiform in appearance, and the latter are fully rounded and similar to wild-type (wt) Prague RSV (Fig. 1). In agar suspension assays for anchorage-independent growth, all mutants induced colony formation at 35°C comparable to that induced by wild-type virus (Table 1). However, the inherent variability of this assay makes comparisons of different viruses less significant than comparison of the same infected cells at different temperatures. The latter comparisons show that LA27 and LA29 are fully temperature sensitive compared with wild type, with ca. 100-fold less colony-forming ability at 41°C, whereas LA32 and LA33 maintain some residual colony-forming ability at a nonpermissive temperature (Table 1). In vitro kinase activity of temperature-sensitive mutants. In vitro kinase assays were performed on the mutant pp60src proteins after immunoprecipitation from RSV-infected chicken cell lysates, and representative results are shown in Fig. 2. The mutants LA27 and LA29 gave a consistently large decrease (more than fivefold) in kinase activity at 41 compared to 35°C. LA32 gave a smaller decrease (up to twofold), whereas LA33 on average gave little or no decrease. wtPrC shows up to a twofold drop in activity at 41°C, although this activity was still fivefold higher than that of all

..27

LA 29

35 41

35 41

_32 35

41

-

; 33

35

uro4

41

.4Ig

FIG. 2. In vitro kinase assays performed on pp60sr( of mutants tsLA27, tsLA29, tsLA32, and tsLA33 as described in the text. The products of the reaction were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and autoradiographed. Ig. 32P-labeled immunoglobulin heavy chains.

the temperature-sensitive mutants (data not shown). As this was an artificial in vitro assay with nonphysiological substrates, we were interested to determine how closely these results reflected the physiological situation. Accordingly, we determined the total phosphotyrosine levels in the infected CEF and also analyzed the phosphorylation of one putative target of pp6O0r(, the 36K protein. Total phosphoamino acid analysis. Transformation of CEF by RSV causes up to a ten-fold increase in the levels of cellular phosphotyrosine over that in normal cells. Temperature shift of temperature-sensitive mutant-infected cells results in parallel changes of total phosphotyrosine levels and the transformed phenotype; the rapidity and temperature dependence of these correlated changes implicates pp6osr' as the primary kinase (55). Total cell phosphoamino acid analysis on CEF infected with the four temperature-sensitive mutants under study revealed that LA27, LA29, and LA33 showed a clear two- to threefold decrease in phosphotyrosine to normal cell levels upon shift up to 41°C, and thus they are mutants with a temperature-labile kinase activity (Fig. 3 and Table 2). With LA32 however, there was little change in phosphotyrosine levels upon shift to 41°C, and the levels remained two- to threefold higher than those of noninfected cells. Phosphorylation of the 36K protein. The total phosphotyrosine levels reflect only the gross level of tyrosine kinase activity in the cell population. In LA32, a small number of critical targets may not be phosphorylated at 41°C, perhaps hidden by the high nonspecific activity of the kinase. Our initial approach to this question was to look at the pattern of 36K protein phosphorylation in the temperature-sensitive mutant-infected CEF. The 36K protein was the first cellular protein identified as containing elevated phosphotyrosine levels in RSV-transformed cells. It is a relatively abundant protein, and upon transformation of the host cells by RSV, this protein becomes up to 10-fold more phosphorylated on tyrosine than in normal cells. Recent studies by several groups have revealed that the 36K protein is localized in the plasma membrane or the detergent insoluble matrix of the cell or both (8, 14, 19, 32, 41). In the transformed cell, only 5% of the total 36K protein is phosphorylated on tyrosine and the localization of this phosphorylated form appears to be no different from the major nonphosphorylated form, within the detection limits of the experiments. Using an anti-36K serum, we studied the phosphorylation levels of 36K protein in CEF infected by the temperaturesensitive mutants. The 36K proteins were immunoprecipitated from 32P-labeled chicken cell lysates and run on polyacrylamide gels. The gels were alkali treated (see above) to preferentially reduce any phosphoserine and phosphothreonine present (13) (Fig. 4). The data clearly show that the wtPrC levels of 36K phosphorylation are relatively much higher than those of the mutants, and that for the mutants the levels are all very similar at a permissive temperature (Table 3). For the mutants LA27, LA29, and LA33, there is a threeto fourfold decrease in 36K phosphorylation upon a shift up which correlates with the reversion to normal phenotype of the infected cells. LA32, however, maintains 36K phosphorylation after a shift up, yet the cells are no longer transformed. pp6wc phosphorylation. In transformed cells pp6Osr( itself is phosphorylated on both serine and tyrosine. The major phosphotyrosine site is at amino acid 416, in the carboxyterminal half of the protein, and the major phosphoserine site is thought to be at amino acid 17, near to the amino terminus

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TABLE 2. Abundance of phosphotyrosine in chicken cells infected with temperature-sensitive mutants of RSV

s -7

Infecting >

virus

Noninfected WtPrC tsLA27

tsLA29

y> P*

tsLA32

tsLA33

1

2

Growth temp (°C)

Relative abundance of phosphotyrosine"

35 41 35 41 35 41 35 41 35 41

0.08 0.12 0.72 NDb 0.27 0.10 0.32

35 41

NDb 0.24 0.31 0.24 0.11

The phosphototyrosine levels are presented as a percentage of the total counts per minute found in phosphoserine, phosphothreonine. and phosphotyrosine from hydrolyzed '2P-labeled total cell protein and are normalized for background counts per minute. These represent the mean values from two independent experiments. bND, Levels not quantitated.

3

4

4

5

6

FIG. 3. Representative autoradiograms of two-dimensional analof total cell phosphoamino acid contents from normal and transformed CEF. Presented here are two-dimensional thin-layer electrophoresis separations of 32P-labeled total cell hydrolysates from cells infected with-: 1, no virus at 35°C; 2, PrC at 41°C; 3, tsLA29 at 35°C; 4, tsLA29 at 41°C; 5, tsLA32 at 35°C; and 6, tsLA32 at 410C. The origins were at the bottom right. Electrophoresis in the first dimension was from right to left, and in the second dimension it was from bottom to top. Phosphoamino acids used were s, phosphosenne; t, phosphothreonine; and y, phosphotyrosine. Arrow heads in panels 2 through 6 indicate the position of phosphotyrosine. yses

(11, 31, 54, 57). V8 cleavage analysis of 32P-labeled pp60Yr( proteins from the temperature-sensitive mutants separated the 36- and 24-Kd major digestion fragments of ppsrs', which contain the major phosphoserine and phosphotyrosine sites, respectively. The results show that for all four temperature-sensitive mutants, a significant decrease in V2 phosphorylation relative to Vl phosphorylation is seen at the

nonpermissive temperature, correlating with the reversion of the transformed morphology to normal (Fig. 5, LA27 data not shown). Densitometer scanning shows this relative reduction to be at least sixfold in each case. vvtPrC shows no such decrease and remains transformed at 41°C. DISCUSSION Our aim in this study was to analyze the functional and structural basis of the lesions found in four temperaturesensitive mutants of the src gene of RSV, chosen after an initial analysis of in vitro kinase activities in a number of mutants. Our interest focused on the low temperature lability of LA32 and LA33 pp6orc kinases. These in vitro results suggest that mutants of this kind may provide examples of temperature-sensitive lesions in a novel functional domain(s) of pp60src. Because of the artificial nature of this assay, caution must be used in extrapolating to the in vivo situation. Therefore, we looked at cellular phosphotyrosine levels in infected CEF as an indicator of in vivo tyrosine kinase activity (Fig. 3, Table 2). Wild-type-transformed CEF have an eightfold elevated level of phosphotyrosine compared to uhinfected cells, agreeing with previous work in this area (13, 55). At a permissive temperature the LA27- and LA29-infected cells contained only three- to fourfold elevated levels of phosphotyrosine, but the higher levels in PrC-transformed cells did not give rise to a more notably transformed phenotype (Fig. 1, Table 1, and unpublished data). LA27 and LA33 show a reduction of phosphotyrosine to unihfected cell levels upon shift up to 41°C, a pattern similar to that previously found for LA29 (54) and confirmed here. The result of interest is the maintenance of phosphotyrosine levels at 41°C in LA32infected cells. There are two possible but not mutually exclusive explanations for this result. Firstly, LA32 pp6Osrc may have a lesion affecting specific substrate availability by either altering pp6Osr(_substrate interaction or altering the cellular compartment in which it is located. Alternatively, a mutation has affected a novel functional domain of the pp65src protein, whereby the kinase activities are unaffected but transformation cannot be maintained. In testing the first explanation, we looked at 36K phosphorylation and found that its temperature sensitivity parallels in vivo phosphotyrosine levels. Thus, unlike the other mutants, LA32 main-

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MOL. CELL. BIOL.

STOKER, ENRIETTO, AND WYKE NI 35

pp36

41

35

LA32

LA 29

PrC

35

41

35

41

41

_M

P,

a b a b a b a b a b a b a b a b FIG. 4. Autoradiograph of 32P-labeled pp36 immune precipitated from CEF transformed by wt'tPrC and the mutants tsLA29 and tsLA32. The 32P-labeling was done at 35 and 41°C (see text). NI. Noninfected CEF. Lanes: a, normal rabbit serum: b. anti-36K serum.

tains 36K phosphorylation at 41°C. We also note the similarity in the relative levels of 36K phosphorylation and total phosphotyrosine seen at the permissive temperature for the four mutants, which is at variance with the observed variation of in vitro kinase activities (Fig. 2). There are two conclusions from these data. Again, we have shown, by looking at a putative target of pp60''', that the kinase of LA32 is not obviously heat labile. We have also divorced 36K phosphorylation from the parameter of morphological transformation, in agreement with Nakamura and Weber (40). We plan to carry out similar studies with other putative substrates of pp6OArc. Clearly, if LA32 exhibits minor, specific changes in substrate specificity, then the search for crucial targets will be more approachable than with the viruses that are temperature sensitive for phosphorylation of many proteins. The V8 cleavage analysis of 32P-labeled pp603c' revealed that all of the mutants had reduced phosphorylation in the 24-Kd fragment at 41°C. Since the major phosphoamino acid in the 24-Kd fragment is tyrosine, it is likely that this is due to loss of phosphate on tyrosine, paralleling a loss of kinase activity. For LA32, however, this result was unexpected, as our other data showed the kinase to be active at 41°C. It is possible that the lesion in LA32 specifically prevents autophosphorylation of pp6O0sr on tyrosine (37, 46), although not affecting its phosphorylation of most other substrates. An alternative explanation is that autophosphorylation does

not occur in vivo but an unidentified transformation-dependent tyrosine kinase is responsible for tyrosine phosphorylation of pp6OSr(. The major v-src phosphotyrosine residue (tyr-416) is not necessary for kinase activity, nor is it necessary for the ability to transform CEF in culture (20, 58). It is known that c-src utilizes a different tyrosine as its major phosphate acceptor and, although this site has not been located exactly, it is possible that v-src is also phosphorylated there and that this is the critical site in regulating kinase activity (43). Our findings with LA32 are consistent with the concept that phosphorylation of the major tyrosine residue is not necessary for pp6O3r( kinase activity, but they leave open the possibility that phosphorylation of the 24-Kd fragment is important for cell transformation. A mutant similar to LA32 has been made recently by Bryant and Parsons (7), in which a src gene deletion, causing loss of amino acids 202 to 255, results in a temperature-sensitive mutation in which infected CEF suffer only a 50% decrease of total cell phosphotyrosine and 36K phosphorylation upon shift up. The position of this deletion is in the region of src in which the LA32 and LA33 lesions lie, delineating a region of pp605'' outside the kinase active site that contains important domains of the protein. Other workers have described N-terminal domains important for membrane binding of pp6O0'' (34, 35) or for cytoplasmic complex

TABLE 3. Levels of pp36 in mutant- and wild-type-infected

_e

cells" Infecting virus

Temp (OC)

Expt 1

Noninfected

35 41 35 41 35 41 35 41 35 41 35 41

< 15 < 15 1,000 480 115