Factor-Independent Fibroblast Growth: Correlation with Kinase

MOLECULAR AND CELLULAR BIOLOGY, Jan. 1989, p. 278-287

Vol. 9, No. 1

0270-7306/89/010278-10$02.00/0 Copyright C) 1989, American Society for Microbiology

Abelson Murine Leukemia Virus Induces Platelet-Derived Growth Factor-Independent Fibroblast Growth: Correlation with Kinase Activity and Dissociation from Full Morphologic Transformation ROBERT W. REES-JONES,l.2* MITCHELL GOLDFARB,2 AND STEPHEN P. GOFF2 Departments of Medicine' and Biochemistry and Molecular Biophysics,2 College of Physicians and Surgeons, Columbia University, New York, New York 10032 Received 12 July 1988/Accepted 14 October 1988

Abelson murine leukemia virus (A-MuLV) encodes a single protein product, a tyrosine-specific protein kinase, whose activity is necessary for cell transformation by this retrovirus. Using a defined medium culture system, we demonstrate that transformation of NIH 3T3 fibroblasts by A-MuLV abrogates their normal requirement for platelet-derived growth factor (PDGF) for cell growth. Analysis of constructed insertional mutant viruses revealed an absolute correlation between A-MuLV-encoded tyrosine kinase activity and PDGF-independent fibroblast growth. Sequences of the provirus not required for kinase activity appeared unnecessary for abrogating the fibroblast requirement for PDGF. Conversely, sequences required for kinase activity appeared necessary, suggesting that induction of PDGF-independent fibroblast growth, like cell transformation, is a function of this tyrosine kinase. Fibroblasts transformed by a partially transformationdefective mutant demonstrated incomplete morphological transformation but were still independent of PDGF for growth. Thus, the processes of full morphological transformation and growth factor independence can be partially dissociated.

Protein phosphorylation and dephosphorylation play a central role in regulating a variety of intracellular processes (for a review, see reference 20). Although the majority of phosphorylation occurs on serine or threonine residues of proteins, one class of protein kinases selectively phosphorylates tyrosine residues (for a review, see reference 17). This enzymatic activity was discovered with analysis of proteins encoded by two acutely transforming retroviruses, Abelson murine leukemia virus (A-MuLV) (40) and Rous sarcoma virus (16). A large subset of viral oncogenes encodes proteins with tyrosine-specific protein kinase activity, localized at the inner face of the plasma membrane (for a review, see reference 35). Cell transformation by these retroviruses requires tyrosine kinase activity (35); however, the relevant intracellular substrate(s) has defied discovery. Tyrosine-specific protein kinases are also contained within the cytoplasmic domain of many growth factor receptors, including those for epidermal growth factor (4), insulin (18), insulin-like growth factor 1 (33), platelet-derived growth factor (PDGF) (26), colony-stimulating factor 1 (30), and bombesin (3). Recent evidence suggests that kinase activity mediates the intracellular effects of at least some of these growth factors (7, 8); again, the intracellular pathways have not been elucidated. These two groups of tyrosine kinases are closely related, as cellular genes encoding growth factors or their receptors are the source of many of the viral oncogene sequences. For example, avian erythroblastosis virus encodes a truncated epidermal growth factor receptor lacking the ligand-binding site but containing a constitutively active tyrosine kinase which mediates cell transformation by this virus (6, 17). The McDonough feline sarcoma virus encodes a tyrosine kinase related to the colony-stimulating factor 1 receptor (30). Finally, all of the tyrosine kinases have sequence homology *

to each other and weaker homology to cAMP-dependent protein kinase (17), suggesting that they all evolved from a common precursor enzyme. As further confirmation of the relatedness of these two groups of tyrosine kinases, transformation of a variety of cell types by the retroviruses results in abrogated requirements of the cells for normal growth factors (34). Transformation of fibroblasts by a variety of sarcoma-inducing retroviruses, including A-MuLV, Kirsten virus, Harvey virus, Rous sarcoma virus, simian sarcoma virus, and Moloney sarcoma

virus, renders the fibroblasts independent of PDGF for growth (34, 43). A-MuLV has been shown to induce growth factor independence in several additional cell types: independence of erythropoietin in erythroid cells (37), interleukin-3 in Friend virus-immortalized myeloid cells (25), and colony-stimulating factor 1 and multi-colony-stimulating factor in other myeloid cell lines (5). A defined medium culture system has recently been developed to determine the PDGF independence of fibroblast growth induced by oncogenes (43). We have used this system to study PDGF independence of fibroblasts transformed by A-MuLV. Although extensive evidence exists that cell transformation by A-MuLV requires the activity of the encoded tyrosine kinase (11, 39), no such evidence exists for the induction of growth factor independence, which is also observed with oncogenes not encoding tyrosine kinases. We therefore tested a battery of insertional mutants of A-MuLV to determine whether this biological activity of the virus is correlated with tyrosine kinase activity. During these studies, we identified a mutant A-MuLV which has a greatly reduced and incomplete transformation ability but which still renders fibroblasts independent of PDGF for growth. MATERIALS AND METHODS Materials. DNA enzymes were from New England BioLabs, Inc. Polyclonal rabbit antiserum (76S142) to gag of

Corresponding author. 278

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PDGF-INDEPENDENT FIBROBLAST GROWTH INDUCED BY A-MuLV

Moloney murine leukemia virus (M-MuLV) was obtained from the Biological Carcinogenesis Branch Repository, National Cancer Institute, Bethesda, Md. Many artificial substrates previously used for other tyrosine kinases (2, 29) were used as substrates for the A-MuLV kinase: casein and the random copolymers GT (4:1), GT (1:1), GAT (6:3:1), and GAT (1:1:1) (Sigma Chemical Co.); histone H2B (Organon Teknika); and GLAT, GLT5, GT'0, GALT, and GAT10 (kind gifts of P. H. Maurer and A. R. Zeiger, Jefferson Medical College, Philadelphia). Pansorbin (Calbiochem-Behring) was pretreated before use (28). Cell lines. NIH 3T3 fibroblasts, frozen at passage 123, were obtained from the American Type Culture Collection, Rockville, Md., and were grown at 37°C in 5% CO2 in Dulbecco modified Eagle medium (DMEM) supplemented with 10% calf serum (GIBCO Laboratories) and gentamycin base (100 ,ug/ml). Cells were used for focus assays at our passage number 2. Transfection of NIH 3T3 fibroblasts. Transfections for focus assays used the DEAE-dextran method (24) as described previously (28). Mutants were scored as transformation competent if they induced foci comparable in number and morphology to those seen with wild-type A-MuLV. Cultures enriched for A-MuLV-transformed fibroblasts, which contained >50% cells of transformed morphology, were obtained by gently washing plates containing foci with medium which was then transferred to new plates. This method selectively transferred the loosely adherent, rounded-up A-MuLV-transformed cells. Assay for PDGF independence of fibroblast growth. This assay was recently described (43). In brief, 35-mm-diameter culture dishes were treated with 0.1% poly-D-lysine for 2 h at 37°C. After being rinsed with phosphate-buffered saline minus (28), the plates were incubated overnight at 37°C in DMEM plus 10% calf serum and 15 ,ug of human fibronectin per ml. Enriched cultures of A-MuLV-transformed cells were trypsinized, and 1 x 105 cells in DMEM plus serum were plated per 35-mm dish. After cell attachment, the medium was replaced with defined medium containing DMEM:Hams F12 medium (3:1) supplemented with 15 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.4)-4 x 10-6 M MnCI2-1 x 10-5 M ethanolamine-3 mM histidine-8 mM NaHCO3-500-Rug/ml bovine serum albumin-linoleic acid complex-1 x 10-7 M sodium selenite-5-,ug/ml transferrin-20-,ug/ml insulin. Cultures were fed the next day and then every 3 days. Culture dishes were scored for colonies after 7 to 10 days in defined medium. Transformation-defective A-MuLV mutants were assessed for induction of PDGF independence by minor changes in this protocol. Serum was omitted from the medium used in the overnight fibronectin incubation. Untransformed fibroblasts were transferred to defined medium 12 days after DEAE-dextran transfection with mutants and helper virus. To analyze the growth rates of transformed fibroblasts in serum or defined medium, 104 fibroblasts were transferred to fibronectin-coated plates in DMEM plus 10% calf serum. The same day, after attachment, some plates were fed with DMEM plus serum while others were switched to defined medium with or without PDGF (25 ng/ml). The next morning (day 0), triplicate plates were trypsinized and cells were counted (Coulter Counter) to give starting counts; remaining plates were fed to ensure removal of residual serum from the defined medium plates. After 5 days, with feeding on day 3, triplicate plates were again trypsinized and cells were counted to calculate growth in population doublings.

279

Bacterial hosts and plasmids. Escherichia coli HB101 was the recipient for most DNA transformations, by standard protocols (22). Strain CC114 (21) was the recipient in suppressor-linker insertional mutagenesis. pTll contains MMuLV proviral DNA and flanking cellular DNA cloned into the HaeII fragment of pACYC177 (21). pABLpv contains full-length (p160) A-MuLV proviral DNA with flanking cellular DNA cloned into the EcoRI site of pBR322 (27). pTAB1 was constructed from these two plasmids by excising the entire A-MuLV genome from pABLpv with KpnI (sites in long terminal repeats at proviral base pairs [bp] 479 and 6349) and by using this fragment to replace the M-MuLV sequences between the KpnI sites in pTll. Plasmid pAAbl was constructed by using the bacterial expression vector pATH2, the generous gift of T. J. Koerner and A. Tzagaloff (Columbia University). pTAB1 was digested with BstEII and was treated with S1 nuclease to blunt the ends. After the addition of BamHI linkers and digestion with BamHI and HindIII, the fragment of pTAB1 from BstEII-BamHI to HindIII (containing gag-abl) was cloned into the BamHI and Hindlll sites in the polylinker region of pATH2. The resulting plasmid, pAAbl, codes for a fusion protein of trpE ("-40 kilodaltons) (kDa) and gag-abl (-140 kDa). DNA enzymatic reactions and large-scale plasmid DNA preparations were performed as described elsewhere (19, 23). Southern blotting. Southern blots of genomic DNA prepared from enriched cultures of A-MuLV-transformed fibroblasts were performed (12). The probe used, pAB3Sub3 (12), was nick translated (31) with four [at-32P]deoxyribonucleoside triphosphates (Amersham Corp.). Insertional mutagenesis. A library of 12-bp insertional mutants of A-MuLV proviral DNA was previously constructed and characterized (28). A library of 16-bp insertional mutations in pTAB1 was generated with the suppressor-linker technique (21), with an aberrant cloned suppressor-linker DNA which yielded a 16-bp insertion of 5' CTGGAATTCCAGCCAG 3' or its complementary sequence. Bacterial expression of the A-MuLV kinase. Expression of the A-MuLV kinase in bacteria and extraction of the kinase from bacteria have been previously described (28). A-MuLV kinase assay. Activities of mutant A-MuLV kinases extracted from transfected fibroblasts were measured in an immunoprecipitate assay assessing both autophosphorylation and artificial substrate phosphorylation (28). Artificial substrate phosphorylation at 24'C was linear to at least 30 min and was measured at 20 min. Metabolic labeling of A-MuLV gag-abl protein. Labeling of gag-abl proteins with [35S]methionine (Amersham) in trans-

fected fibroblasts, extraction of the labeled proteins, immunoprecipitation with antiserum to gag, and analysis by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and autoradiography were performed as described previously (28). For pulse-chase labeling of gag-abl proteins with [35S]methionine, the protocol was modified. Some plates of fibroblasts were lysed for extraction of gag-abl proteins as usual immediately after the 1-h labeling period (28). Other plates had the labeling medium completely removed, were washed twice with phosphate-buffered saline minus (28), were fed with normal medium (0.2 mM methionine), and were incubated at 37'C for either 1 or 4 h prior to Iysis. [35S]methionine incorporation into proteins was quantitated by densitometry of the autoradiograms.

280

REES-JONES ET AL.

RESULTS A-MuLV induces PDGF-independent fibroblast growth. Defined medium containing only insulin and PDGF as mitogens supports the growth of NIH 3T3 fibroblasts (43). Removal of PDGF from this medium results in growth arrest of the fibroblasts and subsequent cell death (43; data not

shown). We used this defined medium culture system to confirm that fibroblasts transformed by A-MuLV are independent of PDGF for growth, as previously demonstrated (34). NIH 3T3 fibroblasts were transfected with A-MuLV and helper M-MuLV proviral DNA by the DEAE-dextran method (24). After the appearance of foci of cells transformed by AMuLV, enriched cultures of these cells were obtained and transferred to defined medium lacking PDGF. After 10 days without PDGF, colonies of transformed cells were evident (Fig. 1A). In contrast, control plates of fibroblasts transfected with M-MuLV alone contained dead or dying cells, with a few single cells surviving but no colonies (Fig. 1B). Thus, fibroblast transformation by A-MuLV supplants the normal PDGF requirement of these cells for growth. Analysis of 12-bp insertional mutants. To determine which A-MuLV sequences are required to mediate PDGF-independent growth of fibroblasts, we assayed a library of linker insertion mutants which has been previously described (28). Two groups of these mutants were analyzed for their abilities to render fibroblasts independent of PDGF (Fig. 2). The first group (mutants 12-11 to 12-13 and 12-15 to 12-17) contained insertional mutations which define a critical kinase domain of A-MuLV (28). These mutants encode stably expressed but totally inactive tyrosine kinases; viruses containing these mutations are transformation defective (28). Fibroblasts transfected with these mutants and helper virus were transferred to defined medium 10 days after transfection. All of these mutants failed to induce PDGF-independent growth (Fig. 2). Under these conditions, induction of

PDGF-independent growth by wild-type A-MuLV was readily detectable. The second group (mutants 12-6 to 12-10 and 12-18 to 12-23) contained insertional mutations which flank either side of the critical kinase domain. The phenotypes of these mutants (kinase activity, transformation ability) are indistinguishable from that of wild-type A-MuLV (28). Fibroblasts transformed by these mutants all demonstrated PDGF independence (Fig. 2), suggesting that v-abl sequences surrounding the critical kinase domain are not involved in the induction of PDGF independence. In summary, the 12-bp mutants demonstrated an absolute concordance between

tyrosine kinase activity, transformation competence, and induction of PDGF-independent fibroblast growth. Analysis of 16-bp insertional mutants. To determine whether more C-terminal A-MuLV sequences are required to induce PDGF independence, we constructed a library of 16-bp insertional mutations in A-MuLV proviral DNA, using a variation of the suppressor-linker insertional mutagenesis method (21). Six mutants with 16-bp insertions in abl sequences C terminal to the critical kinase domain (Fig. 3; Table 1) were selected for further study. Mutations 16-1 through 16-5 did not affect the kinase activity or transformation ability of the provirus (Table 1), as expected from prior deletion studies (14, 27, 38). Each of these mutations resulted in premature translational termination of the gag-abl protein, with appropriate C-terminal truncation, when visualized by autophosphorylation of the

MOL. CELL. BIOL.

protein extracted from transformed fibroblasts (data not shown). Fibroblasts transformed by A-MuLV mutants 16-1 through 16-5 were fully independent of PDGF for growth (Fig. 3; Table 1), suggesting that the C-terminal sequences distal to nucleotide 3436 (termination point of mutation 16-5) are not required for this function. Again, these frameshift mutants demonstrated a complete concordance between normal tyrosine kinase activity, transformation competence, and PDGF-independent fibroblast growth. Mutation 16-6 resulted in a provirus with diminished transforming ability in the focus assay. Foci induced by mutant 16-6 were delayed in appearance (2 to 3 days) and diminished in number at least 10-fold compared to foci of the other 16-bp mutants and of wild-type A-MuLV. Furthermore, the morphology of fibroblasts transformed by mutant 16-6 was distinct. While wild-type foci contained piles of highly refractile, rounded-up cells (Fig. 4A), fibroblasts transformed by mutant 16-6 appeared mostly fusiform, flatter, and less refractile (Fig. 4B). With time, small numbers of more-refractile rounded-up cells appeared in foci induced by mutant 16-6. Fibroblasts transformed by mutant 16-6 were studied immediately after recovery of the transformed cells. Enriched cultures of transformed fibroblasts were extracted for an immunoprecipitate kinase assay, which demonstrated a phosphorylated band of 91 kDa (Fig. 5, lane 5), the size expected for the prematurely terminated gag-abl protein of the mutant. A phosphorylated band of 75 kDa and minor intermediate bands were also observed in the kinase assays (Fig. 5, lane 5); these may represent kinases truncated further by additional mutations in the 16-6 provirus (see below). Despite the abnormal focus-forming ability of mutant 16-6, fibroblasts recovered from these foci were independent of PDGF for growth (Fig. 3; Table 1) and were quantitatively similar to wild type-transformed fibroblasts in their colony formation in defined medium. To further compare the PDGF independence of fibroblasts transformed by mutant 16-6 and wild-type A-MuLV, we measured their growth rates. In a representative 5-day assay in defined medium without PDGF, mutant 16-6-transformed fibroblasts grew even faster than cells transformed by wild-type A-MuLV (2.6 versus 1.5 population doublings). Growth rates were similar in defined medium with maximal (25 ng/ml) PDGF (4.2 versus 3.8 population doublings) and in DMEM with 10% calf serum (5.6 versus 4.8 population doublings). Thus, mutant 16-6 clearly induced PDGF independence equal to or even greater than that induced by wild-type A-MuLV. To summarize the results for mutant 16-6, there was again correlation between an active tyrosine kinase and PDGFindependent fibroblast growth, in this case despite abnormal focus-forming ability and incomplete morphological transformation of the fibroblasts. Substrate specificity of mutant 16-6. The diminished transformation efficiency of mutant 16-6 raised the possibility that the truncated gag-abl fusion protein encoded by this virus failed to phosphorylate some intracellular substrate(s) required for full transformation or was quantitatively not able to phosphorylate a critical substrate(s) to a required level. As a measure of the substrate specificities of the 16-6 and wild-type kinases, we screened a battery of artificial substrates for their abilities to serve as substrates for the two kinases. To compare the specific activities of these kinases, we first labeled the wild-type and 16-6-encoded gag-abi proteins in

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FIG. 1. NIH 3T3 fibroblasts transformed by wild-type A-MuLV (A) or infected with helper M-MuLV (B) were placed in defined medium without PDGF for 10 days. Phase-contrast photomicrographs of representative areas of these cultures at the end of selection are shown.

transformed fibroblasts with [35S]methionine. After extraction and immunoprecipitation with antiserum to gag, we visualized the proteins by SDS-polyacrylamide gel electrophoresis and autoradiography (Fig. 6). In addition to the

expected 91-kDa band of the 16-6 mutant, another faint specific band of 75 kDa was evident. By densitometry, the 16-6 preparation had 1.28 times as much labeled gag-abl protein as the wild-type preparation; this figure was used to

282

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REES-JONES ET AL.

TABLE 1. Phenotypes of 16-bp insertional mutantsa

12 bp Inserts PDGF Independence +

+

12-7

12~-12_

.

..

+

+

~~~~~~..... L... n ;

T'~~~~~ 't

12-7 12-1l 12 9 12-3

t tt12-20 12-23

12-16

12-18+

+

4'I 4 ~~~2 3 kilobase pairs

1

5

FIG. 2. Locations of the 12-bp insertions in A-MuLV proviral DNA shown on a representation of the encoded gag-abl protein. Positions in kilobase pairs refer to the proviral DNA. The presence (+) or absence (-) of PDGF-independent growth in fibroblasts transformed by the mutant is indicated. The shaded area is the previously defined critical kinase domain, in which 12- or 6-bp insertions totally inactivate the kinase and render the virus fully transformation defective.

obtain specific activities of the two kinases in the artificial substrate assays, which used aliquots of the same preparations. To ensure that metabolic labeling yielded a valid measurement of steady-state gag-abl levels, we used pulsechase labeling with [35S]methionine to measure the half-lives of these proteins (see Materials and Methods). The 16-6 and wild-type gag-abl proteins showed similar half-lives of 3.7 and 4.2 h, respectively. The wild-type and 16-6 kinases showed differences in artificial substrate phosphorylation (Table 2). The 16-6 kinase specific activity averaged about 30% of that of the wild-type kinase, considering those substrates against which the enzymes were most active. The 16-6 kinase was more active toward GAT (6:3:1) and GAT (1:1:1), although still not reaching wild-type activity. Notably, several substrates phosphorylated by the wild-type kinase were essentially not phosphorylated by the 16-6 kinase (e.g., casein, GLAT, GALT, and GLT5). Thus, both quantitative and qualitative differences exist between the enzymatic function of wildtype and 16-6 kinases. Instability of A-MuLV containing mutation 16-6. When fibroblasts transformed by mutant 16-6 were carried in culture for weeks, their distinct morphology became less and less evident, and they eventually resembled fibroblasts transformed by wild-type A-MuLV. To investigate the basis of this change, we analyzed these fibroblasts for alterations in the contained 16-6 proviral DNA. Genomic DNA from fibroblasts transformed by mutant 16-6 was digested with

16 bp Inserts PDGF Independence 16-6 GAG

Slteb Siteb (bp)

Kinase activity" G/T (4:1 Auto

4186 4169 3909 3752

+++ +++ +++ +++ +++ +

Focus-forming activityd (no. of foci/,ug of

DNA)

12-19 12-21

12-6 12-8 12-12 12-15 GAG

Mutation

16-4

16-2

B L t

16-5

3

t

t

16-3 16-1 + +.

4

kilobase pairs FIG. 3. Locations of the 16-bp insertional mutations in A-MuLV proviral DNA and the critical kinase domain (shaded area). The mutants all rendered fibroblasts independent of PDGF (+).

16-1 16-2 16-3 16-4 16-5 16-6

3411

3306

+++ +++ +++ +++ +++ +

>50 >50 >50

>50 >50

50 distinct foci per jLg of proviral DNA 12 to 16 days after transfection. e The morphology of fibroblasts transformed by mutant 16-6 was distinct from that of fibroblasts transformed by wild-type A-MuLV (see Fig. 4). a

EcoRV (sites in the long terminal repeats) with or without EcoRI (site created by the 16-bp insertion). This digested DNA was used to perform Southern blotting experiments with a nick-translated probe for abl sequences, pAB3Sub3 (12). The results (Fig. 7) demonstrate that fibroblasts transformed by mutant 16-6 contained several species of AMuLV proviral DNA. When genomic DNA was prepared from the transformed fibroblasts shortly after recovery (less than 1 month after transfection), the predominant proviral DNA visualized was 6.2 kilobases (kb) (lane 5), the same length as that of wild-type provirus (lane 3). However, minor bands of 5.7 and 4.7 kb, representing truncated proviruses, were evident (lane 5). While the full-length provirus retained the inserted 16-bp EcoRI site, yielding the predicted 3.4- and 2.6-kb bands on digestion with EcoRI, the smaller proviruses did not (lane 6). Genomic DNA was also prepared from fibroblasts transformed by mutant 16-6 which had been passaged for several months. Only the smallest truncated proviral species (4.4 kb) was evident in Southern blots of this genomic DNA (lane 7), and the EcoRI site representing the inserted frameshift mutation was no longer present (lane 8). Thus, loss or mutation of the original linker insertion correlates temporally with the observed change in morphology of fibroblasts transformed by 16-6 to wild type. This instability of the virus was also demonstrated by labeling fibroblasts recently transformed by mutant 16-6 with [35S]methionine (Fig. 5). Extracts were prepared from labeled fibroblasts, immunoprecipitated with antiserum to gag, and visualized by SDS-polyacrylamide gel electrophoresis and autoradiography. Prominent specific bands were observed at the expected 91 kDa and also at 75 kDa (Fig. 5, lane 2). These bands often appeared as doublets. In addition, a fainter band was variably observed at 100 kDa, representing the glycosylated form of the 91-kDa protein (28). Of note, metabolic labeling performed at the earliest time after transfection demonstrated predominantly the 91-kDa species (Fig. 6). Similar experiments with fibroblasts passaged for several months after transformation revealed the disappear-

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FIG. 4. NIH 3T3 focus assays were performed with helper virus and wild-type A-MuLV (A) or A-MuLV mutant 16-6 (B) proviral DNA as described in Materials and Methods. Phase-contrast photomicrographs of representative foci in an assay are shown.

of the 91-kDa (full-length) band, while new bands corresponding to shorter gag-abl proteins appeared (Fig. 5, lane 3). Visualization by autophosphorylation of these same early (Fig. 5, lane 5) and late (Fig. 5, lane 6) gag-abl proteins from fibroblasts transformed by mutant 16-6 confirmed these

ance

results. In addition, several minor bands between the 91- and 75-kDa proteins were seen at early times (lane 5) when labeled by autophosphorylation. When the autophosphorylation and metabolic labeling results were compared, the specific activities of the several species of 16-6 kinase varied considerably. Of particular note, the specific activity of the

284

REES-JONES ET AL.

MOL. CELL. BIOL. TABLE 2. Comparison of activities of wild-type and 16-6-encoded kinasesa

,.74411T 16-6 INSTAHBLITYt 5

3

2

6

Sp actb

Substrate

pTAB1

GT(4: 1)

U_ .-.sem ;

U

6--6 -6 EARLY LATE

lT 1

6.. EARJ

r

-;;

FIG. 5. gag-abl proteins in fibroblasts transfected with helper M-MuLV alone (pT11) or transformed by A-MuLV mutant 16-6 were labeled with [35S]methionine (lanes 1 to 3) or by autophosphorylation (lanes 4 to 6), as described in Materials and Methods. Shown are autoradiograms of gels from a representative experiment. Early (lanes 2 and 5) and late (lanes 3 and 6) 16-6-transformed fibroblasts had been carried for 1 and 3 months after transfection, respectively. Molecular masses (in kilodaltons) are shown at left.

predominant truncated form of the kinase (75 kDa) was increased over that of the full-length product (91 kDa). DISCUSSION PDGF independence requires A-MuLV tyrosine kinase activity. We made use of a panel of A-MuLV mutants to investigate the basis of PDGF-independent fibroblast growth induced by A-MuLV. Our initial goal was to determine whether this A-MuLV effect required the A-MuLV-encoded tyrosine kinase, as is the case for cell transformation (11, 39). The results are straightforward. Insertional mutations in

30 36 24 34

GAT(6:3:1) GAT(1:1:1)

37 19

17 12

46 63

Casein GLAT GALT GLT5 Histone H2B

41 7.3 5.1 2.9 0

l

2 2

200 -b4

11 5 6 0

the proviral abl sequences which had no effect on the activity of the encoded tyrosine kinase failed to affect PDGF-independent growth induced by the virus. Those insertional mutations within the critical kinase domain, which abolished the kinase activity and the transformation ability of the mutant virus, also rendered the virus incapable of mediating PDGF-independent fibroblast growth. Of note, these kinase domain mutants encoded gag-abl proteins which were stably expressed (28), thus leaving the inactive kinase as the probable explanation for the failure to induce PDGF independence. 3 4

5 6

7 8

Size (Kb)

3

*A.

.4 150

116

92

4.5 0.4 0.3 0 0

a The activities of the wild-type (pTAB1) and 16-6-encoded kinases against a battery of artificial substrates were assessed after expression in and extraction from fibroblasts, as described in Materials and Methods. The amounts of kinase used in these assays were determined by [35S]methionine labeling (Fig. 6) to derive the specific activities shown here. Assays were performed in duplicate; these data are representative of three experiments. b In arbitrary units.

1 2

Mr, 103

ratio (%)

109 128 60 7.8

GT(1:1)

I

1,4.+

16-6

365 356 251 23

GT'o GAT'0

-

2

16-6/pTAB1 Activity

9.4 6.6

-.- -62 8

AX "t-. -62

4.4

*0*

-

- W.-*

9]

qPr809og

pTAB- 1

16-61

-347

1-6

,,,

66

_

Pr65909

abl plasmid TABI 16-6 FIG. 6. Fibroblasts were transfected with helper M-MuLV and wild-type (lane 1) or mutant 16-6 (lane 2) A-MuLV proviral DNA. Enriched cultures of transformed cells were recovered and labeled with [35S]methionine. Detergent extracts of 106 fibroblasts were immunoprecipitated with antiserum to gag, and labeled proteins were separated by SDS-polyacrylamide gel electrophoresis. Shown is an autoradiogram of the gel. For identification of M-MuLVencoded gag proteins, lane 3 was derived from fibroblasts transfected with M-MuLV alone. Replicates of the extracts used in this experiment were used for the kinase assays shown in Table 2. Molecular masses (in kilodaltons) are shown at left.

2.3

s

plasmid

pTIl

EcoRl

- +

t

pTABI 16-6 - +

-- +

16-6 - +

FIG. 7. Genomic DNA was prepared from fibroblasts infected with M-MuLV alone (pT11) or transformed by wild-type A-MuLV (pTAB1) or mutant 16-6. The fibroblasts used for lanes 5 and 6 were passaged for less than 1 month after transfection; those used for lanes 7 and 8 were passaged for more than 3 months after transfection. The genomic DNA was digested with EcoRV (all lanes) with (+) or without (-) EcoRI (site of the insertional mutation) as indicated and was used to perform a Southern blot with an ablspecific probe. Shown is an autoradiogram of the blot. Sizes of the major proviral species are shown (in kilobase pairs) to the right of the autoradiogram. The band of 12.8 kb is seen in DNA from cells not containing v-abl and thus represents the c-abl locus.

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Analysis of A-MuLV mutants encoding proteins with truncated C termini showed that the majority of C-terminal abl sequences, from proviral bp 3331 (termination point of mutant 16-6) to the end of the protein, were also not required to induce PDGF-independent fibroblast growth. These sequences have also been shown not to be necessary for kinase function and fibroblast transformation (14, 27, 28, 38). Thus, it appears that PDGF-independent fibroblast growth is closely correlated with morphological transformation by A-MuLV and is most likely a function of the encoded tyrosine kinase activity. Transformation-defective mutant 16-6. One mutant of AMuLV, mutant 16-6, exhibits a unique phenotype. This virus is partially defective in transformation, resulting in 10-fold fewer foci than the wild-type virus. The morphology of fibroblasts transformed by this virus appears distinct from that of wild-type transformants. Their appearance is reminiscent of that of fusiform mutants of Rous sarcoma virus (10, 41). Analysis of these src mutants has revealed less inhibition of fibronectin synthesis and secretion by the fusiform transformants compared with those of wild-type transformants (32). Presumably, these mutant kinases fail to phosphorylate, or phosphorylate to a diminished extent, some critical substrate necessary for full rounding up of the transformed cells. An obvious possibility is cystoskeletal substrates, but one study of fusiform mutant transformants found that phosphorylation of vinculin on tyrosine was not different (32). To assess the kinase function of mutant 16-6, we screened its activities towards artificial substrates and compared them with wild-type kinase activity. We detected significant differences in both specificity and total specific activity between the 16-6-encoded kinase and the wild-type enzyme, the latter being more active. Although this analysis may not reflect activity or specificity towards endogenous substrates, such screens have been able to distinguish the substrate specificities of several other tyrosine kinases with distinct cellular effects (44). We suspect that the 10-fold reduction of transformation efficiency of mutant 16-6 is largely a reflection of diminished total kinase activity, which may prevent critical endogenous substrates from being phosphorylated at all or may allow them to be phosphorylated to a lesser extent than that required for transformation. Likewise, the incomplete morphological changes of these fibroblasts may reflect incomplete action by the 16-6 kinase. In spite of the reduced activity of the 16-6 kinase and its reduced transformation efficiency, fibroblasts recovered from foci formed by this mutant are independent of PDGF for growth. Judging either from colony counts in the defined medium assay or from quantitative growth analysis, the PDGF independence induced by mutant 16-6 is at least equal to that induced by wild-type A-MuLV and may be greater. This suggests that the phenotypes of growth factor independence and full morphological transformation are distinct and can be at least partially dissociated. This may be a simple threshold effect, with transformation requiring the phosphorylation of more substrates or of the same substrates to greater degrees than that required for PDGF independence. Our characterization of mutant 16-6 is notable for the instability of virus containing this mutation when passaged in culture subsequent to our initial phenotypic characterization, with secondary mutations yielding progressive truncation of the provirus and its encoded kinase and loss or mutation of the original 16-bp insertion. The predominant species of truncated kinase is 75 kDa, compared with 91 kDa for the full-length 16-6 kinase. The combination of truncation

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and loss of the 16-bp insertion suggests that the 75-kDa species might result from C-terminal truncation of the 91kDa kinase. The distance from the end of the critical kinase domain (proviral bp 2878) to the translational termination point of the full-length 16-6 mutant (proviral bp 3331) leaves approximately 18 kDa of protein which could be deleted without affecting the kinase domain. In addition to this truncation, the 75-kDa kinase demonstrates an increased specific activity compared with that of the full-length 16-6 kinase. These alterations correlate temporally with the change of the morphology of 16-6-transformed fibroblasts to wild type, again suggesting that the phenotype of mutant 16-6 is a result of diminished kinase activity. Since this mutant is partially transformation defective, this reversion process probably reflects selection for secondary mutations which enhance the transformation efficiency of the virus. Furthermore, the method for enriching transformed fibroblasts may inadvertently favor the recovery of fully roundedup and loosely adherent transformed cells. We doubt that the lethal effect of the A-MuLV C-terminal domain (13, 38, 42) is involved, since in our hands wild-type A-MuLV was not truncated over a similar time course. Proteolysis of the gag-abl protein during extraction of the kinase assay also seems an unlikely explanation for the lower-molecularweight forms of the kinase observed, for the following reasons: the pattern (sizes and relative amounts) of autophosphorylated proteins observed was not variable from experiment to experiment, inclusion of SDS in the extraction buffer did not alter the pattern, and changes in proviral DNA size by Southern blotting paralleled reductions in kinase size. In addition, such truncation has not been observed with other mutants with the same assay conditions or with mutant 16-6 when coexpressed with pSV2neo (linked on the same plasmid) in fibroblasts held under G418 selection (R. W. Rees-Jones and S. P. Goff, unpublished data). Further characterization of this mutant in culture for times longer than those used in this study must take this process into account and will require the use of conditions which diminish or abolish this instability. In summary, PDGF-independent fibroblast growth induced by A-MuLV appears to be mediated by the A-MuLVencoded tyrosine kinase, as previously shown for transformation by A-MuLV. However, it appears that these two processes are not identical, as mutant 16-6 separates the phenotype of PDGF independence from full morphological transformation and full transformation efficiency. The most likely explanation for this observation is a quantitative one, namely, that full morphological transformation of fibroblasts requires a high level of phosphorylation of cellular substrates and that a lower level is necessary for the induction of PDGF independence. Alternatively, different substrates may be involved in these two processes. We are currently beginning an analysis of in vivo substrates of the wild-type and mutant 16-6-encoded enzymes to investigate these possibilities. We hope that further characterization of A-MuLV mutant 16-6 will provide useful information on the processes of growth factor independence and cell transformation induced by this retrovirally encoded tyrosine kinase. For example, the transformation-defective character of mutant 16-6 suggest that some fibroblasts containing this mutant after transfection are not transformed. If we can identify nontransformed fibroblasts containing an active (albeit mutant) kinase, such a line would be extremely useful in looking for substrates relevant for PDGF-independent fibroblast growth and transformation and in determining kinase attributes essential for even partial transformation. The differences

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between the 16-6 kinase in untransformed cells and that which we have characterized in transformed cells would be notable. Finally, such a fibroblast line could be studied for progression to the transformed state, either via further mutations in the provirus or at secondary sites. Possible mechanisms of PDGF-independent fibroblast growth. Two main possibilities exist for the mechanism of PDGF independence induced by A-MuLV. First, A-MuLV could induce expression of PDGF by the fibroblasts with resultant autocrine stimulation. Precedence exists for such an autocrine pathway of PDGF stimulation in fibroblasts transformed by v-sis (1), which expresses the B chain of PDGF, and in the osteosarcoma cell line U-2 OS, which secretes PDGF A-chain homodimers (15). Demonstration of autocrine stimulation in A-MuLV-transformed fibroblasts will require measurement of PDGF mRNA levels in the fibroblasts and PDGF levels in conditioned media, as well as the detection of PDGF receptor activation in the fibroblasts. We are currently pursuing this analysis. Before leaving autocrine mechanisms for growth factor independence, we should mention that previous work demonstrated that fibroblasts transformed by A-MuLV secrete transforming growth factor alpha, which binds to and acts via the epidermal growth factor receptor (36). We consider this potential autocrine mechanism unlikely to be the basis of PDGF independence in our assay, since epidermal growth factor cannot substitute for PDGF in our defined media to support fibroblast growth (43). The second potential mechanism for PDGF independence induced by A-MuLV is that the two pathways of growth stimulation, through the PDGF-stimulated tyrosine kinase and the A-MuLV-encoded tyrosine kinase, have steps in common, perhaps intersecting in a common substrate or both activating the same process distally. A single previous study of intracellular substrates for these two enzymes did not reveal any substrates in common (9). This mechanism seems likely and will require more extensive analysis of in vivo substrates of the Abelson and PDGF receptor kinases for confirmation. ACKNOWLEDGMENTS This work was supported by grant MV 276 from the American Cancer Society. R.W.R.-J. was supported by Physician-Scientist Program Award AM01336 and by Basil O'Connor Starter Research Award 5-637 from the March of Dimes Birth Defects Foundation, Inc. We thank Leslie Lobel for advice in the techniques of insertional mutagenesis and especially for the aberrant suppressor-linker used in this study, and the members of the Goldfarb laboratory for help with the assay for PDGF-independent fibroblast growth.

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