Autocrine growth and anchorage independence: two complementing Jun-controlled genetic programs of cellular transformation - PubMed (original) (raw)

Autocrine growth and anchorage independence: two complementing Jun-controlled genetic programs of cellular transformation

H van Dam et al. Genes Dev. 1998.

Abstract

Cellular transformation can be achieved by constitutive activation of growth-regulatory signaling pathways, which, in turn, activate nuclear transcription factors thought to execute a transformation-specific program of gene expression. Members of the dimeric transcription factor family AP-1 are at the receiving end of such growth-regulating pathways and the viral form of the AP-1 subunit Jun establishes one important aspect of transformation in chick embryo fibroblasts (CEFs): enhanced growth in agar and in low serum. Enhanced Jun activity is likely to target several different genetic programs as Jun forms heterodimers with one of several members of the Fos and ATF2 subfamilies, resulting in transcription factors with different sequence specificities. To identify the programs relevant for transformation, we have reduced the complexity of AP-1 factors by constructing Jun bZip mutants that can efficiently dimerize and transactivate with only a restricted set of partner subunits. Upon introduction into CEFs, a Jun mutant selective for the Fos family induced anchorage-independent growth but no growth factor-independence. In contrast, a c-Jun mutant with preference for ATF2-like proteins caused growth factor-independence, but no growth in agar. Coexpression of both mutants reestablished the combined transformation program as induced by wild-type Jun. These data show that Jun-dependent cell transformation can be resolved into at least two distinct and independent processes, anchorage and growth factor independence, obviously triggered by two classes of Jun heterodimers likely regulating different sets of target genes.

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Figures

Figure 1

Figure 1

c-Jun mutants with altered Jun:Fos and Jun:ATF2 dimerization specificities. (Top) Helical wheel representations of the c-Jun:c-Fos and c-Jun:ATF2 leucine zipper dimers. The amino acids at the e and g positions determining dimer specificity are located adjacent to the hydrophobic interface that is formed by the leucines at the d positions and the a residues. (Bottom) c-Jun mutants m0 and m1 were constructed by replacement of amino acid residues at the e and/or g positions as depicted. In m0, Glu-281 of c-Jun is replaced by a lysine. In m1, Lys-283, Lys-288, Arg-302, and Gln-304 are replaced by four glutamates.

Figure 1

Figure 1

c-Jun mutants with altered Jun:Fos and Jun:ATF2 dimerization specificities. (Top) Helical wheel representations of the c-Jun:c-Fos and c-Jun:ATF2 leucine zipper dimers. The amino acids at the e and g positions determining dimer specificity are located adjacent to the hydrophobic interface that is formed by the leucines at the d positions and the a residues. (Bottom) c-Jun mutants m0 and m1 were constructed by replacement of amino acid residues at the e and/or g positions as depicted. In m0, Glu-281 of c-Jun is replaced by a lysine. In m1, Lys-283, Lys-288, Arg-302, and Gln-304 are replaced by four glutamates.

Figure 2

Figure 2

The interaction of mutant c-Jun leucine zipper domains with c-Fos and ATF2–VP16 as measured by mammalian one-hybrid analysis. Undifferentiated F9 cells were transiently transfected with 2 μg of 5×GAL4–E4–luciferase reporter plasmid together with 0.5 μg of the indicated wild-type or mutant RSV–Gal4–DBD–c-Jun–bZip expression vectors or Gal4–DBD or c-Jun–bZip control constructs in the presence of the indicated amounts of RSV–c-Fos, RSV–ATF2–VP16 expression vectors and/or an RSV control plasmid (pUC–RSV:−) to equalize the total amount of RSV–LTR sequences. Fold activation represents luciferase activity induced by the Gal4 fusion proteins in the presence of the c-Fos or ATF2–VP16 expression vectors. The Gal4 fusion proteins themselves did not significantly affect the activity of the reporter. The data represent the mean of four independent experiments in which the constructs were tested in parallel. Standard deviations are <25%. Relative induction ratios for m0: (m0 + Fos/m0 + ATF2–VP16)/(wt + Fos/wt + ATF2–VP16) = 1.5; for m1: (m1 + Fos/m1 + ATF2–VP16)/(wt + Fos/wt + ATF2–VP16) = 0.2.

Figure 3

Figure 3

Dimer-specific DNA-binding properties of c-Jun leucine zipper mutants m0 and m1 in vitro. Gel retardation assays showing the DNA-binding affinities for the collagenase AP-1 binding site (coll–TRE) and the jun2 site of in vitro-translated wild-type and mutant c-Jun proteins in the absence or presence of in vitro-translated c-Fos or bZip–ATF2 (amino acids 335–505). c-Jun proteins were quantified by measurement of the amount of incorporated [35S]methionine, and similar amounts were mixed on ice with control reticulocyte lysate (−) or lysates containing c-Fos or bZip–ATF2 after which 32P-labeled DNA probe was added. The DNA–protein complexes were resolved on 4% polyacrylamide gels and visualized by autoradiography. Note that the increased mobility of the c-Jun–m1:ATF2–DNA complex as compared with that of c-Jun wild-type is explained by a higher negative charge of the c-Jun–m1 protein than of wild-type c-Jun, owing to the replacement of two lysines, an arginine, and a glutamine by four glutamic acid residues.

Figure 4

Figure 4

Jun:Fos and Jun:ATF2-specific transactivation by c-Jun leucine zipper mutants c-Jun m0 and m1 in vivo. Comparison of transactivation by wild-type c-Jun and by the Jun mutants m0 and m1 of Jun:Fos- and Jun:ATF2-dependent minimal promoters. F9 teratocarcinoma cells were transiently transfected with 2 μg of either the 5×coll–TRE–tata–luciferase, 5×jun2–tata–luciferase, or tata–luciferase reporter plasmids together with 0.5 μg of wild-type or mutant RSV–c-Jun expression vector in the presence or absence of 25 ng pRSV–c-Fos, or an RSV control plasmid (pUC–RSV). Fold activation represents activity obtained in the presence of the c-Jun expression vectors relative to the activity in the presence of the pUC–RSV control vector, and normalized to the activation on the tata-luciferase control. The experiments were performed at least three times and each time had very similar results. One of them is shown.

s.d.

< 25%.

Figure 5

Figure 5

Promoter-selective activation by c-Jun mutants in CEF cells. CEFs (5 × 105) were transiently cotransfected with 2 μg of either the 5×coll–TRE–tata–luciferase, 5×jun2–tata–luciferase, or tata–luciferase reporter plasmids together with 1 μg of either wild-type or mutant RSV–c-Jun expression vector in the presence or absence of 50 ng of pRSV–c-Fos, 25 ng of pCMV–ATF2 or empty control plasmid (pUC18 carrier up to 13 μg per plate). Activity relative to that achieved in CEF transfected by wild-type Jun is plotted. Five independent experiments, using three independently generated series of primary cultures, were performed. One of these is shown.

Figure 5

Figure 5

Promoter-selective activation by c-Jun mutants in CEF cells. CEFs (5 × 105) were transiently cotransfected with 2 μg of either the 5×coll–TRE–tata–luciferase, 5×jun2–tata–luciferase, or tata–luciferase reporter plasmids together with 1 μg of either wild-type or mutant RSV–c-Jun expression vector in the presence or absence of 50 ng of pRSV–c-Fos, 25 ng of pCMV–ATF2 or empty control plasmid (pUC18 carrier up to 13 μg per plate). Activity relative to that achieved in CEF transfected by wild-type Jun is plotted. Five independent experiments, using three independently generated series of primary cultures, were performed. One of these is shown.

Figure 6

Figure 6

c-Jun:ATF2 and c-Jun:Fra2 heterodimeric complexes in CEF cells. Immunoprecipitation of DNA-bound c-Jun, Fra2, and ATF2 from untransformed (CEF) and human c-Jun-transformed CEFs [wild-type c-Jun (cJ-CEF); c-Jun–m0 (m0-CEF); c-Jun–m1 (m1-CEF); c-Jun–eb1 chimera (Castellazzi et al. 1993, eb1-CEF)] extracts after covalent cross-linking to either the jun2 or coll–TRE oligonucleotides. CEF cell extracts were incubated with BrdU- and 32P-labeled DNA probes, cross-linked by UV-irradiation, and diluted in a mild (B) or stringent (A,B) immunoprecipitation buffer (see Materials and Methods). Immunocomplexes with antibodies as indicated were resolved on 12% SDS-PAGE and visualized by autoradiography. (A) The addition of antibody or nonimmune control antibody is shown above the gel plot. Note that endogenous chicken c-Jun migrates faster than human c-Jun. In cJ-CEF, the endogenous expression is repressed (see also Fig. 7). (B) To show the difference in c-Jun levels coprecipitating with Fra2 under nonstringent condition (right), relatively short exposures of the nonstringent precipitations are presented. Note that anti-Fra2 coprecipitates heterodimers of Fra2 with endogenous chicken c-Jun if the cross-links were done with CEF or m1-CEF extracts, heterodimers with human c-Jun only with cJ-CEF and m0-CEF extracts. No heterodimers with Fra2 are formed in the control extracts from Jun–eb1-CEF transformants as eb1–bZip mediates only homodimerization (Castellazzi et al. 1993). (−) Nonimmune serum.

Figure 6

Figure 6

c-Jun:ATF2 and c-Jun:Fra2 heterodimeric complexes in CEF cells. Immunoprecipitation of DNA-bound c-Jun, Fra2, and ATF2 from untransformed (CEF) and human c-Jun-transformed CEFs [wild-type c-Jun (cJ-CEF); c-Jun–m0 (m0-CEF); c-Jun–m1 (m1-CEF); c-Jun–eb1 chimera (Castellazzi et al. 1993, eb1-CEF)] extracts after covalent cross-linking to either the jun2 or coll–TRE oligonucleotides. CEF cell extracts were incubated with BrdU- and 32P-labeled DNA probes, cross-linked by UV-irradiation, and diluted in a mild (B) or stringent (A,B) immunoprecipitation buffer (see Materials and Methods). Immunocomplexes with antibodies as indicated were resolved on 12% SDS-PAGE and visualized by autoradiography. (A) The addition of antibody or nonimmune control antibody is shown above the gel plot. Note that endogenous chicken c-Jun migrates faster than human c-Jun. In cJ-CEF, the endogenous expression is repressed (see also Fig. 7). (B) To show the difference in c-Jun levels coprecipitating with Fra2 under nonstringent condition (right), relatively short exposures of the nonstringent precipitations are presented. Note that anti-Fra2 coprecipitates heterodimers of Fra2 with endogenous chicken c-Jun if the cross-links were done with CEF or m1-CEF extracts, heterodimers with human c-Jun only with cJ-CEF and m0-CEF extracts. No heterodimers with Fra2 are formed in the control extracts from Jun–eb1-CEF transformants as eb1–bZip mediates only homodimerization (Castellazzi et al. 1993). (−) Nonimmune serum.

Figure 7

Figure 7

Expression of wild-type and of mutant human c-Jun proteins in infected CEF cells measured by Western blot analysis. Total cell extract (10 μg) was subjected to SDS-PAGE, blotted onto nitrocellulose and incubated with a polyclonal antibody raised against bacterially expressed mouse c-Jun. The two arrows indicate the positions of the endogenous avian c-Jun protein (c-cJun, 314 amino acids; calculated molecular mass of 34.4 kD) and the virally expressed human c-Jun proteins (h-c-Jun, 331 amino acids; calculated molecular mass of 35.7 kD), respectively.

Figure 8

Figure 8

Promoter-selective activation by v-Jun mutants in stably transformed CEF cells. CEFs (3 × 105) chronically infected with either R–v-Jun–m0 (hatched bar) or R–v-Jun–m1 (solid bar) were transiently transfected with 2 μg of either 5×coll–TRE–TK–luciferase, 5×jun2–TK–luciferase, or TK–luciferase reporter plasmid together with 10 μg of pUC18 as a carrier. Fold activation is the ratio of the activation on 5×jun2–TK/TK or on 5×coll–TRE–TK/TK.

Figure 9

Figure 9

Different transformed phenotypes induced by dimer-specific Jun mutants in CEF cells. (A) Representative microscopic fields from wild-type and mutant c-Jun-expressing CEF cells grown for 2 weeks in agar (2 × 103 cells per 60 mm petri dish were seeded). Bar, 0.1 mm. (B) Serum-dependence of R–Jun-, R–ATF2-, and R–Fra2-infected CEF cultures overexpressing ATF2, Fra2, wild-type Jun or the mutants m0 and m1 in a v-Jun background. (R) Control cells infected with empty retrovirus. (6% serum; left) Infected CEF cells were plated at 1.5 × 105 cells per plate in duplicates and supplied with medium containing 6% FCS (Castellazzi et al. 1990). (0.6% serum; right) CEF cultures were plated at 6 × 105 cells per plate in medium containing 0.6% serum for 4 days to ascertain correct depletion of serum; the cultures were then replated at day 0 in the same medium at 1.5 × 105 cells per plate in duplicates. In each experiment, viable cells were counted by use of trypan blue at the days indicated. R control cells behaved identically to uninfected CEF (data not shown). (C) Induction of serum-independent growth in Jun–m0 cells superinfected by Jun–m1. The procedures were as in B except that CEFs were preinfected with either RD–m0 or RD–m1 as indicated. Success of the double infection was ascertained by Western blotting (not shown). Note that the ability of an m0 infectant is not improved much by reinfection with m0 but is enhanced by m1. Jun–m1-transformed cells grow in low serum (as shown in B). This cannot be further improved by additional m1 and only slightly by m0.

Figure 9

Figure 9

Different transformed phenotypes induced by dimer-specific Jun mutants in CEF cells. (A) Representative microscopic fields from wild-type and mutant c-Jun-expressing CEF cells grown for 2 weeks in agar (2 × 103 cells per 60 mm petri dish were seeded). Bar, 0.1 mm. (B) Serum-dependence of R–Jun-, R–ATF2-, and R–Fra2-infected CEF cultures overexpressing ATF2, Fra2, wild-type Jun or the mutants m0 and m1 in a v-Jun background. (R) Control cells infected with empty retrovirus. (6% serum; left) Infected CEF cells were plated at 1.5 × 105 cells per plate in duplicates and supplied with medium containing 6% FCS (Castellazzi et al. 1990). (0.6% serum; right) CEF cultures were plated at 6 × 105 cells per plate in medium containing 0.6% serum for 4 days to ascertain correct depletion of serum; the cultures were then replated at day 0 in the same medium at 1.5 × 105 cells per plate in duplicates. In each experiment, viable cells were counted by use of trypan blue at the days indicated. R control cells behaved identically to uninfected CEF (data not shown). (C) Induction of serum-independent growth in Jun–m0 cells superinfected by Jun–m1. The procedures were as in B except that CEFs were preinfected with either RD–m0 or RD–m1 as indicated. Success of the double infection was ascertained by Western blotting (not shown). Note that the ability of an m0 infectant is not improved much by reinfection with m0 but is enhanced by m1. Jun–m1-transformed cells grow in low serum (as shown in B). This cannot be further improved by additional m1 and only slightly by m0.

Figure 9

Figure 9

Different transformed phenotypes induced by dimer-specific Jun mutants in CEF cells. (A) Representative microscopic fields from wild-type and mutant c-Jun-expressing CEF cells grown for 2 weeks in agar (2 × 103 cells per 60 mm petri dish were seeded). Bar, 0.1 mm. (B) Serum-dependence of R–Jun-, R–ATF2-, and R–Fra2-infected CEF cultures overexpressing ATF2, Fra2, wild-type Jun or the mutants m0 and m1 in a v-Jun background. (R) Control cells infected with empty retrovirus. (6% serum; left) Infected CEF cells were plated at 1.5 × 105 cells per plate in duplicates and supplied with medium containing 6% FCS (Castellazzi et al. 1990). (0.6% serum; right) CEF cultures were plated at 6 × 105 cells per plate in medium containing 0.6% serum for 4 days to ascertain correct depletion of serum; the cultures were then replated at day 0 in the same medium at 1.5 × 105 cells per plate in duplicates. In each experiment, viable cells were counted by use of trypan blue at the days indicated. R control cells behaved identically to uninfected CEF (data not shown). (C) Induction of serum-independent growth in Jun–m0 cells superinfected by Jun–m1. The procedures were as in B except that CEFs were preinfected with either RD–m0 or RD–m1 as indicated. Success of the double infection was ascertained by Western blotting (not shown). Note that the ability of an m0 infectant is not improved much by reinfection with m0 but is enhanced by m1. Jun–m1-transformed cells grow in low serum (as shown in B). This cannot be further improved by additional m1 and only slightly by m0.

Figure 10

Figure 10

Deoxyglucose uptake by Src and Jun transformants. Conditions are as in Figs. 9 and 11.

Figure 11

Figure 11

Jun-induced uPA and 72-kD gelatinase activities. Zymographs showing uPA and 72-kD gelatinase activity were obtained with conditioned medium of CEF cultures infected with either the empty R vector, or with R vector encoding v-Jun, v-Jun–m0, v-Jun–m1, or v-Src (NY72-4 variant; Mayer et al. 1986). (m0 + m1) and (m1 + m0) are cultures coexpressing v-Jun–m0 and v-Jun–m1 generated by sequential infection (see Table 1 and Fig. 9C). The gels were evaluated by densitometry. Maximum induction was obtained with v-Src and set to 100.

Figure 12

Figure 12

Model of Jun-dependent transformation. Note that Fos stands for Fos-like. In CEFs the major Jun partner is Fra2.

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