Chapter 9 : Malignant Transformation of Cells by the v-Rel Oncoprotein

Comparison of v-Rel and c-Rel. (Top) Generalized structure of class II Rel proteins: the hatched area is the RH domain that contains sequences important for DNA binding, dimerization, inhibitor (IN) (I?B) binding, nuclear localization (N), and phosphorylation by protein kinase A (P); the C-terminal half (open box) contains multiple transcription activation domains (Trans. Act.). (Bottom) Comparative structures of v-Rel and c-Rel. The boxes at the ends of v-Rel indicate Env-derived amino acids (2 at the N terminus and 19 at the C terminus). The lines in v-Rel indicate small amino acid differences between v-Rel and turkey c-Rel. A truncated form of c-Rel (c-Rel Δ55 36 ]), missing 55 C-terminal amino acids, is shown at the bottom. In c-Rel and c-Rel ?55, the hatched boxes at the ends indicate sequences not present in v-Rel. To the right are indicated the subcellular locations of these Rel proteins in chicken fibroblasts (N, nuclear; C, cytoplasmic; N/C nuclear and cytoplasmic), the relative strength of transcriptional activation (TRANS. ACT.) of the C-terminal sequences in Rel proteins, and the relative in vitro chicken spleen cell transforming efficiencies (SPLEEN TRANSF.) of the Rel proteins.

Expression of Rel proteins in transformed spleen cell lines. A Western blot (immunoblot) using anti-Rel antibody was performed on chicken spleen cell lines transformed by viruses overexpressing the following Rel proteins: v-Rel, a c-Rel protein with 55 C-terminal amino acids deleted (cRel d55 [ Fig. 1 ; 36 ]), and a v-Rel-c-Rel recombinant virus (V/C) containing the RH domain of v-Rel and C-terminal sequences from c-Rel ( 11 ). Lanes CI, C2, C3. and C4 contain independent spleen cell lines derived from spleen cell colonies transformed by a retroviral vector (JDc–Rel [ 11 ]) for the overexpression of chicken c-Rel. Cell lines CI, C2, and C4 express c-Rel proteins that comigrate with full-length c-Rel; C3 expresses a truncated c-Rel protein. The positions of full-length c-Rel and v-Rel are indicated by arrows.

Correlation between the presence of a C-terminal transcriptional activation domain in v-Rel and transformation of chicken spleen cells. The general structure of v-Rel is shown at the top: ▪ Env-derived amino acids; insert image, RH domain. Three C-terminal domains (A, B, and C) are delimited by restriction enzyme sites (H, HincII; P, PvuII; B, BstXI; E, EcoRI), and the numbers above the structure indicate the amino acids in v-Rel. Other structures represent GAL4 (1–147) fusion proteins that were created at a unique Stul site (S) and have been assayed for their relative abilities to activate transcription from a GAL4-site-containing reporter plasmid in chicken fibroblasts. Each protein contains the v-Rel amino acids indicated above the structure (SG3'Mc-Rel is a GAL4 fusion protein containing C-terminal sequences from mouse c-Rel). Spleen cell transformation data are not for GAL4 fusion proteins but for v-Rel proteins that contain intact N-terminal RH domains and the indicated C-terminal deletions. —, Not determined. See Sarkar and Gilmore ( 63 ) for further details.

A heterologous C-terminal transcriptional activation domain cannot substitute for the transcriptional activation domain of v-Rel. As in Fig. 3 , the structures of v-Rel (top) and relevant GAL4 fusion proteins (middle) are shown. SG3'v/Act contains vector-encoded sequences (ACT) from pBluescript SK + (Trp-Gln-Asp-Arg-Phe-Pro-Asp-Trp-Leu-Ala-Gly-Ser-Glu-Arg-Asn-Ala-Ile-Asn-Val-Ser) fused to v-Rel sequences at the unique HincII (H) site. The relative transcriptional activation data were determined in duplicate cotransfection assays in chicken cells as described for Fig. 3 (see also reference 63 ), and the relative spleen cell transformation data are for intact v-Rel proteins containing the RH domain and the indicated C-terminal sequences. A representative CAT transcriptional activation assay for the indicated GAL4 fusion proteins is shown at the bottom. In this assay, the extent of transcriptional activation is determined by quantitating the amount of radioactivity in the upper spot.

Genetic screen to identify activated target genes important for transformation by v-Rel. This screen employs ts v-Rel mutants that transform chicken spleen cells at 36°C but not at 41°C ( 75 ) and that can induce the formation of small, abortively transformed colonies at an intermediate temperature (e.g., 39°C). cDNAs that can restore full transformation to the ts v-Rel-transformed cells at the intermediate temperature will be selected, their sequences will be determined, and genomic clones for these cDNAs will be isolated. With the genomic clones corresponding to these cDNAs, it can be determined whether complementing genes are direct targets for activation by v-Rel. See text for further details.

C-terminal sequences from a human c-Rel-Nrg translocation fusion protein and from chicken p100 do not activate transcription when fused to GAL4. Plasmids expressing the indicated GAL4 (1–147) fusion proteins, whose structures are shown, were cotransfected with a GAL4-site-containing reporter plasmid into chicken fibroblasts. A CAT assay was performed on cell lysates 48 h later as described previously ( 47 , 48 ). (A) At the top are shown the general structures of human c-Rel and a human c-Rel protein (c-Rel-Nrg) that has arisen because of a chromosomal translocation in a pre-T diffuse large-cell lymphoma that results in the fusion of 166 amino acids encoded by an unrelated gene (nrg) onto c-Rel ( 42 ). In the middle are structures of five GAL4 fusion proteins: SG424 (GAL4 amino acids 1 through 147), SG3′Hc-Rel (GAL4–human c–Rel amino acids 137 through 587 [ 60 ]), SG3′Mc-Rel (GAL4-mouse c-Rel amino acids 265 through 588 [ 8 ]), SG3′Hc-Rel/nrg (GAL4-human c-Rel-Nrg fusion protein [ 42 ]), and SG3′Hc-ReldRV (GAL4-human c-Rel fusion protein deleted at an EcoRV site). The relative abilities of these proteins to activate transcription are indicated at the right, and a representative CAT assay is shown at the bottom (see legend to Fig. 4 ). Continued (B) At the top are the structures of the five GAL4 fusion proteins: SG424 (GAL4 amino acids 1 through 147), SG-3′Chc (GAL4-chicken c-Rel amino acids 323 through 598 [ 47 ]), SG-3′Chpl05 (GAL4-chicken pl05 amino acids 437 through 971 [10]), SG-3′Chpl00 (GAL4-chicken p100 amino acids 397 through 906), and SGChp40 (GAL4-chicken p40 [chicken IκB-α:] amino acids 49 through 318 [ 47 ]). A representative CAT assay is shown at the bottom.

C-terminal sequences from a human c-Rel-Nrg translocation fusion protein and from chicken p100 do not activate transcription when fused to GAL4. Plasmids expressing the indicated GAL4 (1–147) fusion proteins, whose structures are shown, were cotransfected with a GAL4-site-containing reporter plasmid into chicken fibroblasts. A CAT assay was performed on cell lysates 48 h later as described previously ( 47 , 48 ). (A) At the top are shown the general structures of human c-Rel and a human c-Rel protein (c-Rel-Nrg) that has arisen because of a chromosomal translocation in a pre-T diffuse large-cell lymphoma that results in the fusion of 166 amino acids encoded by an unrelated gene (nrg) onto c-Rel ( 42 ). In the middle are structures of five GAL4 fusion proteins: SG424 (GAL4 amino acids 1 through 147), SG3′Hc-Rel (GAL4–human c–Rel amino acids 137 through 587 [ 60 ]), SG3′Mc-Rel (GAL4-mouse c-Rel amino acids 265 through 588 [ 8 ]), SG3′Hc-Rel/nrg (GAL4-human c-Rel-Nrg fusion protein [ 42 ]), and SG3′Hc-ReldRV (GAL4-human c-Rel fusion protein deleted at an EcoRV site). The relative abilities of these proteins to activate transcription are indicated at the right, and a representative CAT assay is shown at the bottom (see legend to Fig. 4 ). Continued (B) At the top are the structures of the five GAL4 fusion proteins: SG424 (GAL4 amino acids 1 through 147), SG-3′Chc (GAL4-chicken c-Rel amino acids 323 through 598 [ 47 ]), SG-3′Chpl05 (GAL4-chicken pl05 amino acids 437 through 971 [10]), SG-3′Chpl00 (GAL4-chicken p100 amino acids 397 through 906), and SGChp40 (GAL4-chicken p40 [chicken IκB-α:] amino acids 49 through 318 [ 47 ]). A representative CAT assay is shown at the bottom.