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We describe the construction of a v‐rel estrogen receptor fusion protein (v‐relER) which allows the regulation of v‐rel oncoprotein activity by hormone. In the presence of estrogen, v‐relER readily transformed primary chicken fibroblasts and bone marrow cells in vitro. In both cell types, v‐rel‐specific transformation was critically dependent on the presence of estrogen or the estrogen agonist 4‐hydroxytamoxifen (OHT). Withdrawal of estrogen or application of an estrogen antagonist, ICI164,384 (ICI) caused a reversal of the transformed phenotype. We also demonstrate that the v‐relER protein binds to NF‐kappa B sites in an estrogen‐dependent manner, thereby showing that sequence‐specific DNA binding of v‐relER is critical for the activation of its transforming capacity. In transient transfection experiments, we failed to demonstrate a clear repressor or activator function of the v‐rel moiety in v‐relER. However, in v‐relER‐transformed bone marrow cells, estrogen and OHT induced elevated mRNA levels of two cellular genes whose expression is constitutive and high in v‐rel‐transformed cells. These results suggest that v‐rel might exert part of its activity as an activator of rel‐responsive genes.

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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 cyclic-AMP-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 50-kilodalton (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 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-...