Using a protein interaction cloning technique, we identified cDNAs that encode a novel Bcl-2-binding protein, termed BAG-1. The BAG-1 protein shares no significant homology with Bcl-2 or other Bcl-2 family proteins, which can form homo- and heterodimers. In gene transfer experiments using a human lymphoid cell line, Jurkat, coexpression of BAG-1 and Bcl-2 provided markedly increased protection from cell death induced by several stimuli, including staurosporine, anti-Fas antibody, and cytolytic T cells, relative to cells that contained gene transfer-mediated elevations in either BAG-1 or Bcl-2 protein alone. BAG-transfected 3T3 fibroblasts also exhibited prolonged cell survival in response to an apoptotic stimulus. The findings indicate that bag-1 represents a new type of anti-cell death gene and suggest that some routes of apoptosis induction previously ascribed to Bcl-2-independent pathways may instead reflect a need for the combination of Bcl-2 and BAG-1.

Fas is a cell surface receptor that controls a poorly understood signal transduction pathway that leads to cell death by means of apoptosis. A protein tyrosine phosphatase, FAP-1, capable of interacting with the cytosolic domain of Fas, was identified. The carboxyl terminal 15 amino acids of Fas are necessary and sufficient for interaction with FAP-1. FAP-1 expression is highest in tissues and cell lines that are relatively resistant to Fas-mediated cytotoxicity. Gene transfer-mediated elevations in FAP-1 partially abolished Fas-induced apoptosis in a T cell line. These findings are consistent with an inhibitory effect of FAP-1 on Fas signal transduction.

Interactions of the BcI-2 protein with itself and other members of the Bcl-2 family, including Bcl-X-L, Bcl-X-S, Mci-i, and Bax, were explored with a yeast twohybrid system. Fusion proteins were created by linking BcI-2 family proteins to a LexA DNA-binding domain or a B42 trans-activation domain. Protein-protein interactions were examined by expression of these fusion proteins in Saccharomyces cerevisiae having a lacZ ((-galactosidase) gene under control of a LexA-dependent operator. This approach gave evidence for Bcl-2 protein homodimerization. Bcl-2 also interacted with Bcl-X-L and Mcl-i and with the dominant inhibitors Bax and Bcl-X-S. Bd-X-L displayed the same pattern of combinatorial interactions with Bd-2 family proteins as Bc1-2. Use of deletion mutants of Bc-2 suggested that BcI-2 homodimerization involves interactions between two distinct regions within the Bcl-2 protein, since a LexA protein containing Bcl-2 amino acids 83-218 mediated functional interactions with a B42 fusion protein contaiing Bcl-2 amino acids 1-81 but did not complement a B42 fusion protein containing BcI-2 amino acids 83-218. In contrast to LexA/Bcl-2 fusion proteins, expression of a LexA/Bax protein was lethal to yeast. This cytotoxicity could be abrogated by B42 fusion proteins containing BcI-2, Bcl-X-L, or Mci-i but not those containing Bcl-X-S (an alternatively spliced form of Bcl-X that lacks a well-conserved 63-amino acid region). The findings suggest a model whereby Bax and Bcl-X-S differentially regulate Bcd-2 function, and indicate that requirements for Bcl-2/Bax heterodimerization may be different from those for Bcl-2/Bcl-2 homodimerization.The bcl-2 gene becomes dysregulated in a wide variety of human cancers and contributes to neoplastic cell expansion by prolonging cell survival rather than by accelerating rates of cellular proliferation. Specifically, bcl-2 blocks programmed cell death, a physiological process that normally ensures a homeostatic balance between cell production and cell turnover in most tissues with self-renewal capacity and which often involves characteristic changes in cell morphology termed apoptosis. In fact, Bc1-2 can prevent or delay apoptosis induced by a wide variety of stimuli, including growth factor deprivation, alterations in Ca2+, free radicals, cytotoxic lymphokines, some types of viruses, radiation, and most chemotherapeutic drugs, suggesting that this oncoprotein controls a common final pathway involved in cell death regulation (reviewed in refs. 1 and 2).The mechanism by which Bcl-2 prevents cell death remains enigmatic, as the predicted amino acid sequence of the 26-kDa human Bcl-2 protein (239 aa) has no significant homology with other proteins whose biochemical activity is known. Recently, however, Bcl-2 has been shown to interact with a low molecular weight GTPase member of the Ras family, p23-R-Ras (3), and also can be coimmunoprecipitated with the serine/threonine-specific protein kinase Raf-1 (4). Thus, Bcl-2 may somehow regulate a signal transduction pathway involving...