Cancer Letters

Cancer Letters

Volume 188, Issues 1–2, 15 December 2002, Pages 25-32
Cancer Letters

Mini-review
What's in the ‘BAG’? – a functional domain analysis of the BAG-family proteins

https://doi.org/10.1016/S0304-3835(02)00456-1Get rights and content

Abstract

Bcl-2-associated athanogene (BAG)-family proteins are BAG domain-containing proteins that interact with the heat shock proteins 70, both constitutive Hsc70 and inducible Hsp70. BAG-family proteins bind through the BAG domain to the ATPase domain of Hsc70/Hsp70. The BAG domain, approximately 110 amino acids in length, is a conserved region at the carboxyl terminus and consists of three anti-parallel α helices based on X-ray crystallography and NMR studies. The second and third α-helices of the BAG domain interact with the ATP-binding pocket of Hsc70/Hsp70. Currently, six human BAG proteins have been reported, four of which have been shown to functionally bind Hsc70/Hsp70. BAG-family proteins regulate chaperone protein activities through their interaction with Hsc70/Hsp70. Over-expression of BAG-family proteins is found in several cancers and has been demonstrated in the laboratory to enhance cell survival and proliferation. The anti-apoptotic activities of BAG-family proteins may be dependent on their interactions with Hsc70/Hsp70 and/or binding to Bcl-2. Both BAG-1 and BAG-3/CAIR-1 interact with Bcl-2 and have been shown to have a supra-additive anti-apoptotic effect with Bcl-2. Several N-terminal domains or motifs have been identified in BAG-family proteins as well. These domains enable BAG-family proteins to partner with other proteins and potentially alter the activity of those target proteins by recruiting Hsc70/Hsp70. BAG-family proteins participate in a wide variety of cellular processes including cell survival (stress response), proliferation, migration and apoptosis.

Introduction

Bcl-2-associated athanogene (BAG)-family proteins were originally identified by their ability to associate with the anti-apoptotic protein, Bcl-2 [1], [2]. BAG-family proteins were also found to interact with heat shock proteins 70 (Hsc70/Hsp70) and can modulate, either positively or negatively, the functions of these chaperone proteins. Therefore, BAG-family proteins are characterized as co-chaperones [3], [4]. Currently, six human BAG proteins have been reported. However, only four of them (BAG-1, -3, -4 and -6) have been confirmed in vivo and shown to interact with Hsc70/Hsp70. BAG-family proteins contain a single BAG domain, except for BAG-5 which has four BAG repeats (Fig. 1). The BAG domain is a conserved region located at the C-terminus of the BAG-family proteins that binds the ATPase domain of Hsc70/Hsp70 [3], [4]. The BAG domain is evolutionarily conserved, and BAG domain containing proteins have been described and/or proven in a variety of organisms including mice, Xenopus, Drosophila, Bombyx mori (silk worm), Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Arabidopsis thaliana. [4], [5], [6], [7], [8].

Human BAG-1, the founding member of this family, was initially discovered through a screen for Bcl-2 binding proteins. The BAG-1 gene encodes four isoforms of the BAG-1 proteins, expressed through alternative translation initiation sites (Table 1). BAG-1L (52 kDa) starts at a non-canonical CUG codon upstream of the AUG-driven mRNA transcript. Two in-frame downstream AUG codons give rise to BAG-1M (46 kDa) and BAG-1 (34 kDa), while BAG-1S (29 kDa) is believed to be generated by post-translational modification. BAG-1L is located primarily in the nucleus, while BAG-1M, -1, and -1S proteins are found mainly in the cytoplasm. BAG-1 and BAG-1M were occasionally found in the nucleus depending on the cell type and whether the cells were exposed to stress conditions [9], [10].

BAG-3, a 74 kDa cytoplasmic protein, was identified by three different groups using three different approaches. BAG-3 was identified through a screen for ATPase domain-interacting proteins using a yeast two-hybrid system. The ATPase domain of Hsc70/Hsp70 was used as bait to pull down Hsc70/Hsp70 interacting proteins [4]. Bcl-2 interacting death suppressor (Bis) was discovered when a second group used a cDNA library to screen for Bcl-2 binding proteins [2]. In contrast, CAIR-1 (CAI-stress-1) was identified by subtraction hybridization between cells conditioned to grow in otherwise growth limiting concentrations of a calcium influx inhibitor, CAI [11].

BAG-4 was also identified through a yeast two-hybrid screen for proteins that interact with the ATPase domain of Hsc70/Hsp70 [4]. BAG-4 was also identified as silencer of death domains (SODD) [8]. It is a 60 kDa cytosolic protein that interacts with the death domain of tissue necrosis factor receptor-1 (TNF-R1) [12], [13]. Recent data indicates that the TNF-R1 death domain contains an ATPase domain, and both Hsc70/Hsp70 and TNF-R1 interact with the BAG domain of BAG-4/SODD [14]. BAG-6, known as Scythe or HLA-B-associated transcript-3 (BAT-3), was initially purified from Xenopus egg extracts and has been shown to interact in the nucleus with the Drosophila apoptosis-inducing factor, reaper [5], [15]. hBAG-6, which resides in the nucleus, has been confirmed to interact with Hsc70/Hsp70 through the BAG domain [16], [17]. BAG-family proteins may play roles in both the cytoplasm and the nucleus as indicated by their localization and their partner proteins (Table 1).

Section snippets

The BAG domain

The BAG domain of BAG-family proteins was first demonstrated to interact with Hsc70/Hsp70 using glutathione-S-transferase (GST) fusion proteins containing the BAG domain of BAG-1 [4]. The BAG domain was initially described as a conserved region of about 50 amino acids at the C-terminus of the BAG-1 protein [4]. However, recent studies using X-ray crystallography, multidimensional nuclear magnetic resonance (NMR), and limited proteolysis indicate that the BAG domain has 110–124 amino acids [7],

BAG domain function

The BAG domain has been shown to contribute to the anti-apoptotic activity of BAG-family proteins. BAG-1 has been demonstrated to enhance cell resistance to apoptotic stimuli [1]. Although the detailed mechanism remains incompletely understood, some pathway dissection has been done. BAG-1 binds to the serine/threonine kinase Raf-1 or Hsc70/Hsp70 in a mutually exclusive interaction. BAG-1 promotes cell growth by binding to and stimulating Raf-1 activity [24]. The activated Raf-1 turns on its

Additional domains

BAG-family proteins also interact with other proteins through their N-terminal domains or motifs. All four BAG-1 isoforms and BAG-6/Scythe have an ubiquitin-like (UBL) domain at their amino-terminus (Fig. 1). BAG-1 and BAG-1M have been demonstrated to co-immunoprecipitate with the 20S core and the 19S subunit of the proteasome [27]. However, no function for this interaction has been demonstrated. The BAG-1 proteins may serve as adapter molecules linking the proteasome to the Hsp70 chaperone

BAG-family proteins and cancers

BAG-family proteins express differentially in a variety of human cancers and tumor cell lines, notably leukemias and breast, prostate, and colon cancers [10], [28], [47], [48], [49], [50]. BAG-family proteins have been shown to regulate cell growth and block apoptosis [10], [25]. Over-expression of either BAG-1 or BAG-1L can enhance survival of serum-deprived breast cancer cells. Serum-deprived BAG-1 or BAG-1L stably transfected ZR-75-1 cells remained viable (100%), while 80% of the neo control

Conclusions

In summary, BAG-family proteins may be related to tumorgenecity and can inhibit apoptosis through several mechanisms, most of which require a functional BAG domain. This protein family presents a series of provocative biochemical and cellular events ripe for targeting for novel molecular therapeutics.

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