Abstract
Abstract The BAG family of anti-apoptotic co-chaperones regulates protein folding and aggregation during cellular stress. The C-terminal BAG domain directly binds the ATPase domain of Hsp70/Hsc70 and prevents the release of client proteins that are otherwise directed to the proteasome for degradation. BAG3 is a unique member of the BAG family that also contains an evolutionarily conserved N-terminal WW-domain with undefined function. We removed the BAG3 WW-domain and observed a marked change in cell shape and cell cycle progression. This led us to hypothesize the BAG3 WW-domain is critical in regulating cellular division through a cytoskeletal association in a cell cycle-dependent fashion. Immunoblot analysis of synchronized HeLa (p53-null) and U2OS (human osteosarcoma, wild-type p53) cell lysates revealed that BAG3 expression was significantly increased during S phase and precipitated in the insoluble cell fraction at the G2/M transition. Indirect immunofluorescence demonstrated that BAG3 colocalized with the Golgi apparatus in the perinuclear region throughout interphase, and with leading lamellipodia exclusively during G2 phase. Co-immunoprecipitation of BAG3 with F-actin confirmed association with microfilaments and was increased during G2 phase relative to asynchronous lysates. A GST-hBAG3-WW-domain recombinant protein pulldown confirmed a specific F-actin association, suggesting a unique BAG3-WW-domain interaction with the cytoskeleton. We also generated a series of N-terminal, V5 tag-labeled domain-deletion clones of BAG3 with the WW (ΔWW) domain removed. ΔWW-BAG3 cells contained degenerate F-actin organization, abnormal morphology, and loss of BAG3 in lamellipodia. A 3.2-fold increase in DNA content of ΔWW-BAG3 cells was shown by flow cytometry, relative to empty vector (pCI Neo) and BAG3 overexpressing (FL-BAG3) transfected controls (p<0.001, n=3). Furthermore, centrosome amplification was increased in asynchronous cells: 2.43 ± 0.12/ΔWW-BAG3 cell, 1.86 ± 0.06/pCI Neo cell, and 1.79 ± 0.04/FL-BAG3 (p<0.05, n=4). Cell division in real time was followed using GFP-histone 2B-tagged live cell imaging. We confirmed polyploidy development and absence of cytokinesis in ΔWW-BAG3 cells, and also observed mitotic delay relative to pCI Neo (1260 min. vs. 110 min.) and FL-BAG3 cells (vs. 90 min., n=2). These data indicate a novel requirement for BAG3 for normal mitotic progression via BAG3 WW-domain association with the actin cytoskeleton. To our knowledge, this is the first reported activity of differential co-chaperone protein behavior based on cell cycle phase, independent of p53 status. These data identify a novel regulatory point of G2/M progression and suggest the WW-domain of BAG3 is a potential therapeutic target to deter cancer proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-190. doi:10.1158/1538-7445.AM2011-LB-190
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