Bax Inhibitor 1 Increases Cell Adhesion through Actin Polymerization: Involvement of Calcium and Actin Binding

Bax inhibitor 1 (BI-1), a transmembrane protein with Ca²⁺ channel-like activity, has antiapoptotic and anticancer activities. Cells overexpressing BI-1 demonstrated increased cell adhesion. Using a proteomics tool, we found that BI-1 interacted with γ-actin via leucines 221 and 225 and could control actin polymerization and cell adhesion. Among BI-1^−/− cells and cells transfected with BI-1 small interfering RNA (siRNA), levels of actin polymerization and cell adhesion were lower than those among BI-1^+/+ cells and cells transfected with nonspecific siRNA. BI-1 acts as a leaky Ca²⁺ channel, but mutations of the actin binding sites (L221A, L225A, and L221A/L225A) did not change intra-endoplasmic reticulum Ca²⁺, although deleting the C-terminal motif (EKDKKKEKK) did. However, store-operated Ca²⁺ entry (SOCE) is activated in cells expressing BI-1 but not in cells expressing actin binding site mutants, even those with the intact C-terminal motif. Consistently, actin polymerization and cell adhesion were inhibited among all the mutant cells. Compared to BI-1^+/+ cells, BI-1^−/− cells inhibited SOCE, actin polymerization, and cell adhesion. Endogenous BI-1 knockdown cells showed a similar pattern. The C-terminal peptide of BI-1 (LMMLILAMNRKDKKKEKK) polymerized actin even after the deletion of four or six charged C-terminal residues. This indicates that the actin binding site containing L221 to D231 of BI-1 is responsible for actin interaction and that the C-terminal motif has only a supporting role. The intact C-terminal peptide also bundled actin and increased cell adhesion. The results of experiments with whole recombinant BI-1 reconstituted in membranes also coincide well with the results obtained with peptides. In summary, BI-1 increased actin polymerization and cell adhesion through Ca²⁺ regulation and actin interaction.In metastasis, tumor cells migrate from primary tumor sites into the lymphatic or circulatory system and then attach to the basal matrix of the target tissue (16). Cell adhesion and migration contribute to the metastatic process. Adhesion assembly and turnover are highly dynamic, coordinated processes essential for cell migration (16, 26). Adhesions serve as traction points for cell translocation and mediate a network of signaling events that regulate protrusion, contractility, and attachment (16, 29, 30). In migrating cells, protrusions are generated by actin polymerization at the front of the cell (22). Actin exists as monomers (G-actin) and polymers (F-actin), which transform into each other, and the transformation has a major contribution to cell physiology and dynamics. In the cell under physiological conditions, both G- and F-actin contain Mg²⁺ at the high-affinity binding site. The actin dynamic state contributes to cancer metastasis environments, including that of increased cell adhesion.The antiapoptotic protein Bax inhibitor 1 (BI-1) was identified through a functional yeast screen designed to select for human cDNAs that inhibit Bax-induced apoptosis (39). BI-1 regulates Ca²⁺ levels in the endoplasmic reticulum (ER) and cytosol (19) via a C-terminal amino acid sequence of EKDKKKEKK. The antiapoptotic function of BI-1 contributes to the development of cancer and resistance to antitumor therapies (12, 14, 17), but the roles of BI-1 in regulating cell adhesion and actin polymerization are unclear. This study examines the role of BI-1 in cell adhesion through Ca²⁺ regulation and actin polymerization.