Phosphorylation of Bok at Ser-8 blocks its ability to suppress IP₃R-mediated calcium mobilization

Bok is a poorly characterized Bcl-2 protein family member with roles yet to be clearly defined. It is clear, however, that Bok binds strongly to inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs), which govern the mobilization of Ca²⁺ from the endoplasmic reticulum, a signaling pathway required for many cellular processes. Also known is that Bok has a highly conserved phosphorylation site for cAMP-dependent protein kinase at serine-8 (Ser-8). Whether Bok, or phosphorylated Bok, has any direct impact on the Ca²⁺ mobilizing function of IP₃Rs remains to be established.

Bok Ser-8 phosphorylation was characterized using purified proteins, G-protein coupled receptor agonists that increase cAMP levels in intact cells, mass spectrometry, and immunoreactivity changes. Also, using mammalian cells that exclusively or predominately express IP₃R1, to which Bok binds strongly, and a fluorescent Ca²⁺-sensitive dye or a genetically-encoded Ca²⁺ sensor, we explored how endogenous and exogenous Bok controls the Ca²⁺ mobilizing function of IP₃R1, and whether Bok phosphorylation at Ser-8, or replacement of Ser-8 with a phosphomimetic amino acid, is regulatory.

Our results confirm that Ser-8 of Bok is phosphorylated by cAMP-dependent protein kinase, and remarkably that phosphorylation can be detected with Bok specific antibodies. Also, we find that Bok has suppressive effects on IP₃R-mediated Ca²⁺ mobilization in a variety of cell types. Specifically, Bok accelerated the post-maximal decline in G-protein coupled receptor-induced cytosolic Ca²⁺ concentration, via a mechanism that involves suppression of IP₃R-dependent Ca²⁺ release from the endoplasmic reticulum. These effects were dependent on the Bok-IP₃R interaction, as they are only seen with IP₃Rs that can bind Bok (e.g., IP₃R1). Surprisingly, Bok phosphorylation at Ser-8 weakened the interaction between Bok and IP₃R1 and reversed the ability of Bok to suppress IP₃R1-mediated Ca²⁺ mobilization.

For the first time, Bok was shown to directly suppress IP₃R1 activity, which was reversed by Ser-8 phosphorylation. We hypothesize that this suppression of IP₃R1 activity is due to Bok regulation of the conformational changes in IP₃R1 that mediate channel opening. This study provides new insights on the role of Bok, its interaction with IP₃Rs, and the impact it has on IP₃R-mediated Ca²⁺ mobilization.