The New Role of BOK Protein in Calcium Signaling Regulation

Background Introduction

BOK (Bcl-2-related ovarian killer) is a member of the Bcl-2 protein family and has long been thought to play a role in cell apoptosis. However, recent studies suggest that BOK may also have non-apoptotic functions, particularly in the regulation of calcium ion (Ca²⁺) signaling. Calcium ions are important secondary messengers within cells, involved in various cellular processes, including cell proliferation, differentiation, and apoptosis. The endoplasmic reticulum (ER) is the primary intracellular calcium storage site, and the IP3 receptor (IP3R) is a key channel regulating calcium release from the ER. The binding of BOK to IP3R has been confirmed, but its direct impact on IP3R function remains unclear. Additionally, the serine-8 (Ser-8) site of BOK is a highly conserved phosphorylation site, but its functional significance has not been fully explored.

The goal of this study is to investigate the regulatory role of BOK and its phosphorylation on IP3R-mediated calcium release, particularly how phosphorylation at the Ser-8 site affects the interaction between BOK and IP3R and its influence on calcium signaling.

Source of the Paper

This paper was co-authored by Caden G. Bonzerato, Katherine R. Keller, and Richard J. H. Wojcikiewicz, all from the Department of Pharmacology at SUNY Upstate Medical University. The paper was published in 2025 in the journal Cell Communication and Signaling, titled Phosphorylation of BOK at Ser-8 blocks its ability to suppress IP3R-mediated calcium mobilization.

Research Process and Results

1. Phosphorylation of BOK

Experimental Process

• In vitro phosphorylation experiments: Researchers used bacterially expressed His-SUMO-tagged BOK protein (HS-BOKδTM) and catalyzed phosphorylation reactions in vitro using cAMP-dependent protein kinase (PKA). Phosphorylation at the Ser-8 site was confirmed by mass spectrometry (MS).
• In vivo phosphorylation experiments: In αT3 cells, researchers treated cells with PKA activators (e.g., forskolin) and phosphatase inhibitors (e.g., calyculin A). Phosphorylation of BOK was detected using Western blot and co-immunoprecipitation (Co-IP) techniques.

Results

• In vitro experiments: Mass spectrometry confirmed that the Ser-8 site of BOK was phosphorylated by PKA, and phosphorylation significantly weakened BOK’s binding ability to IP3R1.
• In vivo experiments: In αT3 cells, PKA activators significantly increased the phosphorylation level of BOK, and phosphorylated BOK showed reduced binding to IP3R1.

2. Regulation of IP3R1-Mediated Calcium Release by BOK

Experimental Process

• Calcium concentration measurement: Researchers used fluorescent calcium dyes (e.g., Calcium 6) and genetically encoded calcium sensors (e.g., R-CEPIA1er) to measure intracellular calcium concentration ([Ca²⁺]c) and ER calcium concentration ([Ca²⁺]er).
• Cell models: Various cell models were used, including HEK-3KO cells (lacking all IP3R isoforms) and HEK-IP3R1 cells (expressing only IP3R1), as well as αT3 cells and SH-SY5Y cells (primarily expressing IP3R1).

Results

• Inhibitory effect of BOK on calcium release: In HEK-IP3R1 cells, BOK significantly accelerated the decline in [Ca²⁺]c following G protein-coupled receptor (GPCR)-induced calcium release. This effect depended on the binding of BOK to IP3R1, as no similar effect was observed in IP3R mutants that do not bind BOK.
• Regulation of BOK function by phosphorylation: When the Ser-8 site of BOK was phosphorylated or mutated to a phosphomimetic (S8E), the inhibitory effect of BOK on IP3R1-mediated calcium release was reversed.

3. Mechanism of BOK and IP3R1 Interaction

Experimental Process

• Co-immunoprecipitation experiments: Researchers used Co-IP to detect the binding ability of BOK to IP3R1 and analyzed the impact of phosphorylation on this binding.
• ER calcium leak experiments: Thapsigargin (TG) was used to inhibit the ER calcium pump (SERCA), and the rate of calcium leakage was measured.

Results

• Phosphorylation weakens BOK-IP3R1 binding: Phosphorylated BOK or the S8E mutant showed significantly reduced binding to IP3R1, indicating that Ser-8 phosphorylation regulates calcium release by weakening the interaction between BOK and IP3R1.
• BOK inhibits ER calcium leakage: BOK significantly suppressed IP3R1-dependent ER calcium leakage, while phosphorylated BOK or the S8E mutant lost this inhibitory effect.

Conclusions and Significance

This study is the first to reveal that BOK regulates IP3R1-mediated calcium release through phosphorylation at its Ser-8 site. Specifically, BOK inhibits calcium release by binding to IP3R1, while Ser-8 phosphorylation weakens this inhibitory effect. This discovery not only expands the functional role of BOK in calcium signaling regulation but also provides new insights into the non-apoptotic roles of Bcl-2 family proteins.

Research Highlights

1. New function of BOK: For the first time, BOK has been shown to influence calcium release by regulating IP3R1 activity, revealing its non-apoptotic role in calcium signaling.
2. Regulatory mechanism of phosphorylation: Ser-8 phosphorylation reverses the inhibitory effect of BOK on calcium release by weakening its binding to IP3R1.
3. Diversity of cell models: The use of multiple cell models ensures the broad applicability of the findings.

Application Value

The findings of this study provide new targets for developing therapeutic strategies for diseases related to calcium signaling dysregulation. For example, modulating the phosphorylation state of BOK may help treat diseases associated with calcium signaling disorders, such as neurodegenerative and cardiovascular diseases.

Other Valuable Information

• Technological innovation: The high-sensitivity mass spectrometry and genetically encoded calcium sensors used in this study provide powerful tools for future calcium signaling research.
• Interdisciplinary collaboration: This study combines techniques from molecular biology, cell biology, and biochemistry, demonstrating the importance of interdisciplinary research in addressing complex biological questions.