Pannexin 3 ER Ca2+ channel gating is regulated by phosphorylation at the Serine 68 residue in osteoblast differentiation
Pannexin 3 ER Ca2+ channel gating is regulated by phosphorylation at the Serine 68 residue in osteoblast differentiation
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DOI：
10.1038/s41598-019-55371-9
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发表时间：
2019-12-10
影响因子：
4.6
通讯作者：
Yamada, Yoshihiko
中科院分区：
文献类型：
--
作者：
Ishikawa, Masaki;Williams, Geneva;Yamada, Yoshihiko
Pannexin 3（Panx 3）是骨形成的调节因子。Panx 3形成三种不同的功能通道：半通道、间隙连接和内质网（ER）Ca 2+通道。然而，Panx 3通道的门控机制仍不清楚。在这里，我们表明，Panx 3 ER Ca 2+通道的丝氨酸68残基（Ser 68）的磷酸化，以促进成骨细胞分化的调制。在鉴定的17个候选磷酸化位点中，Ser 68突变为Ala（Ser 68 Ala）足以通过减少Osterix和ALP表达来抑制Panx 3介导的成骨细胞分化。使用Ser 68磷酸化特异性抗体（P-Panx 3）显示Panx 3在新生儿生长板的前肥大、肥大软骨细胞和骨区域中被磷酸化。在成骨细胞C2 C12中，P-Panx 3定位于ER膜上。重要的是，Ser 68 Ala突变仅影响Panx 3 ER Ca 2+通道功能。Panx 3上的Ser 68在ATP刺激和PI 3 K/Akt信号转导作用下磷酸化。最后，实时FRET成像和比率分析显示，Panx 3通道构象对ATP敏感。总之，Panx 3在Ser 68处的磷酸化是控制Panx 3 ER Ca 2+通道的门控以促进骨生成的重要步骤。
Pannexin 3 (Panx3) is a regulator of bone formation. Panx3 forms three distinct functional channels: hemichannels, gap junctions, and endoplasmic reticulum (ER) Ca2+ channels. However, the gating mechanisms of the Panx3 channels remain unclear. Here, we show that the Panx3 ER Ca2+ channel is modulated by phosphorylation of the serine 68 residue (Ser68) to promote osteoblast differentiation. Among the 17 candidate phosphorylation sites identified, the mutation of Ser68 to Ala (Ser68Ala) was sufficient to inhibit Panx3-mediated osteoblast differentiation via reduction of Osterix and ALP expression. Using a Ser68 phospho-specific antibody (P-Panx3) revealed Panx3 was phosphorylated in prehypertrophic, hypertrophic chondrocytes, and bone areas of the newborn growth plate. In osteogenic C2C12 cells, P-Panx3 was located on the ER membranes. Importantly, the Ser68Ala mutation only affected Panx3 ER Ca2+ channel function. Ser68 on Panx3 was phosphorylated by ATP stimulation and PI3K/Akt signaling. Finally, real-time FRET imaging and ratio analysis revealed that the Panx3 channel conformation was sensitive to ATP. Together, the phosphorylation of Panx3 at Ser68 is an essential step controlling the gating of the Panx3 ER Ca2+ channel to promote osteogenesis.