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Inhibition of Pyk2 and Src activity improves Cx43 gap junction intercellular communication.
Journal of Molecular and Cellular Cardiology ( IF 5 ) Pub Date : 2020-09-18 , DOI: 10.1016/j.yjmcc.2020.09.004
Li Zheng 1 , Andrew J Trease 1 , Kenichi Katsurada 2 , Gaelle Spagnol 1 , Hanjun Li 1 , Wen Shi 3 , Bin Duan 3 , Kaushik P Patel 2 , Paul L Sorgen 1
Affiliation  

Identification of proteins that interact with Cx43 has been instrumental in the understanding of gap junction (GJ) regulation. An in vitro phosphorylation screen identified that Protein tyrosine kinase 2 beta (Pyk2) phosphorylated purified Cx43CT and this led us to characterize the impact of this phosphorylation on Cx43 function. Mass spectrometry identified Pyk2 phosphorylates Cx43 residues Y247, Y265, Y267, and Y313. Western blot and immunofluorescence staining using HeLaCx43 cells, HEK 293 T cells, and neonatal rat ventricular myocytes (NRVMs) revealed Pyk2 can be activated by Src and active Pyk2 interacts with Cx43 at the plasma membrane. Overexpression of Pyk2 increases Cx43 phosphorylation and knock-down of Pyk2 decreases Cx43 phosphorylation, without affecting the level of active Src. In HeLaCx43 cells treated with PMA to activate Pyk2, a decrease in Cx43 GJ intercellular communication (GJIC) was observed when assayed by dye transfer. Moreover, PMA activation of Pyk2 could be inhibited by the small molecule PF4618433. This partially restored GJIC, and when paired with a Src inhibitor, returned GJIC to the no PMA control-level. The ability of Pyk2 and Src inhibitors to restore Cx43 function in the presence of PMA was also observed in NRVMs. Additionally, an animal model of myocardial infarction induced heart failure showed a higher level of active Pyk2 activity and increased interaction with Cx43 in ventricular myocytes. Src inhibitors have been used to reverse Cx43 remodeling and improve heart function after myocardial infarction; however, they alone could not fully restore proper Cx43 function. Our data suggest that Pyk2 may need to be inhibited, in addition to Src, to further (if not completely) reverse Cx43 remodeling and improve intercellular communication.



中文翻译:

Pyk2 和 Src 活性的抑制改善了 Cx43 间隙连接细胞间通讯。

与 Cx43 相互作用的蛋白质的鉴定有助于理解间隙连接 (GJ) 调节。体外磷酸化筛选确定蛋白酪氨酸激酶 2 β (Pyk2) 磷酸化纯化的 Cx43CT,这使我们能够表征这种磷酸化对 Cx43 功能的影响。质谱鉴定了 Pyk2 磷酸化 Cx43 残基 Y247、Y265、Y267 和 Y313。使用 HeLa Cx43细胞、HEK 293 T 细胞和新生大鼠心室肌细胞 (NRVM) 进行的蛋白质印迹和免疫荧光染色显示 Pyk2 可以被 Src 激活,并且活性 Pyk2 与质膜上的 Cx43 相互作用。Pyk2 的过度表达会增加 Cx43 磷酸化,而 Pyk2 的敲低会降低 Cx43 磷酸化,而不影响活性 Src 的水平。在海拉CX43用 PMA 处理细胞以激活 Pyk2,当通过染料转移测定时观察到 Cx43 GJ 细胞间通讯 (GJIC) 减少。此外,Pyk2 的 PMA 激活可以被小分子 PF4618433 抑制。这部分恢复了 GJIC,当与 Src 抑制剂配对时,将 GJIC 返回到无 PMA 控制水平。在 NRVM 中也观察到 Pyk2 和 Src 抑制剂在 PMA 存在下恢复 Cx43 功能的能力。此外,心肌梗塞诱发心力衰竭的动物模型显示出更高水平的活跃 Pyk2 活性,并增加了与心室肌细胞中 Cx43 的相互作用。Src 抑制剂已被用于逆转 Cx43 重构并改善心肌梗塞后的心脏功能;然而,仅靠它们无法完全恢复正确的 Cx43 功能。

更新日期:2020-09-23
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