Li Jia, Wang Ruping, Tang Dale D
Department of Molecular and Cellular Physiology, Albany Medical College, 47 New Scotland Avenue, MC-8, Albany, New York, USA.
Respir Res. 2016 Jul 25;17(1):91. doi: 10.1186/s12931-016-0415-7.
The intermediate filament protein vimentin undergoes reversible phosphorylation and dephosphorylation at Ser-56, which plays an important role in regulating the contraction-relaxation cycles of smooth muscle. The protein phosphatases that mediate vimentin dephosphorylation in smooth muscle have not been previously investigated.
The associations of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) with vimentin in mouse tracheal rings was evaluated by co-immunoprecipitation. Lentivirus-mediated shRNA against PP1 was used to assess the role of PP1 in vimentin dephosphorylation and the vimentin-associated process in smooth muscle.
Co-immunoprecipitation analysis showed that vimentin interacted with PP1, but barely with PP2A, in airway smooth muscle. Knockdown of PP1 by lentivirus-mediated shRNA increased the acetylcholine-induced vimentin phosphorylation and smooth muscle contraction. Because vimentin phosphorylation is able to modulate p130 Crk-associated substrate (p130CAS) and actin polymerization, we also evaluated the role of PP1 in the biological processes. Silencing of PP1 also enhanced the agonist-induced the dissociation of p130CAS from vimentin and F/G-actin ratios (an index of actin polymerization). However, PP1 knockdown did not affect c-Abl tyrosine phosphorylation, an important molecule that controls actin dynamics.
Taken together, these findings suggest that PP1 is a key protein serine/threonine phosphatase that controls vimentin Ser-56 dephosphorylation in smooth muscle. PP1 regulates actin polymerization by modulating the dissociation of p130CAS from vimentin, but not by affecting c-Abl tyrosine kinase.
中间丝蛋白波形蛋白在丝氨酸-56处发生可逆的磷酸化和去磷酸化，这在调节平滑肌的收缩-舒张周期中起重要作用。此前尚未对介导平滑肌中波形蛋白去磷酸化的蛋白磷酸酶进行研究。
通过免疫共沉淀评估蛋白磷酸酶1（PP1）和蛋白磷酸酶2A（PP2A）与小鼠气管环中波形蛋白的关联。使用慢病毒介导的针对PP1的短发夹RNA（shRNA）来评估PP1在波形蛋白去磷酸化和平滑肌中与波形蛋白相关过程中的作用。
免疫共沉淀分析表明，在气道平滑肌中，波形蛋白与PP1相互作用，但与PP2A几乎不相互作用。慢病毒介导的shRNA敲低PP1可增加乙酰胆碱诱导的波形蛋白磷酸化和平滑肌收缩。由于波形蛋白磷酸化能够调节p130 Crk相关底物（p130CAS）和肌动蛋白聚合，我们还评估了PP1在这些生物学过程中的作用。沉默PP1还增强了激动剂诱导的p130CAS与波形蛋白的解离以及F/G-肌动蛋白比率（肌动蛋白聚合的指标）。然而，敲低PP1并不影响c-Abl酪氨酸磷酸化，c-Abl是控制肌动蛋白动力学的重要分子。
综上所述，这些发现表明PP1是一种关键的蛋白丝氨酸/苏氨酸磷酸酶，可控制平滑肌中波形蛋白丝氨酸-56的去磷酸化。PP1通过调节p130CAS与波形蛋白的解离来调节肌动蛋白聚合，而不是通过影响c-Abl酪氨酸激酶。
