Department of Physiology and Cell Biology, University of Nevada, Reno, School of Medicine, Reno, NV, 89557, USA.
Department of Internal Medicine, Gangneung Asan Hospital, University of Ulsan College of Medicine, Gangneung, Republic of Korea.
J Physiol. 2018 Jun;596(11):2131-2146. doi: 10.1113/JP275406. Epub 2018 Apr 6.
Activation of focal adhesion kinase (FAK) by integrin signalling facilitates smooth muscle contraction by transmitting the force generated by myofilament activation to the extracellular matrix and throughout the smooth muscle tissue. Here we report that electrical field stimulation (EFS) of cholinergic motor neurons activates FAK in gastric fundus smooth muscles, and that FAK activation by EFS is atropine-sensitive but nicardipine-insensitive. PDBu and calyculin A contracted gastric fundus muscles Ca -independently and also activated FAK. Inhibition of FAK activation inhibits the contractile responses evoked by EFS, and inhibits CPI-17 phosphorylation at T38. This study indicates that mechanical force or tension is sufficient to activate FAK, and that FAK appears to be involved in the activation of the protein kinase C-CPI-17 Ca sensitization pathway in gastric fundus smooth muscles. These results reveal a novel role for FAK in gastric fundus smooth muscle contraction by facilitating CPI-17 phosphorylation.
Smooth muscle contraction involves regulating myosin light chain phosphorylation and dephosphorylation by myosin light chain kinase and myosin light chain phosphatase. C-kinase potentiated protein phosphatase-1 inhibitor of 17 kDa (CPI-17) and myosin phosphatase targeting subunit of myosin light-chain phosphatase (MYPT1) are crucial for regulating gastrointestinal smooth muscle contraction by inhibiting myosin light chain phosphatase. Integrin signalling involves the dynamic recruitment of several proteins, including focal adhesion kinase (FAK), to focal adhesions. FAK tyrosine kinase activation is involved in cell adhesion to the extracellular matrix via integrin signalling. FAK participates in linking the force generated by myofilament activation to the extracellular matrix and throughout the smooth muscle tissue. Here, we show that cholinergic stimulation activates FAK in gastric fundus smooth muscles. Electrical field stimulation in the presence of N -nitro-l-arginine methyl ester and MRS2500 contracted gastric fundus smooth muscle strips and increased FAK Y397 phosphorylation (pY397). Atropine blocked the contractions and prevented the increase in pY397. The FAK inhibitor PF-431396 inhibited the contractions and the increase in pY397. PF-431396 also inhibited the electrical field stimulation-induced increase in CPI-17 T38 phosphorylation, and reduced MYPT1 T696 and T853, and myosin light chain S19 phosphorylation. Ca influx was unaffected by PF-431396. Nicardipine inhibited the contractions but had no effect on the increase in pY397. Phorbol 12,13-dibutyrate or calyculin A contracted gastric fundus smooth muscle strips Ca independently and increased pY397. Our findings suggest that FAK is activated by mechanical forces during contraction and reveal a novel role of FAK in the regulation of CPI-17 phosphorylation.
整合素信号激活粘着斑激酶（FAK），通过将肌球蛋白丝激活产生的力传递到细胞外基质并传递到整个平滑肌组织，从而促进平滑肌收缩。在这里，我们报告电刺激（EFS）胆碱能运动神经元可激活胃底平滑肌中的 FAK，并且 EFS 诱导的 FAK 激活对阿托品敏感但对尼卡地平不敏感。PDBu 和 calyculin A 使胃底平滑肌不依赖 Ca2+收缩，并激活 FAK。FAK 激活的抑制抑制了由 EFS 引起的收缩反应，并抑制了 CPI-17 在 T38 的磷酸化。本研究表明，机械力或张力足以激活 FAK，并且 FAK 似乎参与了胃底平滑肌中蛋白激酶 C-CPI-17 Ca 敏化途径的激活。这些结果揭示了 FAK 在胃底平滑肌收缩中的新作用，通过促进 CPI-17 的磷酸化。
平滑肌收缩涉及通过肌球蛋白轻链激酶和肌球蛋白轻链磷酸酶调节肌球蛋白轻链磷酸化和去磷酸化。C-激酶增强的蛋白磷酸酶-17 kDa 抑制剂（CPI-17）和肌球蛋白轻链磷酸酶靶向亚单位的肌球蛋白磷酸酶（MYPT1）对于通过抑制肌球蛋白轻链磷酸酶来调节胃肠道平滑肌收缩至关重要。整合素信号涉及包括粘着斑激酶（FAK）在内的几种蛋白质的动态募集。FAK 酪氨酸激酶的激活涉及通过整合素信号将细胞附着到细胞外基质。FAK 参与将肌球蛋白丝激活产生的力传递到细胞外基质并传递到整个平滑肌组织。在这里，我们显示胆碱能刺激可激活胃底平滑肌中的 FAK。在 N-硝基-L-精氨酸甲酯和 MRS2500 的存在下进行的电场刺激收缩了胃底平滑肌条并增加了 FAK Y397 磷酸化（pY397）。阿托品阻断收缩并阻止 pY397 的增加。FAK 抑制剂 PF-431396 抑制收缩和 pY397 的增加。PF-431396 还抑制了电场刺激诱导的 CPI-17 T38 磷酸化的增加，并减少了 MYPT1 T696 和 T853 以及肌球蛋白轻链 S19 的磷酸化。Ca2+内流不受 PF-431396 的影响。尼卡地平抑制收缩，但对 pY397 的增加没有影响。佛波醇 12,13-二丁酸酯或 calyculin A 独立地使胃底平滑肌条 Ca2+收缩并增加 pY397。我们的发现表明 FAK 在收缩过程中被机械力激活，并揭示了 FAK 在调节 CPI-17 磷酸化中的新作用。
