Nonmuscle Myosin II and Upstream and Downstream Signaling

配体诱导的表皮生长因子受体(EGFR)内化是调节信号转导、细胞动力学和细胞间通讯的重要过程。然而，到目前为止，非肌肉肌球蛋白II(NM II)在EGFR内化中的分子功能还没有得到充分的研究。我们证明，NM II是EGFR内化所必需的，以触发依赖于EGFR的ERK和AKT的激活。质谱学分析表明，EGFR是一种与NM II-B相互作用的蛋白质。这种相互作用既需要NM II的调控轻链20(RLC20)，也需要富含小窝蛋白的膜(CEM)中EGFR的激动域。通过Alex 488-EGF内化、生物素化和125I-EGF标记，非肌肉肌球蛋白重链II(NMHC II)的丢失减弱了EGFR的内化。沉默NMHC II对EGFR内化的调节在COS-7细胞和A431细胞中是不同的，这两种细胞表现出不同的表达模式。抑制或丢失NM II-A或NM II-B都会削弱依赖于EGFR的ERK和AKT的激活。Blebbistatin是NM II ATPase活性的抑制剂，它减少了EGFR的内化以及EGFR依赖的ERK和AKT的激活。在NMHC II siRNA处理的A431细胞中，与野生型NM II不同，用运动受损的NM II-A或II-B恢复NM II的表达不能挽救依赖于EGFR的ERK和AKT的激活。综上所述，这些结果表明，NM II是EGFR及其介导的信号通路内化所必需的。

Ligand-induced internalization of the epidermal growth factor receptor (EGFR) is an important process for regulating signal transduction, cellular dynamics, and cell-cell communication. However, until now, the molecular functions of nonmuscle myosin II (NM II) in the internalization of the EGFR have not been fully explored. We demonstrated that NM II is required for the internalization of the EGFR to trigger the EGFR-dependent activation of ERK and AKT. The EGFR was identified as a protein that interacts with NM II-B as determined by mass spectrometry analysis. This interaction requires both the regulatory light chain 20 (RLC20) of NM II and the kinase domain of the EGFR in a caveolin-enriched membrane (CEM). Loss of nonmuscle myosin heavy chain II (NMHC II) attenuates the internalization of the EGFR as determined by Alex 488-EGF internalization, biotinylation, and 125I-EGF labeling. Regulation of the internalization of the EGFR by silencing of NMHC II paralogs is distinct in COS-7 cells and A431 cells which show different expression patterns of these paralogs. Inhibition or loss of either NM II-A or NM II-B impairs the EGFR-dependent activation of ERK and AKT. Blebbistatin, an inhibitor of NM II ATPase activity, decreases the internalization of the EGFR as well as EGFR-dependent activation of ERK and AKT. In NMHC II siRNA treated A431 cells, restoration of the expression of NM II with motor impaired paralogs of NM II-A or II-B unlike wild type NM II failed to rescue EGFR-dependent activation of ERK and AKT. Taken together, these results suggest NM II is required for the internalization of the EGFR and EGFR-mediated signaling pathways.