Craig David H, Haimovich Beatrice, Basson Marc D
Department of Surgery, John D Dingell Veterans Affairs Medical Center, Wayne State University, and Karmanos Cancer Institute, Detroit, MI, USA.
Am J Physiol Cell Physiol. 2007 Dec;293(6):C1862-74. doi: 10.1152/ajpcell.00118.2007. Epub 2007 Sep 26.
Physical forces including pressure, strain, and shear can be converted into intracellular signals that regulate diverse aspects of cell biology. Exposure to increased extracellular pressure stimulates colon cancer cell adhesion by a beta(1)-integrin-dependent mechanism that requires an intact cytoskeleton and activation of focal adhesion kinase (FAK) and Src. alpha-Actinin facilitates focal adhesion formation and physically links integrin-associated focal adhesion complexes with the cytoskeleton. We therefore hypothesized that alpha-actinin may be necessary for the mechanical response pathway that mediates pressure-stimulated cell adhesion. We reduced alpha-actinin-1 and alpha-actinin-4 expression with isoform-specific small interfering (si)RNA. Silencing of alpha-actinin-1, but not alpha-actinin-4, blocked pressure-stimulated cell adhesion in human SW620, HT-29, and Caco-2 colon cancer cell lines. Cell exposure to increased extracellular pressure stimulated alpha-actinin-1 tyrosine phosphorylation and alpha-actinin-1 interaction with FAK and/or Src, and enhanced FAK phosphorylation at residues Y397 and Y576. The requirement for alpha-actinin-1 phosphorylation in the pressure response was investigated by expressing the alpha-actinin-1 tyrosine phosphorylation mutant Y12F in the colon cancer cells. Expression of Y12F blocked pressure-mediated adhesion and inhibited the pressure-induced association of alpha-actinin-1 with FAK and Src, as well as FAK activation. Furthermore, siRNA-mediated reduction of alpha-actinin-1 eliminated the pressure-induced association of alpha-actinin-1 and Src with beta(1)-integrin receptor, as well as FAK-Src complex formation. These results suggest that alpha-actinin-1 phosphorylation at Y12 plays a crucial role in pressure-activated cell adhesion and mechanotransduction by facilitating Src recruitment to beta(1)-integrin, and consequently the association of FAK with Src, to enhance FAK phosphorylation.
包括压力、应变和剪切力在内的物理力可转化为细胞内信号，从而调节细胞生物学的各个方面。细胞外压力增加会通过一种β(1)整合素依赖性机制刺激结肠癌细胞黏附，该机制需要完整的细胞骨架以及粘着斑激酶（FAK）和Src的激活。α-辅肌动蛋白促进粘着斑形成，并将整合素相关的粘着斑复合物与细胞骨架物理连接起来。因此，我们推测α-辅肌动蛋白可能是介导压力刺激细胞黏附的机械反应途径所必需的。我们用亚型特异性小干扰（si）RNA降低了α-辅肌动蛋白-1和α-辅肌动蛋白-4的表达。沉默α-辅肌动蛋白-1而非α-辅肌动蛋白-4可阻断人SW620、HT-29和Caco-2结肠癌细胞系中压力刺激的细胞黏附。细胞暴露于增加的细胞外压力会刺激α-辅肌动蛋白-1酪氨酸磷酸化以及α-辅肌动蛋白-1与FAK和/或Src的相互作用，并增强FAK在Y397和Y576位点的磷酸化。通过在结肠癌细胞中表达α-辅肌动蛋白-1酪氨酸磷酸化突变体Y12F，研究了压力反应中α-辅肌动蛋白-1磷酸化的需求。Y12F的表达阻断了压力介导的黏附，并抑制了压力诱导的α-辅肌动蛋白-1与FAK和Src的结合以及FAK激活。此外，siRNA介导的α-辅肌动蛋白-1减少消除了压力诱导的α-辅肌动蛋白-1和Src与β(1)整合素受体的结合以及FAK-Src复合物的形成。这些结果表明，Y12位点的α-辅肌动蛋白-1磷酸化通过促进Src募集到β(1)整合素，进而促进FAK与Src的结合，增强FAK磷酸化，在压力激活的细胞黏附和机械转导中起关键作用。
