Actin filament assembly and actin-myosin contractility are necessary for anchorage- and EGF-dependent activation of phospholipase Cgamma.

Suzuki K; Takahashi K

摘要

Formation of actin stress fibers and the focal adhesion complex between cell and the substratum are crucial for nonmalignant cells to achieve anchorage-dependent growth. We show here that the adhesion complex formed in normal human mammary epithelial (HME) cells which adhered to type IV collagen, involved the EGF receptor (EGFR) and phospholipase Cgamma (PLCgamma) as signaling molecules, in addition to integrin beta1, alpha-actinin, and actin even before stimulation of the cells with EGF. Stimulation of cells with EGF induced tyrosine phosphorylation of EGFR and activation of PLCgamma, as assessed by the production of a second messenger diacylglycerol (DAG), without any significant increase in the amount of EGFR-bound PLCgamma. Disruption of either actin filaments by cytochalasin D (CD) or actin-myosin contractility by ML-7, an inhibitor of myosin light chain kinase (MLCK), altered the flattened morphology of quiescent cells to a retracted one, without affecting the association between EGFR and PLCgamma. Stimulation of CD- or ML-7-treated cells with EGF failed to inhibit tyrosine phosphorylation of EGFR and its association and colocalization with PLCgamma, but inhibited the PLCgamma activation. Phosphatidylinositol 4,5-bisphosphate (PtdInsP2), substrate of PLCgamma, was tightly associated with alpha-actinin and the content of alpha-actinin-bound PtdInsP2 was reduced by treatment of cells with ML-7 but not with CD. The amount of PtdInsP2 bound to alpha-actinin was increased by the addition of EGF and this EGF-induced increase was blocked by either CD or ML-7. The present results suggest that anchorage-dependent EGF signaling in HME cells may require both actin filament assembly and actin-myosin contractility for the PLCgamma activation. Copyright 2001 Wiley-Liss, Inc.

机译：压力的形成肌动蛋白纤维和焦点细胞之间的粘附复杂和基础非恶性的细胞来实现的关键吗anchorage-dependent增长。粘附复杂形成正常的人类乳腺上皮细胞(HME)坚持IV型胶原蛋白,包括表皮生长因子受体(EGFR)和磷脂酶Cgamma (PLCgamma)信号分子,除了整合素beta1,alpha-actinin和肌动蛋白刺激之前与表皮生长因子的细胞。EGF诱导表皮生长因子受体酪氨酸磷酸化和激活PLCgamma,评估的第二信使甘油二酯的生产(DAG),没有任何显著增加数量的EGFR-bound PLCgamma。肌动蛋白丝通过细胞松弛素D (CD)或ML-7 actin-myosin收缩性的抑制剂肌球蛋白轻链的激酶(MLCK),改变了扁平的静止细胞的形态收回,而不影响协会表皮生长因子受体和PLCgamma之间。ML-7-treated细胞EGF未能抑制表皮生长因子受体及其酪氨酸磷酸化协会和colocalization PLCgamma,但是抑制了PLCgamma激活。磷脂酰肌醇4,5-bisphosphate (PtdInsP2),基质PLCgamma紧密相关alpha-actinin和内容alpha-actinin-bound PtdInsP2降低了细胞治疗与ML-7但不是CD。数量的PtdInsP2绑定到alpha-actinin增加了添加EGF和这个EGF-induced被CD或增加ML-7。anchorage-dependent EGF在HME细胞信号可能需要两个肌动蛋白丝装配和PLCgamma actin-myosin收缩性激活。

Actin filament assembly and actin-myosin contractility are necessary for anchorage- and EGF-dependent activation of phospholipase Cgamma.

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