Matrix-independent activation of phosphatidylinositol 3-kinase, Stat3, and cyclin A-associated Cdk2 Is essential for anchorage-independent growth of v-Ros-transformed chicken embryo fibroblasts.
Matrix-independent activation of phosphatidylinositol 3-kinase, Stat3, and cyclin A-associated Cdk2 Is essential for anchorage-independent growth of v-Ros-transformed chicken embryo fibroblasts.
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磷脂酰肌醇 3-激酶、Stat3 和细胞周期蛋白 A 相关 Cdk2 的基质依赖性激活对于 v-Ros 转化鸡胚成纤维细胞的贴壁依赖性生长至关重要。
DOI：
10.1074/jbc.m211522200
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Wang,Lu-Hai
中科院分区：
文献类型：
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作者：
Uttamsingh,Shailaja;Zong,CongS;Wang,Lu-Hai
问题仍然是开放的信号转导途径显示，是重要的生长和转化在贴壁培养进行类似的，并发挥类似的作用，为细胞生长在锚定非依赖性条件下。鸡胚成纤维细胞（CEF）感染禽肉瘤病毒UR 2，编码致癌受体蛋白酪氨酸激酶（RPTK）的v-Ros，或与它的两个转化受损的突变体生长在非粘附条件下的甲基纤维素（MC）含培养基中，和信号转导功能必不可少的罗斯诱导锚定非依赖性生长进行了分析。我们发现，在非粘附条件下，v-Ros或两种致癌的非受体蛋白酪氨酸激酶（PTKs），v-Src和v-Yes转化的CEF中细胞蛋白的整体酪氨酸磷酸化显著降低，表明细胞粘附到细胞外基质在这些组成性激活的PTKs的有效底物磷酸化中起着重要作用。在非粘附条件下，与UR 2相比，UR 2转化缺陷型突变体在磷脂酰肌醇3-激酶（PI 3-激酶）和Stat 3的激活中受到不同程度的损害。一致地，组成型激活的PI 3-激酶和Stat 3突变体拯救了UR 2突变体促进锚定非依赖性生长的能力。相反，PI 3-激酶和Stat 3的显性负突变体抑制UR 2诱导的锚定非依赖性生长。在非贴壁条件下生长的UR 2感染的CEF显示出比对照或UR 2 mux感染的细胞更快的细胞周期进程，这似乎与PI 3-激酶依赖性增加细胞周期蛋白A相关的Cdk 2活性相关。用Cdk 2抑制剂处理UR 2感染的细胞导致UR 2的锚定非依赖性生长促进活性的丧失。总之，我们已经通过了一个实验系统，使我们能够研究在锚定非依赖性条件下生长的细胞中的信号传导途径，并确定了PI 3-激酶和Stat 3信号功能的基质非依赖性激活，以及PI 3-激酶依赖性增加细胞周期蛋白A相关的Cdk 2激酶活性，对于Ros-PTK诱导的锚定非依赖性生长至关重要。
The question remains open whether the signaling pathways shown to be important for growth and transformation in adherent cultures proceed similarly and play similar roles for cells grown under anchorage-independent conditions. Chicken embryo fibroblasts (CEF) infected with the avian sarcoma virus UR2, encoding the oncogenic receptor protein-tyrosine kinase (RPTK) v-Ros, or with two of its transformation-impaired mutants were grown in nonadherent conditions in methylcellulose (MC)-containing medium, and the signaling functions essential for Ros-induced anchorage-independent growth were analyzed. We found that the overall tyrosine phosphorylation of cellular proteins in CEF transformed by v-Ros or by two oncogenic nonreceptor protein-tyrosine kinases (PTKs), v-Src and v-Yes, was dramatically reduced in nonadherent conditions compared with that in adherent conditions, indicating that cell adhesion to the extracellular matrix plays an important role in efficient substrate phosphorylation by these constitutively activated PTKs. The UR2 transformation-defective mutants were differentially impaired compared with UR2 in the activation of phosphatidylinositol 3-kinase (PI 3-kinase) and Stat3 in nonadherent conditions. Consistently, the constitutively activated mutants of PI 3-kinase and Stat3 rescued the ability of the UR2 mutants to promote anchorage-independent growth. Conversely, dominant negative mutants of PI 3-kinase and Stat3 inhibited UR2-induced anchorage-independent growth. UR2-infected CEF grown in nonadherent conditions displayed faster cell cycle progression than the control or the UR2 mutant-infected cells, and this appeared to correlate with a PI 3-kinase-dependent increase in cyclin A-associated Cdk2 activity. Treatment of UR2-infected cells with Cdk2 inhibitors led to the loss of the anchorage-independent growth-promoting activity of UR2. In conclusion, we have adopted an experimental system enabling us to study the signaling pathways in cells grown under anchorage-independent conditions and have identified matrix-independent activation of PI 3-kinase and Stat3 signaling functions, as well as the PI 3-kinase-dependent increase of cyclin A-associated Cdk2 kinase activity, to be critical for the Ros-PTK-induced anchorage-independent growth.