Yip Shu-Chin, El-Sibai Mirvat, Hill Karen M, Wu Haiyan, Fu Zheng, Condeelis John S, Backer Jonathan M
Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Cell Motil Cytoskeleton. 2004 Nov;59(3):180-8. doi: 10.1002/cm.20032.
Phosphoinositide 3-kinase (PI 3-kinase) activity is required for growth factor-induced cytoskeletal regulation and cell migration. We previously found that in MTLn3 rat adenocarcinoma cells, EGF-stimulated induction of actin barbed ends and lamellipod extension specifically requires the p85/p110alpha isoform of PI 3-kinase. To further characterize signaling by distinct PI 3-kinase isoforms, we have developed MTLn3 cells that transiently or stably overexpress either p110alpha or p110beta. Transient overexpression of p110beta inhibited EGF-stimulated lamellipod extension, whereas p110alpha-transfected cells showed normal EGF-stimulated lamellipod extension. Similar results were obtained by overexpression of kinase-dead p110beta, suggesting that effects on cytoskeletal signaling were due to competition with p85/p110alpha complexes. Stable overexpression of p110alpha appeared to be toxic, based on the difficulty in obtaining stable overexpressing clones. In contrast, cells expressing a 2-fold increase in p110beta were readily obtainable. Interestingly, cells stably expressing p110beta showed a marked inhibition of EGF-stimulated lamellipod extension. Using computer-assisted analysis of time-lapse images, we found that overexpression of p110beta caused a nearly complete inhibition of motility. Cells overexpressing p110beta showed normal activation of Akt and Erk, suggesting that overall PI 3-kinase signaling was intact. A chimeric p110 molecule containing the p85-binding and Ras-binding domains of p110alpha and the C2, helical, and kinase domains of p110beta, was catalytically active yet also inhibited EGF-stimulated lamellipod extension. These data highlight the differential signaling by distinct p110 isoforms. Identification of effectors that are differently regulated by p110alpha versus p110beta will be important for understanding cell migration and its role in metastasis.
磷脂酰肌醇3激酶（PI 3激酶）活性是生长因子诱导的细胞骨架调节和细胞迁移所必需的。我们之前发现，在MTLn3大鼠腺癌细胞中，表皮生长因子（EGF）刺激诱导肌动蛋白丝的刺端和片状伪足延伸特别需要PI 3激酶的p85/p110α亚型。为了进一步表征不同PI 3激酶亚型的信号传导，我们构建了瞬时或稳定过表达p110α或p110β的MTLn3细胞。p110β的瞬时过表达抑制了EGF刺激的片状伪足延伸，而转染p110α的细胞显示出正常的EGF刺激的片状伪足延伸。通过过表达激酶失活的p110β也获得了类似的结果，这表明对细胞骨架信号传导的影响是由于与p85/p110α复合物的竞争。基于难以获得稳定过表达克隆，p110α的稳定过表达似乎具有毒性。相比之下，p110β表达增加两倍的细胞很容易获得。有趣的是，稳定表达p110β的细胞显示出EGF刺激的片状伪足延伸受到明显抑制。使用延时图像的计算机辅助分析，我们发现p110β的过表达导致运动性几乎完全受到抑制。过表达p110β的细胞显示Akt和Erk的正常激活，这表明整体PI 3激酶信号传导是完整的。一种嵌合p110分子，包含p110α的p85结合和Ras结合结构域以及p110β的C2、螺旋和激酶结构域，具有催化活性，但也抑制了EGF刺激的片状伪足延伸。这些数据突出了不同p110亚型的差异信号传导。鉴定受p110α与p110β不同调节的效应器对于理解细胞迁移及其在转移中的作用将是重要的。
