Uberall F, Hellbert K, Kampfer S, Maly K, Villunger A, Spitaler M, Mwanjewe J, Baier-Bitterlich G, Baier G, Grunicke H H
Institute of Medical Chemistry and Biochemistry, University of Innsbruck, A-6020 Innsbruck, Austria.
J Cell Biol. 1999 Feb 8;144(3):413-25. doi: 10.1083/jcb.144.3.413.
Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound morphological alterations which include a disassembly of actin stress fibers. The Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations which inhibit the activity of the atypical aPKC isotypes lambda and zeta, whereas lower concentrations of the inhibitor which block conventional and novel PKC isotypes are ineffective. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant-negative (DN) mutants of aPKC-lambda and aPKC-zeta, as well as antisense constructs encoding RNA-directed against isotype-specific 5' sequences of the corresponding mRNA, abrogates the Ha-Ras-induced reorganization of the actin cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-alpha was unable to counteract Ras with regard to the dissolution of actin stress fibers. Transfection of cells with constructs encoding constitutively active (CA) mutants of atypical aPKC-lambda and aPKC-zeta lead to a disassembly of stress fibers independent of oncogenic Ha-Ras. Coexpression of (DN) Rac-1 N17 and addition of the phosphatidylinositol 3'-kinase (PI3K) inhibitors wortmannin and LY294002 are in agreement with a tentative model suggesting that, in the signaling pathway from Ha-Ras to the cytoskeleton aPKC-lambda acts upstream of PI3K and Rac-1, whereas aPKC-zeta functions downstream of PI3K and Rac-1. This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-lambda or aPKC-zeta results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-zeta was abrogated by coexpression of DN Rac-1 N17.
在NIH3T3细胞中表达转化型Ha-Ras L61会引起深刻的形态学改变，其中包括肌动蛋白应力纤维的解聚。Ras诱导的肌动蛋白应力纤维溶解被特异性PKC抑制剂GF109203X阻断，该抑制剂在抑制非典型aPKC亚型lambda和zeta活性的浓度下发挥作用，而阻断传统和新型PKC亚型的较低浓度抑制剂则无效。将转化型Ha-Ras L61与aPKC-lambda和aPKC-zeta的激酶缺陷型显性负性（DN）突变体共表达，以及编码针对相应mRNA的亚型特异性5'序列的RNA的反义构建体，可消除Ha-Ras诱导的肌动蛋白细胞骨架重组。cPKC-α的激酶缺陷型DN突变体的表达在肌动蛋白应力纤维溶解方面无法对抗Ras。用编码非典型aPKC-lambda和aPKC-zeta组成型活性（CA）突变体的构建体转染细胞会导致应力纤维解聚，且与致癌性Ha-Ras无关。（DN）Rac-1 N17的共表达以及磷脂酰肌醇3'-激酶（PI3K）抑制剂渥曼青霉素和LY294002的添加与一个初步模型相符，该模型表明，在从Ha-Ras到细胞骨架的信号通路中，aPKC-lambda在PI3K和Rac-1的上游起作用，而aPKC-zeta在PI3K和Rac-1的下游起作用。该模型得到了研究的支持，这些研究表明，与编码L61Ras和aPKC-lambda或aPKC-zeta的质粒共转染会刺激这两种酶的激酶活性。此外，DN Rac-1 N17的共表达消除了Ras介导的PKC-zeta激活。
