Suen K L, Bustelo X R, Barbacid M
Department of Molecular Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA.
Oncogene. 1995 Sep 7;11(5):825-31.
We have isolated cDNA clones encoding the rodent 14-3-3 zeta and epsilon isoforms by screening bacteriophage expression libraries with a probe derived from the carboxy-terminus of the Vav oncoprotein. These isoforms, however, did not recognize the full length Vav protein under physiological conditions. In agreement with previous studies (see D Morrison, Science 266, 56-57, 1994), these 14-3-3 proteins bound very efficiently to Raf. The interaction between 14-3-3 zeta and Raf involves the central region of 14-3-3 zeta which includes a motif related to annexins. 14-3-3 zeta binds to Raf independently of Ras and forms stable ternary complexes with these two molecules. In contrast to published reports, we have observed that the catalytic activity of Raf was not activated in Raf/14-3-3 zeta immunocomplexes. Likewise, purified preparations of 14-3-3 zeta had no effect on the kinase activity of Raf immunoprecipitates. In addition, Ras activated Raf regardless of whether it was bound or not to 14-3-3 zeta. Finally, overexpression of 14-3-3 zeta cDNA clones in NIH3T3 cells did not result in detectable morphologic transformation even when co-transfected with plasmids encoding Raf and/or Ras proteins. These observations argue against a critical regulatory role of the 14-3-3 proteins in the Raf mitogenic pathway.
我们通过用源自Vav癌蛋白羧基末端的探针筛选噬菌体表达文库，分离出了编码啮齿动物14-3-3 ζ和ε亚型的cDNA克隆。然而，在生理条件下，这些亚型无法识别全长Vav蛋白。与先前的研究一致（见D·莫里森，《科学》266卷，56 - 57页，1994年），这些14-3-3蛋白能非常有效地与Raf结合。14-3-3 ζ与Raf之间的相互作用涉及14-3-3 ζ的中央区域，该区域包含一个与膜联蛋白相关的基序。14-3-3 ζ独立于Ras与Raf结合，并与这两个分子形成稳定的三元复合物。与已发表的报告相反，我们观察到在Raf/14-3-3 ζ免疫复合物中，Raf的催化活性并未被激活。同样，纯化的14-3-3 ζ制剂对Raf免疫沉淀物的激酶活性没有影响。此外，无论Ras是否与14-3-3 ζ结合，它都能激活Raf。最后，即使与编码Raf和/或Ras蛋白的质粒共转染，在NIH3T3细胞中过表达14-3-3 ζ cDNA克隆也不会导致可检测到的形态转化。这些观察结果表明14-3-3蛋白在Raf促有丝分裂途径中不具有关键的调节作用。
