Identification of the 14.3.3 ζ Domains Important for Self-association and Raf Binding (*)
Identification of the 14.3.3 ζ Domains Important for Self-association and Raf Binding (*)
复制标题
14.3.3 对自关联和 Raf 结合很重要的 z 域的识别 (*)
DOI：
--
复制
发表时间：
1995
影响因子：
4.8
通讯作者：
J. Avruch
中科院分区：
文献类型：
--
作者：
Z. Luo;Xian;U. Rapp;J. Avruch
14.3.3 ζ蛋白是一种普遍存在的丰富的花生四烯酮选择性酰基转移酶和假定的磷脂酶A2，它在体外和完整细胞中自组装成二聚体并与c-Raf-1和其他多肽结合。Sf9细胞内源性的14.3.3多肽与活性和非活性重组Raf就地结合，并以相当可重复的摩尔比（可能≥1）共化。纯化的杆状病毒重组Raf，尽管其预先关联14.3.3多肽，在体外可饱和和特异性反应中结合额外的重组14.3.3 ζ多肽，形成一个对1 M LiCl耐药的复合物。双杂交分析表明，14.3.3 ζ主要与Raf非催化序列结合，不同于与Ras-GTP结合的序列，并且在体外14.3.3 ζ与Raf结合而不抑制Ras-Raf结合或Raf催化的MEK磷酸化。14.3.3 ζ(1-245)的缺失分析表明，14.3.3结构域负责与Raf结合，延伸超过羧基末端100个氨基酸，而14.3.3二聚化是由氨基末端序列介导的。与Ras一样，14.3.3 ζ多肽在体外不能直接激活纯化的Raf。此外，通过共转染的Erk-1报告细胞的活性判断，在COS细胞中表达重组14.3.3 ζ，超过内源性14.3.3蛋白的大量水平，不会改变内源性Raf激酶。重组14.3.3与重组myc标记的Raf在COS细胞中共表达，确实显著提高了短暂表达过程中Myc-Raf激酶的活性，这主要是由于Myc-Raf多肽水平的增加，而Myc-Raf特异性活性或对表皮生长因子或12- o - tetradecanoylphorbol13 -acetate的反应没有改变。然而，14.3.3积极参与Raf原位激活的证据是通过发现，尽管全长14.3.3 ζ与活性Raf原位结合，14.3.3的截短版本，其中一些与Raf多肽原位结合几乎和全长14.3.3 ζ一样，仅与非活性Raf多肽相关联。因此，14.3.3多肽与c-Raf上的一个或多个位点紧密结合。14.3.3 ζ的过表达可能通过稳定Raf多肽来增强重组Raf的表达。此外，与截断的14.3.3多肽结合的Raf多肽无法原位激活，这表明14.3.3参与了Raf的激活过程，其机制尚待阐明。
The 14.3.3 ζ protein is a ubiquitous and abundant arachidonate-selective acyltransferase and putative phospholipase A2, which self-assembles into dimers and binds to c-Raf-1 and other polypeptides in vitro and in intact cells. The 14.3.3 polypeptides endogenous to Sf9 cells associate in situ with both active and inactive recombinant Raf and copurify at a fairly reproducible molar ratio that is probably ≥1. Purified baculoviral recombinant Raf, despite its preassociated 14.3.3 polypeptide, binds additional recombinant 14.3.3 ζ polypeptide in vitro, in a saturable and specific reaction, forming a complex that is resistant to 1 M LiCl. A two-hybrid analysis indicates that 14.3.3 ζ binds primarily to Raf noncatalytic sequences distinct from those that bind Ras-GTP, and in vitro 14.3.3 ζ binds to Raf without inhibiting the Ras-Raf association or Raf-catalyzed MEK phosphorylation. Deletion analysis of 14.3.3 ζ (1-245) indicates that the 14.3.3 domain responsible for binding to Raf extends over the carboxyl-terminal 100 amino acids, whereas 14.3.3 dimerization is mediated by amino-terminal sequences. As with Ras, the 14.3.3 ζ polypeptide does not activate purified Raf directly in vitro. Moreover, expression of recombinant 14.3.3 ζ in COS cells beyond the substantial level of endogenous 14.3.3 protein does not alter endogenous Raf kinase, as judged by the activity of a cotransfected Erk-1 reporter. Coexpression of recombinant 14.3.3 with recombinant Myc-tagged Raf in COS cells does increase substantially the Myc-Raf kinase activity achieved during transient expression, which is attributable primarily to an increased level of Myc-Raf polypeptide, without alteration of Myc-Raf specific activity or the activation that occurs in response to epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate. Nevertheless, evidence that 14.3.3 actively participates in Raf activation in situ is provided by the finding that although full-length 14.3.3 ζ binds active Raf in situ, truncated versions of 14.3.3, some of which bind Raf polypeptide in situ nearly as well as full-length 14.3.3 ζ, are recovered in association only with inactive Raf polypeptides. Thus, 14.3.3 polypeptides bind tightly to one or more sites on c-Raf. Overexpression of 14.3.3 ζ enhances the expression of recombinant Raf, perhaps by stabilizing the Raf polypeptide. In addition, Raf polypeptides bound to truncated 14.3.3 polypeptides are unable to undergo activation in situ, indicating that 14.3.3 participates in the process of Raf activation by mechanisms that remain to be elucidated.
登录
查看更多内容
DOI：
10.1073/pnas.90.6.2320
发表时间：
1993-03-15
影响因子：
11.1
作者：
FU, H;COBURN, J;COLLIER, RJ
通讯作者：
COLLIER, RJ
影响因子：
2.9
作者：
GUAN, KL;DIXON, JE
通讯作者：
DIXON, JE
影响因子：
10.5
作者：
DURFEE, T;BECHERER, K;ELLEDGE, SJ
通讯作者：
ELLEDGE, SJ
raf-1 激酶富含半胱氨酸的区域含有锌，易位至脂质体，并与结合 GTP-ras 的片段相邻。
DOI：
--
发表时间：
1994
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Ghosh,S;Xie,WQ;Quest,AF;Mabrouk,GM;Strum,JC;Bell,RM
通讯作者：
Bell,RM
p21ras 和 pp60v-src 都是激活 Raf-1 激酶所必需的，但单独使用两者都不足以激活 Raf-1 激酶。
DOI：
10.1073/pnas.89.7.2922
发表时间：
1992
影响因子：
11.1
作者：
Williams,NG;Roberts,TM;Li,P
通讯作者：
Li,P