Aronheim A, Broder Y C, Cohen A, Fritsch A, Belisle B, Abo A
Department of Molecular Genetics, The Rappaport Family Institute of Research in the Medical Sciences, Faculty of Medicine Technion-Israel Institute of Technology, P.O. Box 9649, Bat-Galim, Haifa, 31096, Israel.
Curr Biol. 1998 Oct 8;8(20):1125-8. doi: 10.1016/s0960-9822(98)70468-3.
The p21-activated protein kinases (PAKs) are activated through direct interaction with the GTPases Rac and Cdc42Hs, which are implicated in the control of the mitogen-activated protein kinase (MAP kinase) c-Jun N-terminal kinase (JNK) and the reorganization of the actin cytoskeleton [1-3]. The exact role of the PAK proteins in these signaling pathways is not entirely clear. To elucidate the biological function of Pak2 and to identify its molecular targets, we used a novel two-hybrid system, the Ras recruitment system (RRS), that aims to detect protein-protein interactions at the inner surface of the plasma membrane (described in the accompanying paper by Broder et al. [4]). The Pak2 regulatory domain (PakR) was fused at the carboxyl terminus of a RasL61 mutant protein and screened against a myristoylated rat pituitary cDNA library. Four clones were identified that interact specifically with PakR and three were subsequently shown to encode a previously unknown homologue of the GTPase Cdc42Hs. This approximately 36 kDa protein, designated Chp, exhibits an overall sequence identity to Cdc42Hs of approximately 52%. Chp contains two additional sequences at the amino and carboxyl termini that are not found in any known GTPase. The amino terminus contains a polyproline sequence, typically found in Src homology 3 (SH3)-binding domains, and the carboxyl terminus appears to be important for Pak2 binding. Results from the microinjection of Chp into cells implicated Chp in the induction of lamellipodia and showed that Chp activates the JNK MAP kinase cascade.
p21激活蛋白激酶（PAKs）通过与GTP酶Rac和Cdc42Hs直接相互作用而被激活，Rac和Cdc42Hs参与有丝分裂原激活蛋白激酶（MAP激酶）c-Jun氨基末端激酶（JNK）的调控以及肌动蛋白细胞骨架的重组[1-3]。PAK蛋白在这些信号通路中的具体作用尚不完全清楚。为了阐明Pak2的生物学功能并确定其分子靶点，我们使用了一种新型的双杂交系统——Ras招募系统（RRS），该系统旨在检测质膜内表面的蛋白质-蛋白质相互作用（见Broder等人[4]在随附论文中的描述）。将Pak2调节结构域（PakR）融合到RasL61突变蛋白的羧基末端，并针对肉豆蔻酰化的大鼠垂体cDNA文库进行筛选。鉴定出四个与PakR特异性相互作用的克隆，随后证明其中三个编码一种先前未知的GTP酶Cdc42Hs同源物。这种约36 kDa的蛋白质，命名为Chp，与Cdc42Hs的总体序列同一性约为52%。Chp在氨基和羧基末端含有两个在任何已知GTP酶中都未发现的额外序列。氨基末端包含一个多聚脯氨酸序列，通常存在于Src同源3（SH3）结合结构域中，羧基末端似乎对与Pak2结合很重要。将Chp显微注射到细胞中的结果表明Chp参与片状伪足的诱导，并表明Chp激活JNK MAP激酶级联反应。
