Saxena Anjana, Morozov Pavel, Frank Dale, Musalo Raymond, Lemmon Mark A, Skolnik Edward Y, Tycko Benjamin
Institute for Cancer Genetics, Department of Pathology, Columbia University, 1150 St. Nicholas Avenue, New York, NY 10032, USA.
J Biol Chem. 2002 Dec 20;277(51):49935-44. doi: 10.1074/jbc.M206497200. Epub 2002 Oct 8.
The Ipl protein consists of a single pleckstrin homology (PH) domain with short N- and C-terminal extensions. This protein is highly conserved among vertebrates, and it acts to limit placental growth in mice. However, its biochemical function is unknown. The closest paralogue of Ipl is Tih1, another small PH domain protein. By sequence comparisons, Ipl and Tih1 define an outlying branch of the PH domain superfamily. Here we describe phosphatidylinositol phosphate (PIP) binding by these proteins. Ipl and Tih1 bind to immobilized PIPs with moderate affinity, but this binding is weaker and more promiscuous than that of prototypical PH domains from the general receptor for phosphoinositides (GRP1), phospholipase C delta1, and dual adaptor for phosphoinositides and phosphotyrosine 1. In COS7 cells exposed to epidermal growth factor, green fluorescent protein (GFP)-Ipl and GFP-Tih1 accumulate at membrane ruffles without clearing from the cytoplasm, whereas control GFP-GRP1 translocates rapidly to the plasma membrane and clears from the cytoplasm. Ras*-Ipl and Ras*-Tih1 fusion proteins both rescue cdc25ts Saccharomyces cerevisiae, but Ras*-Ipl rescues more efficiently in the presence of phosphatidylinositol 3-kinase (PI3K), whereas PI3K-independent rescue is more efficient with Ras*-Tih1. Site-directed mutagenesis defines amino acids in the beta1-loop1-beta2 regions of Ipl and Tih1 as essential for growth rescue in this assay. Thus, Ipl and Tih1 are bona fide PH domain proteins, with broad specificity and moderate affinity for PIPs.
Ipl蛋白由一个单一的普列克底物蛋白同源（PH）结构域以及较短的N端和C端延伸序列组成。该蛋白在脊椎动物中高度保守，在小鼠中其作用是限制胎盘生长。然而，其生化功能尚不清楚。Ipl最接近的旁系同源物是Tih1，另一种小的PH结构域蛋白。通过序列比较，Ipl和Tih1定义了PH结构域超家族的一个外围分支。在此我们描述了这些蛋白与磷脂酰肌醇磷酸（PIP）的结合情况。Ipl和Tih1以中等亲和力与固定化的PIP结合，但这种结合比来自磷脂酰肌醇通用受体（GRP1）、磷脂酶Cδ1以及磷脂酰肌醇和磷酸酪氨酸双重衔接蛋白1的典型PH结构域的结合更弱且更具混杂性。在暴露于表皮生长因子的COS7细胞中，绿色荧光蛋白（GFP）-Ipl和GFP-Tih1在膜皱褶处积累且未从细胞质中清除，而对照GFP-GRP1则迅速转运至质膜并从细胞质中清除。Ras*-Ipl和Ras*-Tih1融合蛋白均可拯救cdc25ts酿酒酵母，但在磷脂酰肌醇3激酶（PI3K）存在的情况下，Ras*-Ipl的拯救效率更高，而Ras*-Tih1在不依赖PI3K的拯救中效率更高。定点诱变确定Ipl和Tih1的β1-环1-β2区域中的氨基酸对于该实验中的生长拯救至关重要。因此，Ipl和Tih1是真正的PH结构域蛋白，对PIP具有广泛的特异性和中等亲和力。
