Shisheva A, Sbrissa D, Ikonomov O
Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Mol Cell Biol. 1999 Jan;19(1):623-34. doi: 10.1128/MCB.19.1.623.
Signaling by phosphorylated species of phosphatidylinositol (PI) appears to regulate diverse responses in eukaryotic cells. A differential display screen for fat- and muscle-specific transcripts led to identification and cloning of the full-length cDNA of a novel mammalian 2,052-amino-acid protein (p235) from a mouse adipocyte cDNA library. Analysis of the deduced amino acid sequence revealed that p235 contains an N-terminal zinc-binding FYVE finger, a chaperonin-like region in the middle of the molecule, and a consensus for phosphoinositide 5-kinases at the C terminus. p235 mRNA appears as a 9-kb transcript, enriched in insulin-sensitive cells and tissues, likely transcribed from a single-copy gene in at least two close-in-size splice variants. Specific antibodies against mouse p235 were raised, and both the endogenously and heterologously expressed proteins were biochemically detected in 3T3-L1 adipocytes and transfected COS cells, respectively. Immunofluorescence microscopy analysis of endogenous p235 localization in 3T3-L1 adipocytes with affinity-purified anti-p235 antibodies documented a punctate peripheral pattern. In COS cells, the expressed p235 N-terminal but not the C-terminal region displayed a vesicular pattern similar to that in 3T3-L1 adipocytes that became diffuse upon Zn2+ chelation or FYVE finger truncation. A recombinant protein comprising the N-terminal but not the C-terminal region of the molecule was found to bind 2.2 mole equivalents of Zn2+. Determination of the lipid kinase activity in the p235 immunoprecipitates derived from 3T3-L1 adipocytes or from COS cells transiently expressing p235 revealed that p235 displayed unique preferences for PI substrate over already phosphorylated PI. In conclusion, the mouse p235 protein determines an important novel class of phosphoinositide kinases that seems to be targeted to specific intracellular loci by a Zn-dependent mechanism.
磷脂酰肌醇（PI）磷酸化产物的信号传导似乎调节真核细胞中的多种反应。一项针对脂肪和肌肉特异性转录本的差异显示筛选，从小鼠脂肪细胞cDNA文库中鉴定并克隆出一种新型哺乳动物2052个氨基酸蛋白（p235）的全长cDNA。对推导的氨基酸序列分析表明，p235包含一个N端锌结合FYVE结构域、分子中部的一个伴侣蛋白样区域以及C端的一个磷酸肌醇5激酶共有序列。p235 mRNA表现为一种9 kb的转录本，在胰岛素敏感细胞和组织中富集，可能由至少两种大小相近的剪接变体中的单拷贝基因转录而来。制备了针对小鼠p235的特异性抗体，分别在3T3-L1脂肪细胞和转染的COS细胞中通过生化方法检测到内源性和异源表达的蛋白。用亲和纯化的抗p235抗体对3T3-L1脂肪细胞中内源性p235定位进行免疫荧光显微镜分析，记录到点状外周模式。在COS细胞中，表达的p235 N端而非C端区域呈现出与3T3-L1脂肪细胞中相似的囊泡模式，在Zn2+螯合或FYVE结构域截短后变为弥散状。发现包含分子N端而非C端区域的重组蛋白能结合2.2摩尔当量的Zn2+。对来自3T3-L1脂肪细胞或瞬时表达p235的COS细胞的p235免疫沉淀物中的脂质激酶活性测定表明，p235对PI底物的偏好独特，优于已磷酸化的PI。总之，小鼠p235蛋白确定了一类重要的新型磷酸肌醇激酶，其似乎通过锌依赖机制靶向特定的细胞内位点。
