Leslie N R, Gray A, Pass I, Orchiston E A, Downes C P
Division of Signal Transduction Therapy, Department of Biochemistry, University of Dundee, Dundee DD1 5EH, U.K.
Biochem J. 2000 Mar 15;346 Pt 3(Pt 3):827-33.
The tumour suppressor protein, PTEN (phosphatase and tensin homolog deleted on chromosome 10), is a phosphatase that can dephosphorylate tyrosine-containing peptides, Shc, focal adhesion kinase and phosphoinositide substrates. In cellular assays, PTEN has been shown to antagonize the PI-3K-dependent activation of protein kinase B (PKB) and to inhibit cell spreading and motility. It is currently unclear, however, whether PTEN accomplishes these effects through its lipid- or protein-phosphatase activity, although strong evidence has demonstrated the importance of the latter for tumour suppression by PTEN. By using a PTEN G129E (Gly(129)-->Glu) mutant that has lost its lipid phosphatase activity, while retaining protein phosphatase activity, we demonstrated a requirement for the lipid phosphatase activity of PTEN in the regulation of PKB activity, cell viability and membrane ruffling. We also made a small C-terminal deletion of PTEN, removing a putative PDZ (PSD95, Dlg and ZO1)-binding motif, with no detectable effect on the phosphatase activity of the protein expressed in HEK293 cells (human embryonic kidney 293 cells) assayed in vitro. Surprisingly, expression of this mutant revealed differential requirements for the C-terminus in the different functional assays. Wild-type and C-terminally deleted PTEN appeared to be equally active in down-regulating PKB activity, but this mutant enzyme had no effect on platelet-derived growth factor (PDGF)-induced membrane ruffling and was only partially active in a cell viability assay. These results stress the importance of the lipid phosphatase activity of PTEN in the regulation of several signalling pathways. They also identify a mutation, similar to mutations that occur in some human tumours, which removes the effect of PTEN on membrane ruffling but not that on PKB.
肿瘤抑制蛋白PTEN（第10号染色体缺失的磷酸酶及张力蛋白同源物）是一种磷酸酶，能够使含酪氨酸的肽、Shc、粘着斑激酶和磷酸肌醇底物去磷酸化。在细胞实验中，PTEN已被证明可拮抗蛋白激酶B（PKB）的PI-3K依赖性激活，并抑制细胞铺展和运动。然而，目前尚不清楚PTEN是通过其脂质磷酸酶活性还是蛋白质磷酸酶活性实现这些作用，尽管有力证据表明后者对PTEN的肿瘤抑制作用至关重要。通过使用PTEN G129E（甘氨酸（129）→谷氨酸）突变体，该突变体已丧失脂质磷酸酶活性，但保留了蛋白质磷酸酶活性，我们证明了PTEN的脂质磷酸酶活性在调节PKB活性、细胞活力和膜皱襞形成中的必要性。我们还对PTEN进行了小的C末端缺失，去除了一个假定的PDZ（PSD95、Dlg和ZO1）结合基序，对体外检测的HEK293细胞（人胚肾293细胞）中表达的该蛋白的磷酸酶活性没有可检测到的影响。令人惊讶的是，该突变体的表达在不同功能实验中显示出对C末端的不同需求。野生型和C末端缺失的PTEN在下调PKB活性方面似乎同样活跃，但这种突变酶对血小板衍生生长因子（PDGF）诱导的膜皱襞形成没有影响，并且在细胞活力实验中仅部分活跃。这些结果强调了PTEN的脂质磷酸酶活性在调节几种信号通路中的重要性。它们还鉴定出一种与某些人类肿瘤中发生的突变相似的突变，该突变消除了PTEN对膜皱襞形成的影响，但没有消除对PKB的影响。
