Analysis of the cellular functions of PTEN using catalytic domain and C-terminal mutations: differential effects of C-terminal deletion on signalling pathways downstream of phosphoinositide 3-kinase
Analysis of the cellular functions of PTEN using catalytic domain and C-terminal mutations: differential effects of C-terminal deletion on signalling pathways downstream of phosphoinositide 3-kinase
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DOI：
10.1042/0264-6021:3460827
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发表时间：
2000-03-15
影响因子：
4.1
通讯作者：
Downes, CP
中科院分区：
文献类型：
--
作者：
Leslie, NR;Gray, A;Downes, CP
肿瘤抑制蛋白，PTEN（在染色体上缺失的磷酸酶和张力蛋白同源物）是一种磷酸酶，其可以使含酪氨酸的肽、She、粘着斑激酶和磷酸肌醇底物去磷酸化。在细胞测定中，已显示PTEN拮抗蛋白激酶B（PE;B）的PI-3 K依赖性活化并抑制细胞铺展和运动。然而，目前尚不清楚PTEN是否通过其脂质或蛋白磷酸酶活性实现这些作用，尽管有力的证据已经证明了后者对PTEN抑制肿瘤的重要性。通过使用失去其脂质磷酸酶活性而保留蛋白磷酸酶活性的PTEN G129 E（Gly（120）-> Glu）突变体，我们证明了在PKB活性、细胞活力和膜皱褶的调节中需要PTEN的脂质磷酸酶活性。我们还进行了一个小的C-末端缺失的PTEN，除去一个假定的PDZ（PSD 95，Dig和ZO 1）结合基序，没有检测到的影响，在HEK 393细胞（人胚肾293细胞）中表达的蛋白磷酸酶活性在体外测定。令人惊讶的是，该突变体的表达揭示了在不同的功能测定中对C-末端的不同要求。野生型和C-末端缺失的PTEN在下调PE;B活性方面表现出同样的活性，但这种突变酶对血小板衍生生长因子（PDGF）诱导的膜皱褶没有影响，并且在细胞活力测定中仅具有部分活性。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。他们还发现了一种突变，类似于一些人类肿瘤中发生的突变，这种突变消除了PTEN对膜皱褶的影响，但没有消除对PKB的影响。
The tumour suppressor protein, PTEN (phosphatase and tensin homolog deleted on chromosome in), is a phosphatase that can dephosphorylate tyrosine-containing peptides, She, focal adhesion kinase and phosphoinositide substrates. In cellular assays, PTEN has been shown to antagonize the PI-3K-dependent activation of protein kinase B (PE;B) 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(120) --> 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, Dig and ZO1)-binding motif, with no detectable effect on the phosphatase activity of the protein expressed in HEK393 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 PE;B 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.