Kortholt Arjan, King Jason S, Keizer-Gunnink Ineke, Harwood Adrian J, Van Haastert Peter J M
Department of Molecular Cell Biology, University of Groningen, 9751 NN Haren, The Netherlands.
Mol Biol Cell. 2007 Dec;18(12):4772-9. doi: 10.1091/mbc.e07-05-0407. Epub 2007 Sep 26.
Generation of a phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] gradient within the plasma membrane is important for cell polarization and chemotaxis in many eukaryotic cells. The gradient is produced by the combined activity of phosphatidylinositol 3-kinase (PI3K) to increase PI(3,4,5)P(3) on the membrane nearest the polarizing signal and PI(3,4,5)P(3) dephosphorylation by phosphatase and tensin homolog deleted on chromosome ten (PTEN) elsewhere. Common to both of these enzymes is the lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], which is not only the substrate of PI3K and product of PTEN but also important for membrane binding of PTEN. Consequently, regulation of phospholipase C (PLC) activity, which hydrolyzes PI(4,5)P(2), could have important consequences for PI(3,4,5)P(3) localization. We investigate the role of PLC in PI(3,4,5)P(3)-mediated chemotaxis in Dictyostelium. plc-null cells are resistant to the PI3K inhibitor LY294002 and produce little PI(3,4,5)P(3) after cAMP stimulation, as monitored by the PI(3,4,5)P(3)-specific pleckstrin homology (PH)-domain of CRAC (PH(CRAC)GFP). In contrast, PLC overexpression elevates PI(3,4,5)P(3) and impairs chemotaxis in a similar way to loss of pten. PI3K localization at the leading edge of plc-null cells is unaltered, but dissociation of PTEN from the membrane is strongly reduced in both gradient and uniform stimulation with cAMP. These results indicate that local activation of PLC can control PTEN localization and suggest a novel mechanism to regulate the internal PI(3,4,5)P(3) gradient.
在许多真核细胞中，质膜内磷脂酰肌醇-3,4,5-三磷酸[PI(3,4,5)P(3)]梯度的产生对于细胞极化和趋化性至关重要。该梯度是由磷脂酰肌醇-3激酶（PI3K）的联合活性产生的，以增加最靠近极化信号的膜上的PI(3,4,5)P(3)，而在其他位置则通过10号染色体缺失的磷酸酶和张力蛋白同源物（PTEN）使PI(3,4,5)P(3)去磷酸化。这两种酶的共同之处在于脂质磷脂酰肌醇-4,5-二磷酸[PI(4,5)P(2)]，它不仅是PI3K的底物和PTEN的产物，而且对于PTEN与膜的结合也很重要。因此，磷脂酶C（PLC）活性的调节（其水解PI(4,5)P(2)）可能对PI(3,4,5)P(3)的定位产生重要影响。我们研究了PLC在盘基网柄菌中PI(3,4,5)P(3)介导的趋化性中的作用。通过CRAC的PI(3,4,5)P(3)特异性普列克底物蛋白同源（PH）结构域（PH(CRAC)GFP）监测，plc基因缺失的细胞对PI3K抑制剂LY294002具有抗性，并且在cAMP刺激后几乎不产生PI(3,4,5)P(3)。相反，PLC过表达会升高PI(3,4,5)P(3)，并以与pten缺失类似的方式损害趋化性。PI3K在plc基因缺失细胞前缘的定位未改变，但在cAMP梯度刺激和均匀刺激下，PTEN从膜上的解离都大大减少。这些结果表明PLC的局部激活可以控制PTEN的定位，并提示了一种调节细胞内PI(3,4,5)P(3)梯度的新机制。
