Das Sudipto, Dixon Jack E, Cho Wonhwa
Department of Chemistry, University of Illinois, Chicago, IL 60607, USA.
Proc Natl Acad Sci U S A. 2003 Jun 24;100(13):7491-6. doi: 10.1073/pnas.0932835100. Epub 2003 Jun 13.
PTEN is a tumor suppressor that reverses the action of phosphoinositide 3-kinase by catalyzing the removal of the 3' phosphate of phosphoinositides. Despite the critical role of PTEN in cell signaling and regulation, the mechanisms of its membrane recruitment and activation is still poorly understood. PTEN is composed of an N-terminal phosphatase domain, a C2 domain, and a C-terminal tail region that contains the PSD-95/Dlg/ZO-1 homology (PDZ) domain-binding sequence and multiple phosphorylation sites. Our in vitro surface plasmon resonance measurements using immobilized vesicles showed that both the phosphatase domain and the C2 domain, but not the C-terminal tail, are involved in electrostatic membrane binding of PTEN. Furthermore, the phosphorylation-mimicking mutation on the C-terminal tail of PTEN caused an approximately 80-fold reduction in its membrane affinity, mainly by slowing the membrane-association step. Subcellular localization studies of PTEN transfected into HEK293T and HeLa cells indicated that targeting of PTEN to the plasma membrane is coupled with rapid degradation and that the phosphatase domain and the C2 domain are both necessary and sufficient for its membrane recruitment. Results also indicated that the phosphorylation regulates the targeting of PTEN to the plasma membrane not by blocking the PDZ domain-binding site but by interfering with electrostatic membrane binding of PTEN. On the basis of these results, we propose a membrane-binding and activation mechanism for PTEN, in which the phosphorylation/dephosphorylation of the C-terminal region serves as an electrostatic switch that controls the membrane translocation of the protein.
PTEN是一种肿瘤抑制因子，它通过催化去除磷酸肌醇的3'磷酸基团来逆转磷酸肌醇3激酶的作用。尽管PTEN在细胞信号传导和调节中起着关键作用，但其膜募集和激活机制仍知之甚少。PTEN由一个N端磷酸酶结构域、一个C2结构域和一个C端尾部区域组成，该区域包含PSD-95/Dlg/ZO-1同源（PDZ）结构域结合序列和多个磷酸化位点。我们使用固定化囊泡进行的体外表面等离子体共振测量表明，磷酸酶结构域和C2结构域而非C端尾部参与了PTEN的静电膜结合。此外，PTEN C端尾部的磷酸化模拟突变导致其膜亲和力降低约80倍，主要是通过减缓膜结合步骤。对转染到HEK293T和HeLa细胞中的PTEN进行的亚细胞定位研究表明，PTEN靶向质膜与快速降解相关，并且磷酸酶结构域和C2结构域对于其膜募集都是必要且充分的。结果还表明，磷酸化调节PTEN靶向质膜不是通过阻断PDZ结构域结合位点，而是通过干扰PTEN的静电膜结合。基于这些结果，我们提出了一种PTEN的膜结合和激活机制，其中C端区域的磷酸化/去磷酸化作为一个静电开关，控制蛋白质的膜易位。
