Várnai Péter, Bondeva Tzvetanka, Tamás Péter, Tóth Balázs, Buday László, Hunyady László, Balla Tamas
Endocrinology and Reproduction Research Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
J Cell Sci. 2005 Oct 15;118(Pt 20):4879-88. doi: 10.1242/jcs.02606.
Several pleckstrin-homology (PH) domains with the ability to bind phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3, PIP3] were expressed as green fluorescent protein (GFP) fusion proteins to determine their effects on various cellular responses known to be activated by PIP3. These proteins comprised the PH domains of Akt, ARNO, Btk or GRP1, and were found to show growth-factor-stimulated and wortmannin-sensitive translocation from the cytosol to the plasma membrane in several cell types, indicating their ability to recognize PIP3. Remarkably, although overexpressed Akt-PH-GFP and Btk-PH-GFP were quite potent in antagonizing the PIP3-mediated activation of the Akt protein kinase, such inhibition was not observed with the other PH domains. By contrast, expression of the PH domains of GRP1 and ARNO, but not of Akt or Btk, inhibited the attachment and spreading of freshly seeded cells to culture dishes. Activation of PLCgamma by epidermal growth factor (EGF) was attenuated by the PH domains of GRP1, ARNO and Akt, but was significantly enhanced by the Btk PH domain. By following the kinetics of expression of the various GFP-fused PH domains for several days, only the PH domain of Akt showed a lipid-binding-dependent self-elimination, consistent with its interference with the anti-apoptotic Akt signaling pathway. Mutations of selective residues that do not directly participate in PIP3 binding in the GRP1-PH and Akt-PH domain were able to reduce the dominant-negative effects of these constructs yet retain their lipid binding. These data suggest that interaction with and sequestration of PIP3 may not be the sole mechanism by which PH domains interfere with cellular responses and that their interaction with other membrane components, most probably with proteins, allows a more specific participation in the regulation of specific signaling pathways.
表达了几种具有结合磷脂酰肌醇(3,4,5)-三磷酸[PtdIns(3,4,5)P3，PIP3]能力的普列克底物蛋白同源(PH)结构域作为绿色荧光蛋白(GFP)融合蛋白，以确定它们对已知由PIP3激活的各种细胞反应的影响。这些蛋白包括Akt、ARNO、Btk或GRP1的PH结构域，发现在几种细胞类型中，它们在生长因子刺激下以及在渥曼青霉素敏感的情况下，会从胞质溶胶转运到质膜，表明它们具有识别PIP3的能力。值得注意的是，尽管过表达的Akt-PH-GFP和Btk-PH-GFP在拮抗PIP3介导的Akt蛋白激酶激活方面相当有效，但其他PH结构域未观察到这种抑制作用。相比之下，GRP1和ARNO的PH结构域（而非Akt或Btk的PH结构域）的表达抑制了新接种细胞在培养皿上的附着和铺展。表皮生长因子(EGF)对PLCγ的激活被GRP1、ARNO和Akt的PH结构域减弱，但被Btk的PH结构域显著增强。通过追踪几种GFP融合的PH结构域的表达动力学数天，只有Akt的PH结构域显示出脂质结合依赖性的自我消除，这与其对抗凋亡Akt信号通路的干扰一致。GRP1-PH和Akt-PH结构域中不直接参与PIP3结合的选择性残基的突变能够降低这些构建体的显性负效应，但仍保留其脂质结合能力。这些数据表明，与PIP3的相互作用和隔离可能不是PH结构域干扰细胞反应的唯一机制，并且它们与其他膜成分（很可能与蛋白质）的相互作用允许更特异性地参与特定信号通路的调节。
