Suppression of Beta-arrestin1 Phosphorylation and Function by Beta-arrestin2

Beta-arrestin2 对 Beta-arrestin1 磷酸化和功能的抑制

• 批准号: 7964066
• 负责人: Rui-Ping Xiao
• 金额: $ 20.33万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7964066
• 关键词: Ablation; Adaptor Signaling Protein; Address; Affect; Arrestins; Binding; Biological; Biological Process; Cell Death; Cell Fate Control; Cell Survival; Cells; Co-Immunoprecipitations; Complex; Data; Dominant-Negative Mutation; Dose; Embryo; Equilibrium; Exhibits; Family member; Fibroblasts; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gene Transfer; Genes; Growth; Heterodimerization; Image; Immunoprecipitation; Knock-out; Knockout Mice; MAP Kinase Gene; Mediating; Mus; Normal Cell; Okadaic Acid; Oxidative Stress; Pathway interactions; Pattern; Phosphorylation; Physiological; Play; Protein Dephosphorylation; Protein Family; Protein Inhibition; Protein Phosphatase 2A Regulatory Subunit PR53; Proteins; Recruitment Activity; Regulation; Resistance; Retinal Cone; Role; Signal Pathway; Signal Transduction; Vertebrate Photoreceptors; Visual; Western Blotting; Wild Type Mouse; arrestin 1; arrestin 2; arrestin3; beta-arrestin; cell growth; intermolecular interaction; mutant; polyclonal antibody; receptor; receptor function; response; retinal rods

In the present study, there are three major findings. First, under physiological conditions, beta-arrestin1 and beta-arrestin2 physically interact with each other. Second, phosphorylation of beta-arrestin1 plays a crucial role in the formation of the beta-arrestin complex and subsequent recruitment of PP2A. Third, ablation of beta-arrestin2 leads to markedly increased phosphorylation of beta-arrestin1 which promotes cell survival and cell growth via elevated activation of ERK1/2. (1) Co-localization and co-immunoprecipitation of beta-arrestin1 and beta-arrestin2 To investigate the potential relationship between beta-arrestin1 and beta-arrestin2, we expressed both beta-arrestin family members at a matched level in cultured mouse embryonic fibroblasts (MEFs) derived from beta-arrestin1 and beta-arrestin2 double knockout mice. Using confocal immunocytochemical imaging, we visualized a similar intracellular distribution pattern of specific immunofluorescent signals of beta-arrestin1 and beta-arrestin2 and an excellent pixel-to-pixel correction when the images were emerged in cultured mouse MEFs, suggesting these beta-arrestins are co-localized. To directly demonstrate physical interaction of beta-arrestin1 with beta-arrestin2, we performed immunoprecipitation and Western blotting. Total cellular proteins containing both beta-arrestin subtypes were first immunoprecipitated with either a beta-arrestin1 or beta-arrestin2 polyclonal antibody. The pull-down of beta-arrestin1 by anti-beta-arrestin2 in the immunoprecipitation was then confirmed by Western blot. (2) Ablation of beta-arrestin2 increases the phosphorylation level of beta-arrestin1 and reduces beta-arrestin1/ PP2A complex formation To explore the functional consequence of the intermolecular interaction of beta-arrestin1 and beta-arrestin2, we examined the potential impact of beta-arrestin2 ablation on beta-arrestin1 phosphorylation in MEFs derived from beta-arrestin2 knockout (KO) mice. To our surprise, the lack of beta-arrestin2 led to a profound increase in beta-arrestin1 phosphorylation level. Adenoviral gene transfer of beta-arrestin2 fully normalized -arrestin1 phosphorylation status, indicating that the hyper-phosphorylation of beta-arrestin1 is specifically attributable to the absence of beta-arrestin2 rather than an adaptive response due to beta-arrestin2 null background. Next, we found that endogenous beta-arrestin1 and PP2A form a complex in MEFs derived from wild type (WT) mice, as evidenced by their abundant co-immunoprecipitation. The association of beta-arrestin1 with PP2A was strikingly impaired in beta-arrestin2 deficient MEFs. Adenoviral gene transfer of beta-arrestin2 rescued the interaction of beta-arrestin1 with PP2A, indicating that beta-arrestin2 is required for the formation of PP2A/beta-arrestin1 complex. These data suggest that beta-arrestin2-dependent interaction of PP2A with beta-arrestin1 may sever as an important mechanism responsible for beta-arrestin1 dephosphorylation and the termination of beta-arrestin1-mediated signaling. We further determined whether altered beta-arrestin1 phosphorylation status affects its interaction with beta-arrestin2 and PP2A. A dominant negative beta-arrestin1 mutant (S412A) markedly impaired beta-arrestin heterodimerization and reduced the recruitment of PP2A by beta-arrestin1. These data corroborate the importance of beta-arrestin1 phosphorylation in its intermolecular interaction with beta-arrestin2 that subsequently recruit PP2A to the beta-arrestin complex. (3) Deficiency of beta-arrestin2 increases cell viability, rendering cells resistant to oxidative stress-induced cell death. The next key question is what is the biological significance of the negative regulation of beta-arrestin1 phosphorylation by beta-arrestin2? To address this question, we characterized cell survival and cell growth in WT MEFs or those lacking either beta-arrestin1 or 2. Interestingly, deficiency of beta-arrestin2, but not beta-arrestin1, rendered cells resistant to H2O2-induced cell death. H2O2-induced reduction in cell viability was fully restored in beta-arrestin2 KO MEFs infected by an adenoviral expression of beta-arrestin2, indicating that the resistance of beta-arrestin2 KO cells to oxidative stress is a direct consequence of the gene ablation instead of an adaptive response. In addition, deficiency of beta-arrestin2 but not beta-arrestin1 markedly enhanced MEF growth rate, suggesting the coexisted beta-arrestin1 and beta-arrestin2 elicit different functional roles. (4) Beta-arrestin1 phosphorylation positively correlates with activation of ERK1/2. To define the mechanism underlying the prosurvival and pro-growth effects of beta-arrestin2 ablation, we examined major signaling pathways involved in the regulation of cell fate, in particular, ERK1/2 MAPK pathway. ERK1/2 phosphorylation level was overtly augmented in beta-arrestin2 deficient cells, and restored to normal level when beta-arrestin2 was reintroduced. Inhibition of protein phosotases with okadaic acid (OA) led to a dose-dependent increase in beta-arrestin1 phosphorylation which was accompanied by a proportional increase in EKR1/2 phosphorylation (activation). In contrast, expression of the dominant negative beta-arrestin1 (S412A) mutant suppressed ERK1/2 activation. These results indicate that beta-arrestin2 ablation-associated increase in ERK1/2 activation is a consequence of hyper-phosphorylation of beta-arrestin1. Importantly, inhibition ERK1/2 activity with PD98059 (10 mM) blocked beta-arrestin2 ablation-induced cell resistance to oxidative stress, indicating that the pro-survival effect of beta-arrestin2 ablation is mediated by enhanced phosphorylation of beta-arrestin1 and subsequent activation of ERK1/2. These findings not only define beta-arrestin2 as a powerful endogenous negative regulator of beta-arrestin1 phosphorylation and biological function, but also highlight the importance of the balance between the coexisted beta-arrestin1 and beta-arrestin2 in maintaining normal cell growth and cell viability.

在本研究中，有三个主要发现。首先，在生理条件下，β-arrestin1 和 beta-arrestin2 彼此发生物理相互作用。其次，β-arrestin1 的磷酸化在 β-arrestin 复合物的形成和随后的 PP2A 募集中起着至关重要的作用。第三，β-arrestin2 的消除会导致 β-arrestin1 的磷酸化显着增加，从而通过 ERK1/2 的激活增强来促进细胞存活和细胞生长。 (1) beta-arrestin1和beta-arrestin2的共定位和免疫共沉淀 为了研究 β-arrestin1 和 β-arrestin2 之间的潜在关系，我们在源自 β-arrestin1 和 β-arrestin2 双敲除小鼠的培养小鼠胚胎成纤维细胞 (MEF) 中以匹配水平表达这两种 β-arrestin 家族成员。使用共聚焦免疫细胞化学成像，我们观察到 β-arrestin1 和 β-arrestin2 特异性免疫荧光信号的相似细胞内分布模式，以及当图像出现在培养的小鼠 MEF 中时出色的像素到像素校正，表明这些 β-arrestin 是共定位的。为了直接证明 β-arrestin1 与 β-arrestin2 的物理相互作用，我们进行了免疫沉淀和蛋白质印迹。首先用 β-arrestin1 或 β-arrestin2 多克隆抗体对含有两种 β-arrestin 亚型的总细胞蛋白进行免疫沉淀。然后通过蛋白质印迹证实了免疫沉淀中抗 β-arrestin2 对 β-arrestin1 的下拉作用。 (2) 消除 beta-arrestin2 会增加 beta-arrestin1 的磷酸化水平并减少 beta-arrestin1/ PP2A 复合物的形成 为了探索 β-arrestin1 和 β-arrestin2 分子间相互作用的功能后果，我们研究了 β-arrestin2 消融对源自 β-arrestin2 敲除 (KO) 小鼠的 MEF 中 β-arrestin1 磷酸化的潜在影响。令我们惊讶的是，β-arrestin2 的缺乏导致 β-arrestin1 磷酸化水平显着增加。 β-arrestin2 的腺病毒基因转移完全标准化了 -arrestin1 磷酸化状态，表明 β-arrestin1 的过度磷酸化具体归因于 β-arrestin2 的缺失，而不是由于 β-arrestin2 无效背景导致的适应性反应。接下来，我们发现内源性 β-arrestin1 和 PP2A 在源自野生型 (WT) 小鼠的 MEF 中形成复合物，正如它们丰富的免疫共沉淀所证明的那样。在 β-arrestin2 缺陷的 MEF 中，β-arrestin1 与 PP2A 的关联显着受损。 β-arrestin2 的腺病毒基因转移挽救了 β-arrestin1 与 PP2A 的相互作用，表明 β-arrestin2 是 PP2A/β-arrestin1 复合物形成所必需的。这些数据表明，PP2A 与 β-arrestin1 的 β-arrestin2 依赖性相互作用可能是导致 β-arrestin1 去磷酸化和终止 β-arrestin1 介导的信号传导的重要机制。 我们进一步确定改变的 beta-arrestin1 磷酸化状态是否会影响其与 beta-arrestin2 和 PP2A 的相互作用。显性失活 β-arrestin1 突变体 (S412A) 显着损害 β-arrestin 异二聚化，并减少 β-arrestin1 对 PP2A 的募集。这些数据证实了 β-arrestin1 磷酸化在其与 β-arrestin2 分子间相互作用中的重要性，β-arrestin2 随后将 PP2A 招募到 β-arrestin 复合物中。 (3) β-arrestin2 缺乏会增加细胞活力，使细胞能够抵抗氧化应激诱导的细胞死亡。 下一个关键问题是 beta-arrestin2 对 beta-arrestin1 磷酸化的负调控有何生物学意义？为了解决这个问题，我们对 WT MEF 或缺乏 beta-arrestin1 或 2 的 MEF 中的细胞存活和细胞生长进行了表征。有趣的是，缺乏 beta-arrestin2 而不是 beta-arrestin1 会使细胞对 H2O2 诱导的细胞死亡具有抵抗力。在被表达β-arrestin2的腺病毒感染的β-arrestin2 KO MEF中，H2O2诱导的细胞活力降低完全恢复，表明β-arrestin2 KO细胞对氧化应激的抵抗是基因消融的直接结果，而不是适应性反应。此外，β-arrestin2 的缺乏而非 β-arrestin1 的缺乏显着提高了 MEF 的生长速度，表明共存的 β-arrestin1 和 β-arrestin2 会引发不同的功能作用。 (4) Beta-arrestin1 磷酸化与 ERK1/2 的激活呈正相关。 为了明确 β-arrestin2 消融促进存活和促生长作用的机制，我们检查了参与细胞命运调节的主要信号通路，特别是 ERK1/2 MAPK 通路。 ERK1/2 磷酸化水平在 β-arrestin2 缺陷细胞中明显增强，当重新引入 β-arrestin2 时，ERK1/2 磷酸化水平恢复到正常水平。用冈田酸 (OA) 抑制蛋白磷酸酶会导致 β-arrestin1 磷酸化呈剂量依赖性增加，同时 EKR1/2 磷酸化（激活）也成比例增加。相反，显性失活β-arrestin1 (S412A) 突变体的表达抑制了ERK1/2 的激活。这些结果表明，β-arrestin2 消融相关的 ERK1/2 激活增加是 β-arrestin1 过度磷酸化的结果。重要的是，用 PD98059 (10 mM) 抑制 ERK1/2 活性可阻断 β-arrestin2 消除诱导的细胞对氧化应激的抵抗力，表明 β-arrestin2 消除的促生存作用是通过增强 β-arrestin1 磷酸化和随后激活 ERK1/2 介导的。 这些发现不仅将β-arrestin2定义为β-arrestin1磷酸化和生物学功能的强大内源性负调节因子，而且强调了共存的β-arrestin1和β-arrestin2之间的平衡在维持正常细胞生长和细胞活力中的重要性。