Threonine 89 Is an Important Residue of Profilin-1 That Is Phosphorylatable by Protein Kinase A.
Threonine 89 Is an Important Residue of Profilin-1 That Is Phosphorylatable by Protein Kinase A.
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DOI：
10.1371/journal.pone.0156313
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Roy P
中科院分区：
文献类型：
--
作者：
Gau D;Veon W;Zeng X;Yates N;Shroff SG;Koes DR;Roy P
肌动蛋白细胞骨架的动态调节是所有基于肌动蛋白的细胞事件的核心。在这项研究中，我们试图确定新的翻译后修饰的Profilin-1（Pfn 1），一个重要的调节肌动蛋白聚合细胞。我们进行了体外蛋白激酶测定，然后通过质谱法来鉴定Pfn 1的蛋白激酶A（PKA）磷酸化位点。通过双向凝胶电泳（2D-GE）分析，我们进一步检测了HEK-293细胞中异位表达Pfn 1对cAMP/PKA通路激活剂forskolin（FSK）的反应。最后，我们结合分子动力学模拟（MDS），GST下拉分析和F-actin分析的哺乳动物细胞表达Pfn 1的位点特异性磷酸化模拟变体预测Pfn 1磷酸化的潜在后果。我们鉴定了Pfn 1的几个PKA磷酸化位点，包括一个新的位点Threatom 89（T89）。与PKA在体外磷酸化Pfn 1的能力一致，FSK刺激增加了HEK-293细胞中最负电荷形式的Pfn 1的库，其可以被PKA抑制剂H89减弱。MDS预测T89磷酸化使Pfn 1的分子内相互作用不稳定，可能增加其对肌动蛋白的亲和力。Pfn 1的T89 D磷酸模拟突变导致了几种变化，这些变化是折叠成替代三维构象的蛋白质的标志，包括去污剂不溶性，蛋白质聚集和加速蛋白水解，这表明T89是Pfn 1的结构上重要的残基。T89 D-Pfn 1的表达诱导肌动蛋白：T89 D-Pfn 1共簇并显著降低细胞中的整体肌动蛋白聚合，表明T89 D-Pfn 1的肌动蛋白螯合作用。最后，使T89非磷酸化导致在细胞提取物的2D凝胶电泳分析中Pfn 1的等电曲线中的正电荷偏移，这一发现与T89残基上的细胞内Pfn 1的某个池的磷酸化一致。总之，我们认为T89磷酸化可能对Pfn 1产生重要的功能影响。本研究为进一步研究Pfn 1磷酸化在PKA介导的肌动蛋白依赖性生物过程调节中的潜在作用铺平了道路。
Dynamic regulation of actin cytoskeleton is at the heart of all actin-based cellular events. In this study, we sought to identify novel post-translational modifications of Profilin-1 (Pfn1), an important regulator of actin polymerization in cells. We performed in vitro protein kinase assay followed by mass-spectrometry to identify Protein Kinase A (PKA) phosphorylation sites of Pfn1. By two-dimensional gel electrophoresis (2D-GE) analysis, we further examined the changes in the isoelectric profile of ectopically expressed Pfn1 in HEK-293 cells in response to forskolin (FSK), an activator of cAMP/PKA pathway. Finally, we combined molecular dynamics simulations (MDS), GST pull-down assay and F-actin analyses of mammalian cells expressing site-specific phosphomimetic variants of Pfn1 to predict the potential consequences of phosphorylation of Pfn1. We identified several PKA phosphorylation sites of Pfn1 including Threonine 89 (T89), a novel site. Consistent with PKA’s ability to phosphorylate Pfn1 in vitro, FSK stimulation increased the pool of the most negatively charged form of Pfn1 in HEK-293 cells which can be attenuated by PKA inhibitor H89. MDS predicted that T89 phosphorylation destabilizes an intramolecular interaction of Pfn1, potentially increasing its affinity for actin. The T89D phosphomimetic mutation of Pfn1 elicits several changes that are hallmarks of proteins folded into alternative three-dimensional conformations including detergent insolubility, protein aggregation and accelerated proteolysis, suggesting that T89 is a structurally important residue of Pfn1. Expression of T89D-Pfn1 induces actin:T89D-Pfn1 co-clusters and dramatically reduces overall actin polymerization in cells, indicating an actin-sequestering action of T89D-Pfn1. Finally, rendering T89 non-phosphorylatable causes a positive charge shift in the isoelectric profile of Pfn1 in a 2D gel electrophoresis analysis of cell extracts, a finding that is consistent with phosphorylation of a certain pool of intracellular Pfn1 on the T89 residue. In summary, we propose that T89 phosphorylation could have major functional consequences on Pfn1. This study paves the way for further investigation of the potential role of Pfn1 phosphorylation in PKA-mediated regulation of actin-dependent biological processes.