Actin directly interacts with phospholipase D, inhibiting its activity
Actin directly interacts with phospholipase D, inhibiting its activity
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DOI：
10.1074/jbc.m008521200
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发表时间：
2001-07-27
影响因子：
4.8
通讯作者：
Ryu, SH
中科院分区：
文献类型：
--
作者：
Lee, S;Park, JB;Ryu, SH
哺乳动物磷脂酶D(PLD)在多种信号转导途径中起关键作用，参与多种功能。为了阐明PLD的复杂分子调控，我们研究了从大鼠脑提取液中获得的PLD结合蛋白。在这里，我们报告了大鼠脑中一种43 kDa的蛋白质，β-肌动蛋白，通过结合基质辅助激光解吸电离飞行时间质谱仪的多肽质量指纹图谱显示，它是主要的PLD2直接结合蛋白。我们还利用细菌表达的PLD2片段的谷胱甘肽S转移酶融合蛋白，确定了PLD2的613-723位氨基酸之间的区域是与β-肌动蛋白直接结合所必需的。有趣的是，纯化的β-肌动蛋白以浓度依赖的方式有效地抑制磷脂酰肌醇-4，5-二磷酸和油酸依赖的PLD2活性(IC50=5 nM)。在之前的一篇论文中，我们报道了α-肌动蛋白以相互作用依赖的和ADP核糖化因子1(ARF1)可逆的方式抑制PLD2的活性(Park，J.B.，Kim，J.H.，Kim，Y.，Ha，S.H.，Kim，J.H.，Yoo，J.S.，Du，G.，Frohman，M.A.，Sub，P.-G.和Ryu，S.H.(2000)J.Biol。化学。275、21295-21301)。体外结合分析表明，β-肌动蛋白可以取代α-肌动蛋白与PLD2的结合，表明细胞骨架蛋白与PLD2之间存在独立的相互作用。此外，无论β-肌动蛋白对PLD2的抑制作用如何，ARF1都可以将PLD2的活性引向正向。我们还观察到，β-肌动蛋白以相似的结合和抑制能力调节PLD1和PLD2。免疫细胞化学。免疫共沉淀研究证实了两种PLD同工酶与细胞内肌动蛋白之间的体内相互作用。综上所述，这些结果表明，细胞骨架蛋白、β-肌动蛋白和α-肌动蛋白以及ARF1对PLD的调控可能在细胞骨架相关的PLD功能中发挥重要作用。
Mammalian phospholipase D (PLD) plays a key role in several signal transduction pathways and is involved in many diverse functions. To elucidate the complex molecular regulation of PLD, we investigated PLD-binding proteins obtained from rat brain extract. Here we report that a 43-kDa protein in the rat brain, beta -actin, acts as a major PLD2 direct-binding protein as revealed by peptide mass fingerprinting in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We also determined that the region between amino acids 613 and 723 of PLD2 is required for the direct binding of beta -actin, using bacterially expressed glutathione S-transferase fusion proteins of PLD2 fragments. Intriguingly, purified beta -actin potently inhibited both phosphaticlylinositol-4,5-bisphosphate and oleate-dependent PLD2 activities in a concentration-dependent manner (IC50 = 5 nM). In a previous paper, we reported that alpha -actinin inhibited PLD2 activity in an interaction-dependent and an ADP-ribosylation factor 1 (ARF1)-reversible manner (Park, J. B., Kim, J. H., Kim, Y., Ha, S. H., Kim, J. H., Yoo, J.-S., Du, G., Frohman, M. A., Sub, P.-G., and Ryu, S. H. (2000) J. Biol. Chem. 275, 21295-21301). In vitro binding analyses showed that beta -actin could displace alpha -actinin binding to PLD2, demonstrating independent interaction between cytoskeletal proteins and PLD2. Furthermore, ARF1 could steer the PLD2 activity in a positive direction regardless of the inhibitory effect of beta -actin on PLD2. We also observed that beta -actin regulates PLD1 and PLD2 with similar binding and inhibitory potencies. Immunocytochemical. and co-immunoprecipitation studies demonstrated the in vivo interaction between the two PLD isozymes and actin in cells. Taken together, these results suggest that the regulation of PLD by cytoskeletal proteins, beta -actin and a-actinin, and ARF1 may play an important role in cytoskeleton-related PLD functions.