Synthesis and dephosphorylation of MARCKS in the late stages of megakaryocyte maturation drive proplatelet formation
Synthesis and dephosphorylation of MARCKS in the late stages of megakaryocyte maturation drive proplatelet formation
复制标题
DOI：
10.1182/blood-2015-08663146
复制
发表时间：
2016-03-17
期刊：
影响因子：
20.3
通讯作者：
Italiano, Joseph E., Jr.
中科院分区：
文献类型：
--
作者：
Machlus, Kellie R.;Wu, Stephen K.;Italiano, Joseph E., Jr.
血小板对于止血至关重要，而血小板减少症是一个主要的临床问题。巨核细胞（MK）通过将长突（前血小板）延伸到正弦血管中来产生血小板。然而，人们对调节前血小板形成的因素知之甚少。为了揭示哪些蛋白质在此过程中动态变化，我们分别通过二维差异凝胶电泳 (DIGE) 和多核糖体分析比较了圆形 MK 与前血小板生成 MK 的蛋白质组和转录组。我们的数据显示，一种特征较差的 MK 蛋白——肉豆蔻酰化富含丙氨酸的 C 激酶底物 (MARCKS) 显着增加，在 2D DIGE 和多核糖体分析中，该底物在产生前血小板的 MK 中分别上调 3.4 倍和 5.7 倍。 MARCKS 是一种结合 PIP2 的蛋白激酶 C (PKC) 底物。在 MK 中，它定位于质膜和分界膜。肽抑制 MARCKS 显着降低前血小板形成 53%。为了检查 MARCKS 在 PKC 途径中的作用，我们用聚甲基丙烯酸酯 (PMA) 处理 MK，这显着增加了 MARCKS 磷酸化，同时显着抑制前血小板形成 84%，表明 MARCKS 磷酸化减少了前血小板形成。我们假设 MARCKS 磷酸化促进 Arp2/3 磷酸化，从而下调前血小板形成； MARCKS 和 Arp2 在生成前血小板的 MK 中均被去磷酸化，并且 Arp2 抑制增强了前血小板的形成。最后，我们使用MARCKS敲除（KO）小鼠来探讨MARCKS在前血小板形成中的直接作用； MARCKS KO MKs 显示前血小板水平显着降低。原代 MK 中的 MARCKS 表达和信号传导是一项新发现。我们认为 MARCKS 充当“分子开关”，结合并调节 PIP2 信号传导，以调节前血小板延伸（微管驱动）与前血小板分支（Arp2/3 和肌动蛋白聚合驱动）等过程。
Platelets are essential for hemostasis, and thrombocytopenia is a major clinical problem. Megakaryocytes (MKs) generate platelets by extending long processes, proplatelets, into sinusoidal blood vessels. However, very little is known about what regulates proplatelet formation. To uncover which proteins were dynamically changing during this process, we compared the proteome and transcriptome of round vs proplatelet-producing MKs by 2D difference gel electrophoresis (DIGE) and polysome profiling, respectively. Our data revealed a significant increase in a poorly-characterized MK protein, myristoylated alanine-rich C-kinase substrate (MARCKS), which was upregulated 3.4- and 5.7 -fold in proplatelet-producing MKs in 2D DIGE and polysome profiling analyses, respectively. MARCKS is a protein kinase C (PKC) substrate that binds PIP2. In MKs, it localized to both the plasma and demarcation membranes. MARCKS inhibition by peptide significantly decreased proplatelet formation 53%. To examine the role of MARCKS in the PKC pathway, we treated MKs with polymethacrylate (PMA), which markedly increased MARCKS phosphorylation while significantly inhibiting proplatelet formation 84%, suggesting that MARCKS phosphorylation reduces proplatelet formation. We hypothesized that MARCKS phosphorylation promotes Arp2/3 phosphorylation, which subsequently downregulates proplatelet formation; both MARCKS and Arp2 were dephosphorylated in MKs making proplatelets, and Arp2 inhibition enhanced proplatelet formation. Finally, we used MARCKS knockout (KO) mice to probe the direct role of MARCKS in proplatelet formation; MARCKS KO MKs displayed significantly decreased proplatelet levels. MARCKS expression and signaling in primary MKs is a novel finding. We propose that MARCKS acts as a "molecular switch," binding to and regulating PIP2 signaling to regulate processes like proplatelet extension (microtubule-driven) vs proplatelet branching (Arp2/3 and actin polymerization-driven).