Machlus Kellie R, Wu Stephen K, Stumpo Deborah J, Soussou Thomas S, Paul David S, Campbell Robert A, Kalwa Hermann, Michel Thomas, Bergmeier Wolfgang, Weyrich Andrew S, Blackshear Perry J, Hartwig John H, Italiano Joseph E
Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Boston, MA; Harvard Medical School, Boston, MA;
Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Boston, MA;
Blood. 2016 Mar 17;127(11):1468-80. doi: 10.1182/blood-2015-08-663146. Epub 2016 Jan 7.
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).
血小板对止血至关重要，而血小板减少是一个主要的临床问题。巨核细胞（MKs）通过向窦状血管延伸长的突起（前血小板）来生成血小板。然而，对于调节前血小板形成的因素知之甚少。为了揭示在此过程中哪些蛋白质发生了动态变化，我们分别通过二维差异凝胶电泳（DIGE）和多核糖体谱分析，比较了圆形MKs与产生前血小板的MKs的蛋白质组和转录组。我们的数据显示，一种特征不明的MK蛋白——肉豆蔻酰化富含丙氨酸的C激酶底物（MARCKS）显著增加，在二维DIGE和多核糖体谱分析中，产生前血小板的MKs中该蛋白分别上调了3.4倍和5.7倍。MARCKS是一种蛋白激酶C（PKC）底物，可结合磷脂酰肌醇-4,5-二磷酸（PIP2）。在MKs中，它定位于质膜和分界膜。用肽抑制MARCKS可使前血小板形成显著减少53%。为了研究MARCKS在PKC途径中的作用，我们用聚甲基丙烯酸酯（PMA）处理MKs，这显著增加了MARCKS的磷酸化，同时显著抑制前血小板形成84%，表明MARCKS磷酸化会减少前血小板形成。我们推测MARCKS磷酸化会促进肌动蛋白相关蛋白2/3（Arp2/3）的磷酸化，进而下调前血小板形成；在产生前血小板的MKs中，MARCKS和Arp2均发生去磷酸化，抑制Arp2可增强前血小板形成。最后，我们使用MARCKS基因敲除（KO）小鼠来探究MARCKS在前血小板形成中的直接作用；MARCKS基因敲除的MKs显示前血小板水平显著降低。MARCKS在原代MKs中的表达和信号传导是一项新发现。我们提出，MARCKS作为一个“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。
