The cell division control protein 42-Src family kinase-neural Wiskott-Aldrich syndrome protein pathway regulates human proplatelet formation.
Essentials The role of the cytoskeleton during megakaryocyte differentiation was examined. Human megakaryocytes are derived from in vitro cultured CD34+cells. Cell division control protein 42 (CDC42) positively regulates proplatelet formation (PPF). Neural Wiskott-Aldrich syndrome protein, the main effector of CDC42 with Src positively regulates PPF.



Background Cytoskeletal rearrangements are essential for platelet release. The RHO small GTPase family, as regulators of the actin cytoskeleton, play an important role in proplatelet formation (PPF). In the neuronal system, CDC42 is involved in axon formation, a process that combines elongation and branching as for PPF. Objective To analyze the role of CDC42 and its effectors of the Wiskott-Aldrich syndrome protein (WASP) family in PPF. Methods Human megakaryocytes (MKs) were obtained from CD34+cells. Inhibition of CDC42 in MKs was performed with the chemical inhibitor CASIN or with an active or a dominant-negative form of CDC42. The knock-down of N-WASP was obtained with a small hairpin RNA strategy Results Herein, we show that CDC42 activity increased during MK differentiation. The use of the chemical inhibitor CASIN or of an active or a dominant-negative form of CDC42 demonstrated that CDC42 positively regulated PPF in vitro. We determined that N-WASP, but not WASP, regulated PPF. We found that N-WASP knockdown led to a marked decrease in PPF, owing to a defect in the demarcation membrane system (DMS). This was associated with RHOA activation, and a concomitant augmentation in the phosphorylation of mysosin light chain 2. Phosphorylation of N-WASP, creating a primed form of N-WASP, increased during MK differentiation. Phosphorylation inhibition by two Src family kinase inhibitors decreased PPF. Conclusions We conclude that N-WASP positively regulates DMS development and PPF, and that the Src family kinases in association with CDC42 regulate PPF through N-WASP.
Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée Ligue Contre le Cancer, Laboratoire d'Excellence GR-Ex, Villejuif, France.|Université Paris-Saclay, UMR 1170, Villejuif, France.|Gustave Roussy, UMR 1170, Villejuif, France.
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Palazzo
Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée Ligue Contre le Cancer, Laboratoire d'Excellence GR-Ex, Villejuif, France.|Université Paris-Saclay, UMR 1170, Villejuif, France.|Gustave Roussy, UMR 1170, Villejuif, France.
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Bluteau
Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée Ligue Contre le Cancer, Laboratoire d'Excellence GR-Ex, Villejuif, France.|Université Paris-Saclay, UMR 1170, Villejuif, France.|Gustave Roussy, UMR 1170, Villejuif, France.
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Messaoudi
Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée Ligue Contre le Cancer, Laboratoire d'Excellence GR-Ex, Villejuif, France.|Université Paris-Saclay, UMR 1170, Villejuif, France.|Gustave Roussy, UMR 1170, Villejuif, France.
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Marangoni
Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée Ligue Contre le Cancer, Laboratoire d'Excellence GR-Ex, Villejuif, France.|Université Paris-Saclay, UMR 1170, Villejuif, France.|Gustave Roussy, UMR 1170, Villejuif, France.
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Chang
Gustave Roussy, Centre Nationale de la Recherche Scientifique, UMR 8122, Gustave Roussy, Villejuif, France.
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Souquere
Gustave Roussy, Centre Nationale de la Recherche Scientifique, UMR 8122, Gustave Roussy, Villejuif, France.
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Pierron
Gustave Roussy, Unité de Thérapie Cellulaire, Villejuif, France.
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Lapierre
Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
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Zheng
Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée Ligue Contre le Cancer, Laboratoire d'Excellence GR-Ex, Villejuif, France.|Université Paris-Saclay, UMR 1170, Villejuif, France.|Gustave Roussy, UMR 1170, Villejuif, France.
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Vainchenker
Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée Ligue Contre le Cancer, Laboratoire d'Excellence GR-Ex, Villejuif, France.|Université Paris-Saclay, UMR 1170, Villejuif, France.|Gustave Roussy, UMR 1170, Villejuif, France.
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Raslova
Institut National de la Santé et de la Recherche Médicale, UMR 1170, Equipe labellisée Ligue Contre le Cancer, Laboratoire d'Excellence GR-Ex, Villejuif, France.|Université Paris-Saclay, UMR 1170, Villejuif, France.|Gustave Roussy, UMR 1170, Villejuif, France.
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Debili
D018952:Antigens, CD34 / Q000378:metabolism*; D001369:Axons / Q000378:metabolism; D001792:Blood Platelets / Q000166:cytology*; D002454:Cell Differentiation; D003599:Cytoskeleton / Q000378:metabolism; D005799:Genes, Dominant; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008533:Megakaryocytes / Q000166:cytology; D009474:Neurons / Q000378:metabolism; D010766:Phosphorylation; D051316:Wiskott-Aldrich Syndrome Protein / Q000378:metabolism*; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000235:genetics; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
D018952:Antigens, CD34; C495767:WAS protein, human; C495771:WASL protein, human; D051316:Wiskott-Aldrich Syndrome Protein; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D019061:src-Family Kinases; D020764:cdc42 GTP-Binding Protein
CDC42 protein; N-WASP protein, human; Src family kinases; megakaryocytes; platelet