herbimycin and Wiskott-Aldrich-Syndrome

Platelets are produced from megakaryocytes differentiated from megakaryoblasts, but the differentiation mechanism still remains unknown. Here, we demonstrate that a tyrosine kinase signaling regulates Wiskott-Aldrich syndrome protein (WASP), which is essential for megakaryocyte differentiation. MEG-01 megakaryoblastic cells differentiate into large multinucleated megakaryocyte-like cells characterized by microvesicle formation with a protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol 13-acetate. With parallel to the differentiation, WASP begins to accumulate at microvesicles with actin filaments and associates with tyrosine-phosphorylated Shc, the phosphorylation of which is induced. Moreover, a tyrosine kinase inhibitor, herbimycin A, blocked not only phosphorylation of Shc but also microvesicle formation without affecting cell enlargement and multinucleation, whereas a PKC inhibitor, H-7, completely inhibited all these processes. Because WASP also binds to Ash/Grb2 SH3 domains and the association of Ash/Grb2 and Shc is induced by 12-O-tetradecanoylphorbol 13-acetate treatment, a signaling pathway, PKC-tyrosine kinase-Shc-Ash/Grb2-WASP, is suggested for regulating megakaryocyte differentiation. In addition, WASP antisense oligonucleotide treatment abolishes both microvesicle formation and gathering of actin filaments. These data clearly show that WASP controls the assembly of actin filaments required for microvesicle formation downstream of PKC-tyrosine kinase.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amino Acid Sequence; Animals; Antibodies; Benzoquinones; Cell Differentiation; Cell Line; Humans; Lactams, Macrocyclic; Megakaryocytes; Molecular Sequence Data; Protein Kinase C; Protein-Tyrosine Kinases; Proteins; Quinones; Rabbits; Rifabutin; Signal Transduction; Tetradecanoylphorbol Acetate; Wiskott-Aldrich Syndrome; Wiskott-Aldrich Syndrome Protein