title: Synthesis and dephosphorylation of MARCKS in the late stages of megakaryocyte maturation drive proplatelet formation.
abstract: 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).
authors.affiliation: Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Boston, MA; Harvard Medical School, Boston, MA;
authors.forename: Kellie R
authors.identifier: http://orcid.org/0000-0002-2155-1050
authors.initials: KR
authors.lastname: Machlus
authors.affiliation: Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Boston, MA;
authors.forename: Stephen K
authors.initials: SK
authors.lastname: Wu
authors.affiliation: Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC;
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Stumpo
authors.affiliation: Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Boston, MA;
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Soussou
authors.affiliation: Department of Biochemistry and Biophysics, McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC;
authors.forename: David S
authors.initials: DS
authors.lastname: Paul
authors.affiliation: Program in Molecular Medicine and Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, UT;
authors.forename: Robert A
authors.initials: RA
authors.lastname: Campbell
authors.affiliation: Cardiovascular Medicine Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA; and.
authors.forename: Hermann
authors.initials: H
authors.lastname: Kalwa
authors.affiliation: Cardiovascular Medicine Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA; and.
authors.forename: Thomas
authors.initials: T
authors.lastname: Michel
authors.affiliation: Department of Biochemistry and Biophysics, McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC;
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Bergmeier
authors.affiliation: Program in Molecular Medicine and Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, UT;
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Weyrich
authors.affiliation: Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC;
authors.forename: Perry J
authors.initials: PJ
authors.lastname: Blackshear
authors.affiliation: Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Boston, MA;
authors.forename: John H
authors.initials: JH
authors.lastname: Hartwig
authors.affiliation: Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Boston, MA; Harvard Medical School, Boston, MA; Vascular Biology Program, Department of Surgery, Children's Hospital Boston, Boston, MA.
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Italiano
mesh_terms: D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism; D051378:Actin-Related Protein 3 / Q000378:metabolism; D000595:Amino Acid Sequence; D000091302:Angiopoietin-Like Protein 2; D000075802:Angiopoietin-like Proteins; D042682:Angiopoietins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D001792:Blood Platelets / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000172:deficiency / Q000502:physiology*; D008099:Liver / Q000166:cytology / Q000196:embryology; D008533:Megakaryocytes / Q000378:metabolism*; D008565:Membrane Proteins / Q000172:deficiency / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction; D034061:Thrombopoiesis / Q000502:physiology*
chemical_list: D051376:Actin-Related Protein 2-3 Complex; D051378:Actin-Related Protein 3; C495929:Actr3 protein, mouse; D000091302:Angiopoietin-Like Protein 2; D000075802:Angiopoietin-like Proteins; D042682:Angiopoietins; C485793:Angptl2 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D010446:Peptide Fragments; D019269:Phosphatidylinositol 4,5-Diphosphate; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C