2004 Fiscal Year Final Research Report Summary
Discovery of protein-to-protein interactions in platelets by a novel methods.
| Project/Area Number |
15590991
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Hematology
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
ODA Atsushi Hokkaido Univ., Grad.School of Med., Lecturer, 大学院・医学研究科, 助教授 (50255436)
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| Co-Investigator(Kenkyū-buntansha) |
FUJITA Hiroyoshi Hokkaido Univ., Grad.School of Med., Professor, 大学院・医学研究科, 教授 (60142931)
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| Project Period (FY) |
2003 – 2004
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| Keywords | platelet / signal transduction / mass spectrometry / actin / rac |
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| Research Abstract |
Searching for proteins in platelets that can interact with the N-terminal SH3 domain of CrkL (using a combination of a pull-down assay followed by mass spectrometry), we have found that human platelets express DOCK5, a putative Rac guanine nucleotide exchanger, as a CrkL-binding protein. We cloned DOCK5 and raised specific antibodies against the molecule. We overexpressed CrkL, DOCK5, or both in combination in COS7 cells. Overexpressed DOCK5 showed diffuse cytoplasmic distribution. However, when co-expressed with wild-type CrkL, both DOCK5 accumulated at CrkL-induced focal adhesions, suggesting a functional implication of the interaction.
Using specific antibodies against isoforms of WAVE (WASP [Wiskott-Aldrich syndrome protein] family Verprolin-homologous protein, also called Scar), we demonstrated that human platelets express all 3 isoforms. With the use of an in vitro pull-down technique, the src homology 3 (SH3) domain of insulin receptor substrate p53 (IRSp53) precipitated WAVE2 from platelet lysates more efficiently than did profilin I. The opposite was true for WAVE1, and neither precipitated WAVE3, suggesting that WAVE isoforms have different affinities to these ligands, while the SH3 domain of abl binds to all 3 isoforms. By mass spectrometry, we found that the proteins, which reportedly interact with WAVE/Scars, are present in platelets.
Thus, we have effectively employed mass spectrometry to demonstrate hitherto unreported proteins in platelets and their interactions.
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Research Products
(12 results)
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[Journal Article] WAVE/Scars in platelets2005
Author(s)
Oda A, Miki H, Wada I, Yamaguchi H, Yamazaki D, Suetsugu S, Nakajima M, Nakayama A, Okawa K, Miyazaki H, Matsuno K, Ochs HD, Machesky LM, Fujita H, Takenawa T.
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Journal Title
Blood 105
Pages: 3141-3148
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Thrombopoietin upregulates nucleolin mRNA and protein in thrombopoietin-dependent megakaryocytic cell line, UT-7/TPO.2003
Author(s)
Ito T, Fujihara M, Oda A, Wakamoto S, Yamaguchi M, Komatsu N, Miyazaki H, Azuma H, Ikeda H, Ikebuchi K.
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Journal Title
Mol Cell Blochem. 247
Pages: 75-82
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Bernard-Soulier syndrome with a homozygous 13-bp deletion in the signal peptide-coding region of platelet glycoprotein Ibβ gene.2003
Author(s)
Watanabe R, Ishabshi T, Saitoh Y, Shichishima T, Maruyama Y, Enomoto Y, Handa M, Oda A, Ambo H, Murata M, Ikeda Y.
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Journal Title
B od Coagulation & Fibrinolysis 14
Pages: 387-394
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Functional phenotype of phosphoinositide 3-kinase p85alpha null platelets characterized by an impaired response to GP VI stimulation.2003
Author(s)
Watanabe N, Hideaki Nakamrna H, Suzuki H, Oda A, Matsubara M, Moroi M, Terauchi Y, Takashi Kadowaki T, Suzuki, Shigeo Koyasu S, Ikeda Y, Handa M.
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Journal Title
Description
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