title: Proteomic analysis on insulin signaling in human hematopoietic cells: identification of CLIC1 and SRp20 as novel downstream effectors of insulin.
abstract: Insulin/IGF-I-dependent signals play important roles for the regulation of proliferation, differentiation, metabolism, and autophagy in various cells, including hematopoietic cells. Although the early protein kinase activation cascade has been intensively studied, the whole picture of intracellular signaling events has not yet been clarified. To identify novel downstream effectors of insulin-dependent signals in relatively early phases, we performed high-resolution two-dimensional electrophoresis (2-DE)-based proteomic analysis using human hematopoietic cells 1 h after insulin stimulation. We identified SRp20, a splicing factor, and CLIC1, an intracellular chloride ion channel, as novel downstream effectors besides previously reported effectors of Rho-guanine nucleotide dissociation inhibitor 2 and glutathione S-transferase-pi. Reduction in SRp20 was confirmed by one-dimensional Western blotting. Moreover, MG-132, a proteasome inhibitor, prevented this reduction. By contrast, upregulation of CLIC1 was not observed in one-dimensional Western blotting, unlike the 2-DE results. As hydrophilic proteins were predominantly recovered in 2-DE, the discrepancy between the 1-DE and 2-DE results may indicate a certain qualitative change of the protein. Indeed, the nuclear localization pattern of CLIC1 was remarkably changed by insulin stimulation. Thus insulin induces the proteasome-dependent degradation of SRp20 as well as the subnuclear relocalization of CLIC1.
authors.affiliation: Department of Hematology, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan.
authors.forename: Kumiko
authors.initials: K
authors.lastname: Saeki
authors.forename: Etsuko
authors.initials: E
authors.lastname: Yasugi
authors.forename: Emiko
authors.initials: E
authors.lastname: Okuma
authors.forename: Samuel N
authors.initials: SN
authors.lastname: Breit
authors.forename: Megumi
authors.initials: M
authors.lastname: Nakamura
authors.forename: Tosifusa
authors.initials: T
authors.lastname: Toda
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kaburagi
authors.forename: Akira
authors.initials: A
authors.lastname: Yuo
mesh_terms: D000595:Amino Acid Sequence; D018118:Chloride Channels / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D018922:HL-60 Cells; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D008969:Molecular Sequence Data; D040901:Proteomics; D016601:RNA-Binding Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: C501966:CLIC1 protein, human; D018118:Chloride Channels; D007004:Hypoglycemic Agents; D007328:Insulin; D016601:RNA-Binding Proteins; C491388:SRSF3 protein, human; D000068103:Serine-Arginine Splicing Factors