Proteomic analysis on insulin signaling in human hematopoietic cells: identification of CLIC1 and SRp20 as novel downstream effectors of insulin

1 Departments of Hematology and 2 Metabolic Disorder, Research Institute, International Medical Center of Japan; 3 Proteomics Collaboration Research Group, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan; and 4 Centre for Immunology, St. Vincent's Hospital, and University of New South Wales, Sydney, Australia Submitted 27 October 2004 ; accepted in final form 11 April 2005 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. HL-60 cells; PDQuest; matrix-assisted laser desorption ionization coupled to time-of-flight mass spectrometry; Mascot Address for reprint requests and other correspondence: A. Yuo, Dept. of Hematology, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan (e-mail: yuoakira{at}ri.imcj.go.jp )