title: Comparative proteomics of primitive hematopoietic cell populations reveals differences in expression of proteins regulating motility.
abstract: Lineage-marker depleted (Lin(-)) murine bone marrow cells expressing stem cell antigen 1 (Sca-1) were sorted on the basis of stem cell factor receptor (c-kit) expression to obtain Lin(-)Sca(+)Kit(+) or Lin(-)Sca(+)Kit(-) cells. Lin(-)Sca(+)Kit(-) cells have a markedly greater chemotactic response to stromal derived factor-1 (SDF-1). Using a novel fluorescent stain, we show that both populations generate similar levels of a key messenger, phosphatidylinositol 3,4,5 trisphosphate (PIP(3)), in response to SDF-1. Differences in motile behavior may therefore lie downstream of phosphatidylinositol 3-kinase (PI3-kinase) activation at the level of cytoskeleton regulation. The 2 cell populations were compared using 2-dimensional difference gel electrophoresis (2D-DIGE), with a maleimide CyDye fluorescent protein labeling technique that has enhanced sensitivity for low abundance samples. Comparative proteomic analysis of Cy3- and Cy5-labeled protein samples allows relative quantification of protein spots present in both cell populations; of these, 73% were common. Key protein differences were adseverin and gelsolin, actin micro-filament splicing proteins, regulated by Rac, downstream of PI3-kinase activation. Adseverin was shown to be acetylated, a novel modification for this protein. Differences in major regulators of cell shape and motility between the 2 populations can explain the differential response to SDF-1.
authors.affiliation: Department of Biomolecular Sciences, UMIST, Manchester, United Kingdom.
authors.forename: Caroline A
authors.initials: CA
authors.lastname: Evans
authors.forename: Robert
authors.initials: R
authors.lastname: Tonge
authors.forename: David
authors.initials: D
authors.lastname: Blinco
authors.forename: Andrew
authors.initials: A
authors.lastname: Pierce
authors.forename: Joanne
authors.initials: J
authors.lastname: Shaw
authors.forename: Yuning
authors.initials: Y
authors.lastname: Lu
authors.forename: Hajja G
authors.initials: HG
authors.lastname: Hamzah
authors.forename: Alexander
authors.initials: A
authors.lastname: Gray
authors.forename: C Peter
authors.initials: CP
authors.lastname: Downes
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Gaskell
authors.forename: Elaine
authors.initials: E
authors.lastname: Spooncer
authors.forename: Anthony D
authors.initials: AD
authors.lastname: Whetton
mesh_terms: D000107:Acetylation; D000818:Animals; D001854:Bone Marrow Cells; D048429:Cell Size / Q000235:genetics; D002478:Cells, Cultured; D054377:Chemokine CXCL12; D019743:Chemokines, CXC / Q000494:pharmacology; D002633:Chemotaxis / Q000187:drug effects / Q000235:genetics*; D018260:Gelsolin / Q000032:analysis; D005786:Gene Expression Regulation; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008840:Microfilament Proteins / Q000032:analysis / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000032:analysis; D011506:Proteins / Q000032:analysis / Q000235:genetics; D040901:Proteomics; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis*
chemical_list: D054377:Chemokine CXCL12; D019743:Chemokines, CXC; C515256:Cxcl12 protein, mouse; D018260:Gelsolin; D008840:Microfilament Proteins; D018129:Phosphatidylinositol Phosphates; D011506:Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; C065395:scinderin; D019869:Phosphatidylinositol 3-Kinases; D019009:Proto-Oncogene Proteins c-kit