title: Proteomic analysis of secretagogue-stimulated neutrophils implicates a role for actin and actin-interacting proteins in Rac2-mediated granule exocytosis.
abstract: BACKGROUND
  Neutrophils are abundant leukocytes that play a primary role in defence against pathogens. Neutrophils enter sites of infection where they eliminate pathogens via phagocytosis and the release of antimicrobial mediators via degranulation. Rho GTPases, particularly Rac2, play a key role in neutrophil degranulation. The purpose of this study was to identify Rac2-dependent changes in protein abundance in stimulated neutrophils.
  
  
  METHODS
  We performed a proteomic analysis on secretagogue-stimulated bone marrow neutrophils that were isolated from wild-type and Rac2-/- mice. Protein abundance was analyzed by 2-dimensional SDS-PAGE of fluorescently labelled samples which allowed the detection ~3500 proteins.
  
  
  RESULTS
  We identified 22 proteins that showed significant changes in abundance after secretagogue-stimulation of wild-type neutrophils, which did not occur in neutrophils isolated from Rac2-/- mice. As expected, the abundance of several granule proteins was reduced in wild-type cells; this did not occur in Rac2-/- neutrophils which confirms the requirement for Rac2 in degranulation. We also found changes in abundance of many actin remodelling proteins including coronin-1A, β-actin and the F-actin capping protein, (CapZ-β). Coronin-1A showed elevated levels of several isoforms after stimulation of neutrophils from wild-type, but not from Rac2-/- mice. These isoforms were immunoreactive with anti-phospho-threonine antibodies, suggesting that neutrophil stimulation triggers a Rac2-dependent kinase cascade that results in the phosphorylation of coronin-1A.
  
  
  CONCLUSIONS
  The control of Rac2-mediated degranulation in neutrophils likely functions through actin remodelling via activation of several actin-binding proteins. We found coronin-1A to be a novel downstream effector protein of this pathway that is threonine phosphorylated in response to secretagogue stimulation.
authors.affiliation: Department of Cell Biology, University of Alberta, Edmonton, AB, Canada. gary.eitzen@ualberta.ca.
authors.forename: Gary
authors.initials: G
authors.lastname: Eitzen
authors.forename: Andrea N
authors.initials: AN
authors.lastname: Lo
authors.forename: Troy
authors.initials: T
authors.lastname: Mitchell
authors.forename: John D
authors.initials: JD
authors.lastname: Kim
authors.forename: Danny V
authors.initials: DV
authors.lastname: Chao
authors.forename: Paige
authors.initials: P
authors.lastname: Lacy
references.citation: Blood. 2004 Aug 1;104(3):832-9
references.pmid: 15073033
references.citation: Subcell Biochem. 2008;48:116-23
references.pmid: 18925376
references.citation: J Immunol. 2002 Dec 1;169(11):6141-8
references.pmid: 12444117
references.citation: PLoS Biol. 2008 Jan;6(1):e1
references.pmid: 18177206
references.citation: Mol Biol Cell. 2005 Jul;16(7):3077-87
references.pmid: 15829569
references.citation: J Immunol. 1995 Apr 15;154(8):4157-65
references.pmid: 7535822
references.citation: J Proteomics. 2010 Jan 3;73(3):552-61
references.pmid: 19580889
references.citation: J Exp Med. 2010 Oct 25;207(11):2507-19
references.pmid: 20937706
references.citation: Curr Biol. 2005 Oct 25;15(20):1867-73
references.pmid: 16243035
references.citation: Nat Protoc. 2006;1(4):1732-42
references.pmid: 17487156
references.citation: Blood. 1999 Aug 1;94(3):1121-30
references.pmid: 10419906
references.citation: Am J Physiol Cell Physiol. 2008 Nov;295(5):C1354-65
references.pmid: 18799653
references.citation: J Immunol. 2007 May 1;178(9):5769-78
references.pmid: 17442961
references.citation: Front Biosci. 2008 May 01;13:5559-70
references.pmid: 18508605
references.citation: Trends Immunol. 2002 Mar;23(3):130-5
references.pmid: 11864840
references.citation: Biol Pharm Bull. 2002 Jul;25(7):837-44
references.pmid: 12132654
references.citation: Methods Mol Biol. 2006;332:343-56
references.pmid: 16878704
references.citation: Cell. 2007 Mar 9;128(5):915-29
references.pmid: 17350576
references.citation: Nat Rev Mol Cell Biol. 2003 Jan;4(1):74-80
references.pmid: 12511871
references.citation: J Cell Biol. 2002 Dec 23;159(6):993-1004
references.pmid: 12499356
references.citation: J Cell Sci. 1997 Dec;110 ( Pt 24):3071-81
references.pmid: 9365277
references.citation: Trends Immunol. 2007 Aug;28(8):340-5
references.pmid: 17627888
references.citation: Immunity. 1999 Feb;10(2):183-96
references.pmid: 10072071
references.citation: J Immunol. 2009 Aug 15;183(4):2718-28
references.pmid: 19625648
references.citation: Trends Cell Biol. 2006 Aug;16(8):421-6
references.pmid: 16806932
references.citation: Mol Biol Cell. 2005 Jun;16(6):2786-98
references.pmid: 15800061
references.citation: Mol Cell Proteomics. 2005 Aug;4(8):1205-9
references.pmid: 15911532
references.citation: Bioessays. 2005 Jun;27(6):625-32
references.pmid: 15892111
references.citation: J Immunol. 2001 Jan 15;166(2):1223-32
references.pmid: 11145705
references.citation: Am J Physiol Cell Physiol. 2007 May;292(5):C1690-700
references.pmid: 17202227
references.citation: Mol Cell Proteomics. 2005 Oct;4(10):1503-21
references.pmid: 15985654
references.citation: J Leukoc Biol. 2009 Aug;86(2):423-34
references.pmid: 19447897
references.citation: Mol Biol Cell. 1995 Dec;6(12):1659-71
references.pmid: 8590796
references.citation: Microbes Infect. 2003 Nov;5(14):1317-27
references.pmid: 14613775
references.citation: J Immunol. 1993 Feb 15;150(4):1535-43
references.pmid: 8381838
references.citation: J Immunol. 1980 Feb;124(2):855-62
references.pmid: 6153206
references.citation: J Immunol. 2008 Apr 15;180(8):5575-81
references.pmid: 18390742
references.citation: J Cell Biol. 2007 Oct 22;179(2):239-45
references.pmid: 17954607
references.citation: J Immunol. 2007 Feb 15;178(4):2421-8
references.pmid: 17277149