title: Endosomal H2O2 production leads to localized cysteine sulfenic acid formation on proteins during lysophosphatidic acid-mediated cell signaling.
abstract: Lysophosphatidic acid (LPA) is a growth factor for many cells including prostate and ovarian cancer-derived cell lines. LPA stimulates H2O2 production which is required for growth. However, there are significant gaps in our understanding of the spatial and temporal regulation of H2O2-dependent signaling and the way in which signals are transmitted following receptor activation. Herein, we describe the use of two reagents, DCP-Bio1 and DCP-Rho1, to evaluate the localization of active protein oxidation after LPA stimulation by detection of nascent protein sulfenic acids. We found that LPA stimulation causes internalization of LPA receptors into early endosomes that contain NADPH oxidase components and are sites of H2O2 generation. DCP-Rho1 allowed visualization of sulfenic acid formation, indicative of active protein oxidation, which was stimulated by LPA and decreased by an LPA receptor antagonist. Protein oxidation sites colocalized with LPAR1 and the endosomal marker EEA1. Concurrent with the generation of these redox signaling-active endosomes (redoxosomes) is the H2O2- and NADPH oxidase-dependent oxidation of Akt2 and PTP1B detected using DCP-Bio1. These new approaches therefore enable detection of active, H2O2-dependent protein oxidation linked to cell signaling processes. DCP-Rho1 may be a particularly useful protein oxidation imaging agent enabling spatial resolution due to the transient nature of the sulfenic acid intermediate it detects.
authors.affiliation: Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA. Electronic address: cklomsir@wakehealth.edu.
authors.forename: Chananat
authors.initials: C
authors.lastname: Klomsiri
authors.affiliation: Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA. Electronic address: rlrogers@wakehealth.edu.
authors.forename: LeAnn C
authors.initials: LC
authors.lastname: Rogers
authors.affiliation: Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA. Electronic address: lsoito@lib.ucdavis.edu.
authors.forename: Laura
authors.initials: L
authors.lastname: Soito
authors.affiliation: Department of Biology, Wake Forest University, Winston-Salem, NC 27109, USA. Electronic address: mccaulak@wfu.edu.
authors.forename: Anita K
authors.initials: AK
authors.lastname: McCauley
authors.affiliation: Department of Chemistry, Wake Forest University, Winston-Salem, NC 27109, USA. Electronic address: kingsb@wfu.edu.
authors.forename: S Bruce
authors.initials: SB
authors.lastname: King
authors.affiliation: Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA. Electronic address: kinelson@wakehealth.edu.
authors.forename: Kimberly J
authors.initials: KJ
authors.lastname: Nelson
authors.affiliation: Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA. Electronic address: lbpoole@wakehealth.edu.
authors.forename: Leslie B
authors.initials: LB
authors.lastname: Poole
authors.affiliation: Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA. Electronic address: ldaniel@wakehealth.edu.
authors.forename: Larry W
authors.initials: LW
authors.lastname: Daniel
mesh_terms: D001549:Benzamides / Q000737:chemistry; D045744:Cell Line, Tumor; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis / Q000096:biosynthesis; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008246:Lysophospholipids / Q000378:metabolism / Q000494:pharmacology*; D008297:Male; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010666:Phenylpropionates / Q000737:chemistry; D021381:Protein Transport; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1 / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D049368:Receptors, Lysophosphatidic Acid / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013434:Sulfenic Acids / Q000032:analysis; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C513861:3-(2,6-dimethyl-4-carbamoylphenyl)propanoic acid; D001549:Benzamides; D008246:Lysophospholipids; D010666:Phenylpropionates; D049368:Receptors, Lysophosphatidic Acid; D013434:Sulfenic Acids; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; C494921:AKT2 protein, human; D051057:Proto-Oncogene Proteins c-akt; C516607:PTPN1 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; C100870:cysteinesulfenic acid; D003545:Cysteine; C032881:lysophosphatidic acid
keywords: Cysteine modifications; LPA signaling; Lipid mediators; Proliferative signaling; Protein oxidation