Quantitative proteomic analysis of fibroblast nuclear proteins after stimulation with mitogen activated protein kinase inhibiting heparan sulfate

Research output: Contribution to journalArticle

Standard

Quantitative proteomic analysis of fibroblast nuclear proteins after stimulation with mitogen activated protein kinase inhibiting heparan sulfate. / Malmström, Johan; Marchese, Jason; Juhasz, Peter; Pukac, Laurel; Karnovsky, Morris; Marko-Varga, György; Westergren-Thorsson, Gunilla.

In: Journal of Chromatography. B, Vol. 815, No. 1-2, 05.02.2005, p. 333-342.

Research output: Contribution to journalArticle

Harvard

APA

CBE

MLA

Vancouver

Author

RIS

TY - JOUR

T1 - Quantitative proteomic analysis of fibroblast nuclear proteins after stimulation with mitogen activated protein kinase inhibiting heparan sulfate

AU - Malmström, Johan

AU - Marchese, Jason

AU - Juhasz, Peter

AU - Pukac, Laurel

AU - Karnovsky, Morris

AU - Marko-Varga, György

AU - Westergren-Thorsson, Gunilla

N1 - The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Analytical Chemistry (S/LTH) (011001004), Department of Experimental Medical Science (013210000)

PY - 2005/2/5

Y1 - 2005/2/5

N2 - Certain structures of heparan sulfate (HS) inhibit cell proliferation of fibroblasts. Whether this inhibition is dependent on inhibition of mitogenic signaling pathways or nuclear translocation of HS is unknown. In this study we investigated possible mechanism(s) and structural requirements by which antiproliferative glycosaminoglycans exert their effects on mitogen-activated protein kinase (MAP kinase) phosphorylation, a key intermediate in cell signaling, followed by quantitative proteomic analysis of nuclear proteins by stable isotope coded affinity tags, multidimensional chromatography and tandem mass spectrometry. Serum starved human lung fibroblasts were stimulated with serum, platelet derived growth factor (PDGF-BB) or epidermal growth factor (EGF) in the presence of structurally different glycosaminoglycans. Antiproliferative heparan sulfate with a high content of 2-O-sulfated iduronic acid (IdoA-2SO4) and heavily sulfated glucosamine, and the structurally related glycosaminoglycan heparin inhibited significantly serum stimulated MAP kinase phosphorylation, by at least 80% when stimulated by serum and HS6. We hypothesized that the inhibition of the MAP kinase pathway will have effect in the nuclear proteome. Therefore an isotope coded affinity tag (ICAT) reagent labeling of nuclear proteins and tandem mass spectrometry was applied, resulting in the identification and quantification of 206 proteins. Several nuclear proteins were found to be induced or repressed due to HS stimulation, where the repression EBNA-2 co-activator and the induction of PML protein were of special interest. These results show that heparan sulfate with high content of (IdoA-2SO4) and heavily sulfated glucosamine specifically inhibits MAP kinase activation with a subsequent change in the nuclear proteome of the fibroblast.

AB - Certain structures of heparan sulfate (HS) inhibit cell proliferation of fibroblasts. Whether this inhibition is dependent on inhibition of mitogenic signaling pathways or nuclear translocation of HS is unknown. In this study we investigated possible mechanism(s) and structural requirements by which antiproliferative glycosaminoglycans exert their effects on mitogen-activated protein kinase (MAP kinase) phosphorylation, a key intermediate in cell signaling, followed by quantitative proteomic analysis of nuclear proteins by stable isotope coded affinity tags, multidimensional chromatography and tandem mass spectrometry. Serum starved human lung fibroblasts were stimulated with serum, platelet derived growth factor (PDGF-BB) or epidermal growth factor (EGF) in the presence of structurally different glycosaminoglycans. Antiproliferative heparan sulfate with a high content of 2-O-sulfated iduronic acid (IdoA-2SO4) and heavily sulfated glucosamine, and the structurally related glycosaminoglycan heparin inhibited significantly serum stimulated MAP kinase phosphorylation, by at least 80% when stimulated by serum and HS6. We hypothesized that the inhibition of the MAP kinase pathway will have effect in the nuclear proteome. Therefore an isotope coded affinity tag (ICAT) reagent labeling of nuclear proteins and tandem mass spectrometry was applied, resulting in the identification and quantification of 206 proteins. Several nuclear proteins were found to be induced or repressed due to HS stimulation, where the repression EBNA-2 co-activator and the induction of PML protein were of special interest. These results show that heparan sulfate with high content of (IdoA-2SO4) and heavily sulfated glucosamine specifically inhibits MAP kinase activation with a subsequent change in the nuclear proteome of the fibroblast.

KW - Binding Sites

KW - Cell Proliferation

KW - Cells, Cultured

KW - Down-Regulation

KW - Fibroblasts

KW - Glycosaminoglycans

KW - Heparitin Sulfate

KW - Humans

KW - Isotope Labeling

KW - Lung

KW - Mass Spectrometry

KW - Mitogen-Activated Protein Kinases

KW - Nuclear Proteins

KW - Phosphorylation

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1016/j.jchromb.2004.11.054

DO - 10.1016/j.jchromb.2004.11.054

M3 - Article

VL - 815

SP - 333

EP - 342

JO - Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences

JF - Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences

SN - 0378-4347

IS - 1-2

ER -