Quantitative proteomics analysis of human endothelial cell membrane rafts: Evidence of MARCKS and MRP regulation in the sphingosine 1-phosphate-induce barrier enhancement

Yurong Guo, Patrick A. Singleton, Austin Rowshan, Marjan Gucek, Robert N. Cole, David R M Graham, Jennifer E. Van Eyk, Joe GN Garcia

Research output: Contribution to journalArticle

63 Citations (Scopus)

Abstract

Endothelial cell barrier dysfunction results in the increased vascular permeability observed in inflammation, tumor metastasis, angiogenesis, and atherosclerosis. Sphingosine 1-phosphate (S1P), a biologically active phosphorylated lipid growth factor released from activated platelets, enhances the endothelial cell barrier integrity in vitro and in vivo. To begin to identify the molecular mechanisms mediating S1P induced endothelial barrier enhancement, quantitative proteomics analysis (iTRAO™) was performed on membrane rafts isolated from human pulmonary artery endothelial cells in the absence or presence of S1P stimulation. Our results demonstrated that S1P mediates rapid and specific recruitment (1 μm, 5 min) of myristoylated alanine-rich protein kinase C substrate (MARCKS) and MARCKS-related protein (MRP) to membrane rafts. Western blot experiments confirmed these findings with both MARCKS and MRP. Finally, small interfering RNA-mediated silencing of MARCKS or MRP or both attenuates S1P-mediated endothelial cell barrier enhancement. These data suggest the regulation of S1P-mediated endothelial cell barrier enhancement via the cell specific localization of MARCKS and MRP and validate the utility of proteomics approaches in the identification of novel molecular targets.

Original languageEnglish (US)
Pages (from-to)689-696
Number of pages8
JournalMolecular and Cellular Proteomics
Volume6
Issue number4
DOIs
StatePublished - Apr 2007
Externally publishedYes

Fingerprint

Endothelial cells
Cell membranes
Alanine
Proteomics
Protein Kinase C
Endothelial Cells
Cell Membrane
Substrates
Proteins
Membranes
Platelets
Small Interfering RNA
sphingosine 1-phosphate
myristoylated alanine-rich C kinase substrate
Tumors
Intercellular Signaling Peptides and Proteins
Capillary Permeability
RNA Interference
Pulmonary Artery
Lipids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Quantitative proteomics analysis of human endothelial cell membrane rafts : Evidence of MARCKS and MRP regulation in the sphingosine 1-phosphate-induce barrier enhancement. / Guo, Yurong; Singleton, Patrick A.; Rowshan, Austin; Gucek, Marjan; Cole, Robert N.; Graham, David R M; Van Eyk, Jennifer E.; Garcia, Joe GN.

In: Molecular and Cellular Proteomics, Vol. 6, No. 4, 04.2007, p. 689-696.

Research output: Contribution to journalArticle

Guo, Yurong ; Singleton, Patrick A. ; Rowshan, Austin ; Gucek, Marjan ; Cole, Robert N. ; Graham, David R M ; Van Eyk, Jennifer E. ; Garcia, Joe GN. / Quantitative proteomics analysis of human endothelial cell membrane rafts : Evidence of MARCKS and MRP regulation in the sphingosine 1-phosphate-induce barrier enhancement. In: Molecular and Cellular Proteomics. 2007 ; Vol. 6, No. 4. pp. 689-696.
@article{ad70ccdea5d34c0298e1407442844432,
title = "Quantitative proteomics analysis of human endothelial cell membrane rafts: Evidence of MARCKS and MRP regulation in the sphingosine 1-phosphate-induce barrier enhancement",
abstract = "Endothelial cell barrier dysfunction results in the increased vascular permeability observed in inflammation, tumor metastasis, angiogenesis, and atherosclerosis. Sphingosine 1-phosphate (S1P), a biologically active phosphorylated lipid growth factor released from activated platelets, enhances the endothelial cell barrier integrity in vitro and in vivo. To begin to identify the molecular mechanisms mediating S1P induced endothelial barrier enhancement, quantitative proteomics analysis (iTRAO™) was performed on membrane rafts isolated from human pulmonary artery endothelial cells in the absence or presence of S1P stimulation. Our results demonstrated that S1P mediates rapid and specific recruitment (1 μm, 5 min) of myristoylated alanine-rich protein kinase C substrate (MARCKS) and MARCKS-related protein (MRP) to membrane rafts. Western blot experiments confirmed these findings with both MARCKS and MRP. Finally, small interfering RNA-mediated silencing of MARCKS or MRP or both attenuates S1P-mediated endothelial cell barrier enhancement. These data suggest the regulation of S1P-mediated endothelial cell barrier enhancement via the cell specific localization of MARCKS and MRP and validate the utility of proteomics approaches in the identification of novel molecular targets.",
author = "Yurong Guo and Singleton, {Patrick A.} and Austin Rowshan and Marjan Gucek and Cole, {Robert N.} and Graham, {David R M} and {Van Eyk}, {Jennifer E.} and Garcia, {Joe GN}",
year = "2007",
month = "4",
doi = "10.1074/mcp.M600398-MCP200",
language = "English (US)",
volume = "6",
pages = "689--696",
journal = "Molecular and Cellular Proteomics",
issn = "1535-9476",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "4",

}

TY - JOUR

T1 - Quantitative proteomics analysis of human endothelial cell membrane rafts

T2 - Evidence of MARCKS and MRP regulation in the sphingosine 1-phosphate-induce barrier enhancement

AU - Guo, Yurong

AU - Singleton, Patrick A.

AU - Rowshan, Austin

AU - Gucek, Marjan

AU - Cole, Robert N.

AU - Graham, David R M

AU - Van Eyk, Jennifer E.

AU - Garcia, Joe GN

PY - 2007/4

Y1 - 2007/4

N2 - Endothelial cell barrier dysfunction results in the increased vascular permeability observed in inflammation, tumor metastasis, angiogenesis, and atherosclerosis. Sphingosine 1-phosphate (S1P), a biologically active phosphorylated lipid growth factor released from activated platelets, enhances the endothelial cell barrier integrity in vitro and in vivo. To begin to identify the molecular mechanisms mediating S1P induced endothelial barrier enhancement, quantitative proteomics analysis (iTRAO™) was performed on membrane rafts isolated from human pulmonary artery endothelial cells in the absence or presence of S1P stimulation. Our results demonstrated that S1P mediates rapid and specific recruitment (1 μm, 5 min) of myristoylated alanine-rich protein kinase C substrate (MARCKS) and MARCKS-related protein (MRP) to membrane rafts. Western blot experiments confirmed these findings with both MARCKS and MRP. Finally, small interfering RNA-mediated silencing of MARCKS or MRP or both attenuates S1P-mediated endothelial cell barrier enhancement. These data suggest the regulation of S1P-mediated endothelial cell barrier enhancement via the cell specific localization of MARCKS and MRP and validate the utility of proteomics approaches in the identification of novel molecular targets.

AB - Endothelial cell barrier dysfunction results in the increased vascular permeability observed in inflammation, tumor metastasis, angiogenesis, and atherosclerosis. Sphingosine 1-phosphate (S1P), a biologically active phosphorylated lipid growth factor released from activated platelets, enhances the endothelial cell barrier integrity in vitro and in vivo. To begin to identify the molecular mechanisms mediating S1P induced endothelial barrier enhancement, quantitative proteomics analysis (iTRAO™) was performed on membrane rafts isolated from human pulmonary artery endothelial cells in the absence or presence of S1P stimulation. Our results demonstrated that S1P mediates rapid and specific recruitment (1 μm, 5 min) of myristoylated alanine-rich protein kinase C substrate (MARCKS) and MARCKS-related protein (MRP) to membrane rafts. Western blot experiments confirmed these findings with both MARCKS and MRP. Finally, small interfering RNA-mediated silencing of MARCKS or MRP or both attenuates S1P-mediated endothelial cell barrier enhancement. These data suggest the regulation of S1P-mediated endothelial cell barrier enhancement via the cell specific localization of MARCKS and MRP and validate the utility of proteomics approaches in the identification of novel molecular targets.

UR - http://www.scopus.com/inward/record.url?scp=34247338134&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34247338134&partnerID=8YFLogxK

U2 - 10.1074/mcp.M600398-MCP200

DO - 10.1074/mcp.M600398-MCP200

M3 - Article

C2 - 17210631

AN - SCOPUS:34247338134

VL - 6

SP - 689

EP - 696

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

IS - 4

ER -