April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
PEDF-induced Dephosphorylation Of VEGFR1 Requires Presenilin-1 To Act As An Adaptor Protein Between VEGFR1 And Vascular Endothelial Protein Tyrosine Phosphatase (VE-PTP)
Author Affiliations & Notes
  • Zhijuan Chen
    Anatomy and Cell Biology,
    University of Florida, Gainesville, Florida
  • Jun Cai
    Anatomy and Cell Biology,
    University of Florida, Gainesville, Florida
  • Qing Ruan
    Anatomy and Cell Biology,
    University of Florida, Gainesville, Florida
  • Song Han
    Surgery,
    University of Florida, Gainesville, Florida
  • Li Liu
    Pharmacology and Therapeutics,
    University of Florida, Gainesville, Florida
  • Xiaoping Qi
    Anatomy and Cell Biology,
    University of Florida, Gainesville, Florida
  • Lin Wu
    Anatomy and Cell Biology,
    University of Florida, Gainesville, Florida
  • Joshua Haakenson
    Anatomy and Cell Biology,
    University of Florida, Gainesville, Florida
  • Maria B. Grant
    Pharmacology and Therapeutics,
    University of Florida, Gainesville, Florida
  • Michael E. Boulton
    Anatomy and Cell Biology,
    University of Florida, Gainesville, Florida
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 4842. doi:
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      Zhijuan Chen, Jun Cai, Qing Ruan, Song Han, Li Liu, Xiaoping Qi, Lin Wu, Joshua Haakenson, Maria B. Grant, Michael E. Boulton; PEDF-induced Dephosphorylation Of VEGFR1 Requires Presenilin-1 To Act As An Adaptor Protein Between VEGFR1 And Vascular Endothelial Protein Tyrosine Phosphatase (VE-PTP). Invest. Ophthalmol. Vis. Sci. 2011;52(14):4842.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Previously, we have shown that PEDF is able to rapidly downregulate VEGF-induced VEGFR1 phosphorylation in endothelial cells via a presenilin-dependent mechanism (Cai et al, J Biol Chem 2006). The aim of this study was to determine the potential role of phosphatases in this presenilin-mediated PEDF-induced dephosphorylation of VEGFR1.

Methods: : Retinal microvascular endothelial cells were treated with VEGF and/or PEDF in the presence of, or the absence of, γ-secretase inhibitors. The phosphorylation status of VEGFR1was assessed using a sandwich ELISA. The effect of siRNAs against protein tyrosine phosphatases 1 & 2 (PTP1, PTP2), vascular endothelial PTP (VE-PTP) and presenilin-1 (PS1) on VEGFR1 phosphorylation were assessed. Immuno-precipitation/Western blot was used to characterize protein-protein association. A proximity ligation assay (PLA) was employed to detect protein complexes in situ.

Results: : Knockdown VE-PTP abolished PEDF-induced dephosphorylation of VEGFR1 while no effect was observed with knockdown of either PTP1 or PTP2 alone. Co-immunoprecipitation followed by Western blot indicated that full-length PS1 coprecipitation with VE-PTP was significantly increased by PEDF while the level of the PS1 fragment (PS1-CTF, a component of the γ-secretase complex) was unchanged. Furthermore, PEDF treatment resulted in an increase in the association of full-length PS1 with VEGFR1 by 30 minutes. siRNA against PS1 blocked PEDF downregulation of VEGFR1 phosphorylation as well as the association between VE-PTP and VEGFR1 indicating that full-length PS1 couples with VE-PTP and VEGFR1 upon PEDF treatment. γ-secretase inhibitors were unable to inhibit the effect of PEDF on VEGFR1 phosphorylation, suggesting that PS1 was acting independently of the γ-secretase complex. PLA provided direct evidence that while VE-PTP and VEGFR1 complexes exist at basal levels in unstimulated cells, complex formation is greatly increased upon stimulation with PEDF.

Conclusions: : These results indicate a regulatory mechanism for VEGFR1 whereby full-length PS1 acts as an adaptor protein for VE-PTP and VEGFR1 allowing VE-PTP to downregulate VEGF-induced VEGFR1 phosphorylation.

Keywords: growth factors/growth factor receptors • phosphorylation • vascular endothelial growth factor 
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