June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
A Newly Discovered Transglutaminase-2-Paxillin Binding is Important for Adhesion Complex Regulation
Author Affiliations & Notes
  • evelyn png
    Singapore Eye Research Institute, Singapore, Singapore
    Department of Ophthalmology, Yong Loo Lin School of Medicine, Singapore, Singapore
  • Kh Poon
    Singapore Eye Research Institute, Singapore, Singapore
  • Louis Tong
    Singapore Eye Research Institute, Singapore, Singapore
    Department of Ophthalmology, Yong Loo Lin School of Medicine, Singapore, Singapore
  • Footnotes
    Commercial Relationships evelyn png, None; Kh Poon, None; Louis Tong, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 4898. doi:
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    • Get Citation

      evelyn png, Kh Poon, Louis Tong; A Newly Discovered Transglutaminase-2-Paxillin Binding is Important for Adhesion Complex Regulation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4898.

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

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Abstract
 
Purpose
 

Cell adhesion and migration in ocular surface wound healing underpin diseases such as allergies and persistent epithelial defects. Transglutaminase (TG)-2 is a multifunctional protein which plays important role in cell adhesion and migration and can be found in the ocular surface. Previously we showed that reduction of TG-2 by RNA interference reduced cell adhesion, spreading and migration. Also, we found that interference with TG-2 was associated to reduce phosphorylation of paxillin at serine 178, which are important for cell adhesion and migration. We aimed to evaluate the possible interaction between TG-2 and paxillin.

 
Methods
 

In this study, stable human corneal epithelial cell line expressing shRNA targeting TG-2 (shTG) and non-specific scrambled shRNA (control) were used. A combination of techniques such as co-immunoprecipitation, western blotting, in-vitro protein interaction assay based on non-label optic grating sensing, in-vitro autoradiographic kinase assay and immunohistochemistry were performed to evaluate the possible relationship between TG-2 and paxillin.

 
Results
 

Paxillin co-immunoprecipitated with TG-2 in corneal epithelial cell lysates. Other adhesion proteins such as vinculin and focal adhesion kinase were also present in these paxillin containing complexes. In-vitro testing showed that TG-2 interacted significantly with recombinant paxillin in a dose dependent manner. As a positive control, recombinant beta-3 integrin also bind to immobilised paxillin. In our kinase assay, recombinant TG-2 was unable to phosphorylate purified paxillin. A library of 190 known serine/threonine kinases was screened and after accounting for the auto-phosphorylation of the kinases, JNK1 was found to have the highest relative activity. Immunohistochemistry performed on migrating corneal epithelial cell sheet after scratch assay showed that paxillin in shTG cells were not localized in the advancing edge of the leading cell, unlike in control cells.<br />

 
Conclusions
 

TG-2 interacts directly with paxillin in adhesion complexes, but it does not directly phosphorylate paxillin. The relationship between TG-2 and paxillin could be used as a possible target in ocular wound healing. In addition, JNK1 may be the kinase that mediates the phosphorylation of paxillin.<br />

 
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