May 2003
Volume 44, Issue 13
ARVO Annual Meeting Abstract  |   May 2003
Adenovirus-Mediated Activation of Serine/Threonine Protein Kinases in Human Corneal Fibroblasts
Author Affiliations & Notes
  • M.S. Rajala
    Molecular Pathogenesis of Eye Infection Research Center, Dean A. McGee Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States
  • Footnotes
    Commercial Relationships  M.S. Rajala, None.
  • Footnotes
    Support  NIH Grants RO1 EY13124 and P30 EY12190, RPB Wasserman Merit Award
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 4641. doi:
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      M.S. Rajala; Adenovirus-Mediated Activation of Serine/Threonine Protein Kinases in Human Corneal Fibroblasts . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4641.

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

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Abstract: : Purpose: Activation of tyrosine kinases in adenovirus-infected cells may mediate viral internalization and subsequent cellular pro-inflammatory gene expression. We sought to determine if adenovirus type 19 (Ad19) infection of human corneal fibroblasts (HCF) also activates cellular serine/threonine protein kinases. Methods: Primary HCF were cultured from human donor corneas and infected at third passage with cesium chloride gradient-purified Ad19 at an MOI of 50. Adenovirus- and mock-infected HCF were solubilized at various time points post-infection, and cell lysates subjected to SDS-PAGE followed by immunoblot analysis with anti-phosphoserine and anti-phosphothreonine antibodies, or immunoprecipitated for subsequent protein kinase assays. Results: Ad19 induced rapid phosphorylation of serine/threonine protein kinases of 120 kd, 60 kd and 20 kd molecular weights between 8 and 30 minutes post-infection. The 60 kd protein kinase was identified as Akt (protein kinase B) with anti-phospho-Akt antibodies specific to phosphorylation sites on serine 473 and threonine 308. Akt kinase activity was demonstrated at 10-30 minutes post infection using the GSK3 paramyosin fusion protein as a down-stream substrate. Phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI3-K) was demonstrated by immunoprecipitation to be maximal at 5 minutes post-infection, and an inhibitor of PI3-K, LY2942002. blocked Ad19-induced Akt phosphorylation as well as Akt kinase activity. Conclusions: Intracellular serine-threonine protein kinases are activated early after Ad19 infection of primary cultured HCF. Activation of the PI3-K - Akt signaling pathway is known to inhibit cellular apoptosis, and possibly provides a growth advantage for the virus. Future studies will focus on the dynamic between Akt activation and the induction of cellular apoptosis in Ad19-infected HCF.

Keywords: adenovirus • cornea: stroma and keratocytes • signal transduction 

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