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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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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.
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