Activation of focal adhesion kinase in adenovirus-infected human corneal fibroblasts.
OBJECTIVE
Focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase with protean downstream influences on cell cycle regulation, cytoskeletal dynamics, and cell attachment, is activated by integrin binding and aggregation. Adenoviruses, including those associated with human keratitis, enter permissive cells by an integrin-mediated mechanism. Hence, a possible relationship between adenovirus infection and tyrosine phosphorylation of FAK in human corneal cells was explored.


METHODS
Human corneal fibroblasts (HCFs) derived from donor corneas were infected for various periods with adenovirus type 19 (Ad19) or were mock infected with virus-free medium. Parallel experiments included echistatin, which is a snake venom disintegrin and a partial inhibitor of FAK. For immunoblot analysis, Triton-X-soluble and Triton-X-insoluble fractions were analyzed by SDS-PAGE and immunoblotted with phosphospecific antibodies. Expression of the interleukin (IL)-8 gene was analyzed by RT-PCR and ELISA.


RESULTS
Ad19 infection of HCFs induced tyrosine phosphorylation of a protein at 125 kDa, which was evident within 15 minutes after infection and was established by immunoprecipitation to be FAK. Immunoblot with antibody to FAK tyrosine-397 confirmed phosphorylation of this key binding site for downstream signaling proteins. Immunoblot analysis further suggested a shift in the intracellular location of phosphorylated FAK from the cytosol (Triton-X-soluble cell lysate fraction) to the cytoskeleton (Triton-X-insoluble cell lysate pellet) on infection. Finally, treatment of HCFs with echistatin reduced virus-induced expression of the neutrophil chemotaxin IL-8, previously implicated in adenoviral pathogenesis.


CONCLUSIONS
Ad19 infection of HCFs induces rapid phosphorylation of FAK, and a dramatic change in its intracellular distribution. Activation of FAK may contribute to the inflammatory response to adenovirus infection of the human cornea.
Natarajan
Kanchana
K
Molecular Pathogenesis of Eye Infection Research Center, Dean A. McGee Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Ghalayini
Abboud J
AJ
Sterling
Robert S
RS
Holbrook
Robert M
RM
Kennedy
Ronald C
RC
Chodosh
James
J
D000260:Adenoviruses, Human / Q000502:physiology*; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000201:enzymology* / Q000821:virology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000201:enzymology / Q000821:virology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8 / Q000096:biosynthesis / Q000235:genetics; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014443:Tyrosine / Q000378:metabolism
D004791:Enzyme Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8; D010455:Peptides; D012333:RNA, Messenger; C057986:echistatin; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human