May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Inhibition of CDK5 Activity Alters Localization of cSRC and Promotes Corneal Wound Healing in vitro
Author Affiliations & Notes
  • C.Y. Gao
    NEI/NIH, Bethesda, MD, United States
  • M.A. Stepp
    Anatomy and Cell Biology, George Washington University, Washington, DC, United States
  • P.S. Zelenka
    Anatomy and Cell Biology, George Washington University, Washington, DC, United States
  • Footnotes
    Commercial Relationships  C.Y. Gao, None; M.A. Stepp, None; P.S. Zelenka, None.
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 3271. doi:
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      C.Y. Gao, M.A. Stepp, P.S. Zelenka; Inhibition of CDK5 Activity Alters Localization of cSRC and Promotes Corneal Wound Healing in vitro . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3271.

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Abstract

Abstract: : Purpose: cSrc, a signaling protein known to regulate cell adhesion and migration, has been reported to be a substrate of cyclin dependent kinase 5 (Cdk5). Our previous work has shown that Cdk5 overexpression in the corneal epithelial cells inhibits healing of debridement wounds in transgenic mice and retards migration of transfected corneal epithelial cells in vitro. The present study was undertaken to test whether Cdk5 may exert its effect on migration by affecting the activation or localization of cSrc. Methods: Total and active c-Src were analyzed in whole cell lysates and Cdk5 immunoprecipitates from A6(1) mouse corneal epithelial cells following transfection with c-Src, Cdk5 or Cdk5-T33 cDNAs with or without the Cdk5 inhibitor, olomoucine(15 µM). Scrape wounded A6(1) cultures were stimulated with PDGF and immunofluorescence was used to localize active c-Src in the presence and absence of olomoucine. Mouse corneas were subjected to central 1.5mm debridement wounds and eyeballs were cultured overnight in the presence or absence of olomoucine. Wound area was determined by image analysis 12 hours after wounding. Results: Cdk5 and cSrc co-immunoprecipitated from co-transfected A6(1) cells. Immunoblotting with an antibody that recognizes active cSrc (phospho-Y416) detected two forms that co-immunoprecipitate with Cdk5, possibly corresponding to different phosphorylation states. The higher mobility band was selectively reduced by expression of a kinase-dead mutation of Cdk5 (Cdk5-T33) in transfected cells, and was abolished by oloumycin. Oloumycin greatly increased the localization of active cSrc in lamellapodia and filopodia of the leading edge in scrape wounded cultures of A6(1) cells and increased the rate of debridement wound closure in cultured eyes by a factor of two (p<0.003). Conclusions: Inhibition of Cdk5 activity promotes the localization of active cSrc to the leading edge of scrape wounds in vitro and increases the rate of debridement wound closure in cultured eyes. Thus, Cdk5 may regulate corneal wound closure by regulating the subcellular localization of activated cSrc.

Keywords: wound healing • cornea: epithelium • cornea: basic science 
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