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J. K. Klarlund, E. R. Block; PYK2 Activation Triggers EGF Receptor Signaling and Cell Motility after Wounding Sheets of Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):368.
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The presence of wounds is detected by the corneal epithelium, and it reacts by migrating rapidly to cover denuded areas. It has been recognized for some time that activation of the epidermal growth factor receptor (EGFR) is a central event in the transition to motility, and we and others have previously shown that it is activated through signaling by Src-family kinases (SFKs). Here we tested the hypothesis that members of the focal adhesion kinase family are upstream activators of the SFKs after wounding.
Multiple wounds were induced in confluent sheets of a human corneal epithelial cell line (HCLE). Analysis of the activation states of various signaling molecules was performed by immunoblotting with relevant phospho-specific antibodies and comparing to total amounts of the proteins present. The effects of interfering with Pyk2 signaling were studied by reducing Pyk2 levels with siRNA and a dominant negative construct of Pyk2, and the consequences of overexpressing Pyk2 were analyzed using adenovirus vectors. Migration rates were determined by following closure of gaps introduced in confluent monolayers of HCLE cells.
Focal adhesion kinase is not stimulated by wounding, but a different family member, Pyk2, is activated rapidly and potently. Pyk2 interaction with SFKs is increased after wounding, as determined by co-immunoprecipitation experiments, and the activities of two SFK isoforms (Src and Fyn) were increased. Disruption of Pyk2 signaling either by transfection with siRNA or by expression of a dominant-negative mutant led to inhibition of wound-induced activation of the SFKs and the EGFR, and conversely, overexpression of wild-type Pyk2 stimulated SFK and EGFR kinase activities in cells. Pyk2 siRNA or a dominant negative construct of Pyk2 inhibited cell migration. These results show that activation of Pyk2 is an early signal that promotes wound healing by stimulating the SFK/EGFR signaling pathway.
Together with previous results, wounding sheets of epithelial cells stimulates the following sequence of signaling events:Wounding →Unknown Sensor →Pyk2 →SFKs →Release of Ligands→EGFR→ Motility
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