Focal adhesion kinase up-regulation and signaling in activated keratinocytes.
BACKGROUND
During wound healing keratinocytes undergo a process called "activation" that enables the cells to spread and migrate on wound matrix molecules. Focal adhesion kinase (FAK) is a key component of integrin-mediated intracellular signaling. We investigated the induction of FAK and its signaling activity during keratinocyte activation.


METHODS
Keratinocytes were harvested from normal human skin. Previous work has shown that culture of keratinocytes causes activation in a manner similar to reepithelialization. Freshly isolated, unactivated cells were compared with cultured, activated cells. Activated cells were further examined either as growing colonies or after replating on type I collagen. FAK content was assessed by Western blotting. FAK distribution was shown using indirect immunofluorescence. FAK signaling activity was assessed using an antiphosphotyrosine antibody.


RESULTS
FAK was not detectable by Western blotting in freshly isolated cells. In contrast FAK was detected in activated cells. FAK was up-regulated between Days 2 and 4 after cell isolation from skin. Immunostaining of activated, growing keratinocyte colonies in vitro showed a diffuse, cytoplasmic pattern. When these cells were replated on collagen, FAK became concentrated in focal adhesions. Lysates from replated cells showed increased tyrosine phosphorylation of FAK.


CONCLUSIONS
In summary FAK is induced in keratinocytes in a time course comparable to that of activation. FAK is phosphorylated and undergoes redistribution to focal adhesions when cells are plated on the beta(1) integrin ligand collagen. These data suggest that induction of FAK and subsequent FAK-induced signaling may be responsible for changes in integrin-mediated behavior of activated keratinocytes during reepithelialization.
Kim
L T
LT
Surgical Service, Veterans Affairs North Texas Health Care System, Dallas, Texas, USA. Lawrence.Kim@email.swmed.edu
Wu
J
J
Bier-Laning
C
C
Dollar
B T
BT
Turnage
R H
RH
D002478:Cells, Cultured; D003094:Collagen / Q000494:pharmacology; D004789:Enzyme Activation / Q000502:physiology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D016023:Integrins / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology / Q000201:enzymology*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D012867:Skin / Q000166:cytology / Q000201:enzymology*
D016023:Integrins; D003094:Collagen; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human