Fig. 4. FAK catalytic activity, PR domains, and localization in focal adhesion sites are required for proper allocation of FN matrix. (A) Loss-of-function experiments. FAK^+/+ cells were transduced with FAK autophosphorylation mutant (Y397F FAK), kinase-dead FAK (KD FAK), PR domains mutant (PA FAK), FRNK, and L1034S FRNK using adenoviral vectors and plated at confluent density. Forty-eight hours later only non-transduced cells and cells transduced with L1034S FRNK, which does not localize in focal contacts, retained complex FN matrix organization, as assessed with staining of live cells. Western blots show the phosphorylation states of Y397 and Y861 on FAK and FRNK and expression levels of endogenous and exogenous FAK and FRNK proteins. FRNK expression could be detected only with antibodies that recognize an epitope at the C terminus of the full-length FAK protein. (B) Gain-of-function experiments. FAK^-/- cells were transduced with wild-type FAK, Y397F FAK, KD FAK, PA FAK and FRNK using adenoviral vectors. Transduced cells were plated at confluent density. Only wild-type FAK was able to rescue organization of FN matrix in FAK^-/- cells 48 hours later. Whole lysate western blots show the phosphorylation states of Y397 and Y861 on FAK and FRNK, and the expression levels of exogenous FAK and FRNK proteins. Arrows point to the size of FRNK, and arrowheads to the FAK band in all panels. Actin was used as a loading control in both sets of experiments.