Fig. 4. The N terminus and FAT domain of PYK2 are required for PYK2-mediated actin cytoskeleton reorganization. (a) Mapping domains of PYK2 required for actin cytoskeleton reorganization and focal adhesion targeting. Plasmids encoding wild-type PYK2 (A,A') and its deletion mutants including PYK21-88 (B,B'), PYK21-416 (C,C'), PYK21-868 (D,D'), PYK21-902 (E,E') and PYK2936-1009 (F,F') were microinjected into Swiss 3T3 cells. The microinjected cells were fixed 2-4 hours after injection and immunostained with antibodies against PYK2 (polyclonal, against PYK2 amino acids 587-988) (A-F) and paxillin (A'-F'). (b) PYK2 and PYK2 mutants and a summary of the phenotypes of Swiss 3T3 cells expressing these mutants. The numbers on the diagrams represent the amino acid residues in PYK2. The proline-rich sequences, Band 4.1, kinase and FAT domains are indicated. The percentage of cytoskeleton reorganizing activity was determined by counting the number of PYK2-expressing cells with podosome-like focal adhesions and dividing by the total number of PYK2-expressing cells. More than 200 injected cells of each construct were counted. The percentage of total PYK2-expressing cells with normal or podosome-like focal adhesion localization of PYK2 is also listed.