DIFFERENTIAL-EFFECTS OF PAK1-ACTIVATING MUTATIONS REVEAL ACTIVITY-DEPENDENT AND ACTIVITY-INDEPENDENT EFFECTS ON CYTOSKELETAL REGULATION

PARs are serine/threonine protein kinases that are activated by bindin g to Rac or Cdc42hs, Different forms of activated PAK1 have been repor ted to either promote membrane ruffling and focal adhesion assembly or cause focal adhesion disassembly and stress fiber dissolution. To und erstand the basis for these distinct morphological effects, we have ex amined the mechanism of mutational activation of PAK1, and characteriz ed the effects of different active PAK1 proteins on cytoskeletal struc ture in vivo, We find that PAK1 contains an autoinhibitory domain that overlaps with its small G protein binding domain and that two separat e activating mutations within this regulatory region each decrease aut oinhibitory activity. Because only one of these mutations affects Cdc4 2hs binding activity, this indicates that activation of PAK1 by these mutations results from interference with the function of the autoinhib itory domain and not with small G protein binding activity. When we ex amined the morphological effects of these different forms of PAK1 in v ivo, we found that PAK1 kinase activity was associated with disassembl y of focal adhesions and actin stress fibers and that this may require interaction with potential SH3 domain-containing proteins. Lamellipod ia formation and membrane ruffling caused by active PAK1 expression, h owever, was independent of PAK1 catalytic activity and likely requires interaction among multiple proteins binding to the PARI regulatory do main.