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Differential effects of PAK1-activating mutations reveal activity-dependent and -independent effects on cytoskeletal
J A Frost1, A Khokhlatchev, S Stippec
1University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041, USA.
Abstract:
PAKs are serine/threonine protein kinases that are activated by binding to Rac or Cdc42hs. Different forms of activated PAK1 have been reported to either promote membrane ruffling and focal adhesion assembly or cause focal adhesion disassembly and stress fiber dissolution. To understand the basis for these distinct morphological effects, we have examined the mechanism of mutational activation of PAK1, and characterized the effects of different active PAK1 proteins on cytoskeletal structure in vivo. We find that PAK1 contains an autoinhibitory domain that overlaps with its small G protein binding domain and that two separate activating mutations within this regulatory region each decrease autoinhibitory activity. Because only one of these mutations affects Cdc42hs binding activity, this indicates that activation of PAK1 by these mutations results from interference with the function of the autoinhibitory domain and not with small G protein binding activity. When we examined the morphological effects of these different forms of PAK1 in vivo, we found that PAK1 kinase activity was associated with disassembly of focal adhesions and actin stress fibers and that this may require interaction with potential SH3 domain-containing proteins. Lamellipodia formation and membrane ruffling caused by active PAK1 expression, however, was independent of PAK1 catalytic activity and likely requires interaction among multiple proteins binding to the PAK1 regulatory domain.
Insights
Protein kinase PAK1 activation involves relieving autoinhibition, not just G protein binding. Active PAK1 kinase promotes focal adhesion and stress fiber disassembly, while other functions are kinase-independent.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- P21-activated kinases (PAKs) are serine/threonine protein kinases regulated by small GTPases like Rac and Cdc42hs.
- Activated PAK1 has been linked to opposing cellular effects, including membrane ruffling and focal adhesion dynamics.
Purpose of the Study:
- To investigate the mechanism of PAK1 activation by mutations.
- To characterize the distinct effects of different activated PAK1 forms on cytoskeletal organization in vivo.
Main Methods:
- Analysis of PAK1 autoinhibitory domain and its overlap with the small G protein binding domain.
- In vivo characterization of cytoskeletal changes induced by various activated PAK1 mutants.
- Assessment of PAK1 kinase activity and protein-protein interactions.
Main Results:
- PAK1 activation by mutations primarily results from disrupting its autoinhibitory domain, independent of direct effects on Cdc42hs binding.
- PAK1 kinase activity is essential for the disassembly of focal adhesions and actin stress fibers, potentially involving SH3 domain proteins.
- Lamellipodia formation and membrane ruffling induced by active PAK1 are independent of its catalytic activity, suggesting regulation via interactions within the PAK1 regulatory domain.
Conclusions:
- PAK1 activation mechanisms are complex, involving autoinhibition release.
- PAK1 plays dual roles in cytoskeletal regulation, with kinase-dependent and kinase-independent pathways influencing cell morphology.
- Understanding PAK1 regulation provides insights into cellular processes like adhesion and motility.