Activating Mutations in PAK1, Encoding p21-Activated Kinase 1, Cause a Neurodevelopmental Disorder

Area of Science:

• Molecular Biology
• Neuroscience
• Genetics

Background:

• p21-activated kinases (PAKs) are crucial serine/threonine protein kinases that act as effectors for RHO GTPases like CDC42 and RAC1.
• PAK1 activity is regulated by homodimerization, with activation occurring upon binding to CDC42 or RAC1, leading to dimer dissociation.
• PAKs play significant roles in cellular processes, including actin cytoskeleton reorganization and regulation of cell spreading.

Purpose of the Study:

• To investigate the functional consequences of de novo PAK1 mutations identified in individuals with neurodevelopmental disorders.
• To determine if these PAK1 variants exhibit altered kinase activity, dimerization, and cellular phenotypes.
• To explore the therapeutic potential of PAK1 inhibition in reversing the observed cellular defects.

Main Methods:

• Identification and characterization of de novo PAK1 mutations (c.392A>G and c.1286A>G).
• Analysis of PAK1 target phosphorylation (JNK, AKT, c-JUN) and kinase activity in patient-derived fibroblasts.
• Assessment of PAK1 mutant dimerization using co-immunoprecipitation and size-exclusion chromatography.
• Evaluation of cell spreading and filopodia formation in patient fibroblasts, with and without PAK1 inhibitor FRAX486 treatment.

Main Results:

• Two de novo PAK1 mutations (p.Tyr131Cys and p.Tyr429Cys) were identified in unrelated subjects with developmental delay, macrocephaly, seizures, and ataxic gait.
• Fibroblasts from affected individuals showed enhanced phosphorylation of PAK1 targets and a trend toward increased PAK1 kinase activity.
• Both PAK1 mutants exhibited significantly reduced homodimerization compared to wild-type PAK1, indicating gain-of-function alleles.
• Patient-derived fibroblasts displayed an enrichment of filopodia, a phenotype fully reversed by the PAK1 inhibitor FRAX486.

Conclusions:

• Dominantly acting, gain-of-function PAK1 mutations cause a neurodevelopmental phenotype characterized by macrocephaly and seizures, likely due to impaired homodimerization and enhanced kinase activity.
• These findings underscore the critical role of RHO GTPase pathway members and their effectors, such as PAK1, in normal neuronal development.
• The study highlights PAK1 as a potential therapeutic target for neurodevelopmental disorders associated with its dysregulation.