Abstract
p21-activated kinase 1 (PAK1) and PAK3 belong to group I of the PAK family and control cell movement and division. They also regulate dendritic spine formation and maturation in the brain, and play a role in synaptic transmission and synaptic plasticity. PAK3, in particular, is known for its implication in X-linked intellectual disability. The pak3 gene is expressed in neurons as a GTPase-regulated PAK3a protein and also as three splice variants which display constitutive kinase activity. PAK1 regulation is based on its homodimerization, forming an inactive complex. Here, we analyze the PAK3 capacity to dimerize and show that although PAK3a is able to homodimerize, it is more likely to form heterodimeric complexes with PAK1. We further show that two intellectual disability mutations impair dimerization with PAK1. The b and c inserts present in the regulatory domain of PAK3 splice variants decrease the dimerization but retain the capacity to form heterodimers with PAK1. PAK1 and PAK3 are co-expressed in neurons, are colocalized within dendritic spines, co-purify with post-synaptic densities, and co-immunoprecipitate in brain lysates. Using kinase assays, we demonstrate that PAK1 inhibits the activity of PAK3a but not of the splice variant PAK3b in a trans-regulatory manner. Altogether, these results show that PAK3 and PAK1 signaling may be coordinated by heterodimerization.
Highlights
Regulation of group I p21-activated kinases is based on dimeric conformation
The presence of the mutations L102F and R436E-K437D in one PAK3 protein totally abrogates dimer formation (1.39 Ϯ 0.8% of PAK3a homodimer). These results show that L102 and R436-K437 residues are strongly involved in PAK3 dimer formation
To define the precise domains involved in the complex formation, a twohybrid assay was performed with the N-terminal moiety and the C-terminal moiety of PAK3 fused to the Lex binding domain or the GAD transactivation domain
Summary
Regulation of group I p21-activated kinases is based on dimeric conformation. Results: PAK3a forms regulatory heterodimers with PAK1 in vitro and in vivo. The pak gene is expressed in neurons as a GTPase-regulated PAK3a protein and as three splice variants which display constitutive kinase activity. We demonstrate that PAK1 inhibits the activity of PAK3a but not of the splice variant PAK3b in a trans-regulatory manner. These results show that PAK3 and PAK1 signaling may be coordinated by heterodimerization. In an attempt to characterize the mechanisms of regulation of PAK3a and of the three PAK3 splice variants, we analyzed, in vitro, the ability of PAK3 proteins to form dimers and to interact with the other neuronal PAK protein, PAK1. Our data show that PAK3 proteins form heterodimers with PAK1 and suggest that PAKs heterodimerization can coordinate PAK signaling and help to synchronize actin polymerization and spine stabilization
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