A New Constitutively Active Brain PAK3 Isoform Displays Modified Specificities toward Rac and Cdc42 GTPases

Abstract

p21-activated kinases (PAK) are involved in the control of cytoskeleton dynamics and cell cycle progression. Here we report the characterization of a new mammalian PAK3 mRNA that contains a 45-bp alternatively spliced exon. This exon encodes for 15 amino acids that are inserted in the regulatory domain, inside the autoinhibitory domain but outside the Cdc42 and Rac interactive binding domain. The transcript of the 68-kDa new isoform named PAK3b is expressed in various areas of the adult mouse brain. In contrast to PAK3 without the exon b (PAK3a), whose basal kinase activity is weak in resting cells, PAK3b displays a high kinase activity in starved cells that is not further stimulated by active GTPases. Indeed, we demonstrate that the autoinhibitory domain of PAK3b no longer inhibits the kinase activity of PAK3. Moreover, we show that the 15-amino acid insertion within the autoinhibitory domain impedes the ability of PAK3b to bind to the GTPases Rac and Cdc42 and changes its specificity toward the GTPases. Altogether, our results show that the new PAK3b isoform has unique properties and would signal differently from PAK3a in neurons.

The p21-activated Kinase PAK3 Forms Heterodimers with PAK1 in Brain Implementing Trans-regulation of PAK3 Activity

“…The insert was then cloned in the GAD vector. The GST-Cdc42-V12 prokaryotic expression plasmid was previously described (19). All plasmid coding sequences obtained were confirmed by sequencing.…”

“…Plasmid Constructs-The following pcDNA3-HA-based plasmids were previously described: pcDNA3-HA-PAK3a-WT, -PAK3a-K297L (kinase-dead), -PAK3b-WT, -PAK3b-K297L (19), -PAK3a-A365E, -PAK3a-R419X, -PAK3a-R67C (24), -PAK3c-WT, -PAK3cb-WT (17). The following FLAG-based constructs were previously described (5): pFLAG-PAK3a-WT, pFLAGPAK3a-K297L, pFLAG-PAK1-K299R (kinase-dead and named pFLAG-PAK1-KD).…”

“…Unlike PAK3a, these splice variants are constitutively active and their interaction with active GTPases of the Rac/ Cdc42 family are profoundly diminished compared with PAK3a. Moreover, their AID cannot inhibit the kinase activity of a PAK3a kinase domain (19). The molecular basis of the specific functions of PAK3 and its splice variants are largely unknown (20), and their mechanisms of regulation deserve to be extensively studied.…”

pak2a mutations cause cerebral hemorrhage in redhead zebrafish

“…This truncation removes nearly the entire kinase domain (Fig. 2F) and is predicted to be nonfunctional based on the loss of activity that has been reported because of missense mutations in the kinase domains of several PAK homologs (24)(25)(26).…”

Distinct Roles of the Rho-GTPase Associated Kinases PAK and ROCK in the Regulation of Dendritic Spines and Synaptic Plasticity

“…Group I PAKs have also been implicated in important activities such as cellular motility, morphology, apoptosis signaling and gene transcription. PAK2 is widely expressed in most regions in the body, whereas PAK1 and PAK3 are more prominent in the brain [17,[32][33][34][35], including hippocampal dendritic spines [36][37][38]. Both PAK1 and PAK3 are present in neurons at various stages of development in rodents, being more prominent in the hippocampus and cortex [32,[39][40][41].…”