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A new constitutively active brain PAK3 isoform displays modified specificities toward Rac and Cdc42 GTPases
Veronique Rousseau1, Olivier Goupille, Nathalie Morin
1Laboratoire de Neurobiologie Cellulaire et Moléculaire, CNRS, 1 Avenue de la Terrasse, 91198 Gif sur Yvette, France.
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.
Insights
A new p21-activated kinase 3 (PAK3) isoform, PAK3b, exhibits high intrinsic kinase activity and altered GTPase binding due to an alternative exon. This suggests distinct signaling roles for PAK3b in neurons compared to PAK3a.
Area of Science:
- Molecular Biology
- Neuroscience
- Cell Signaling
Background:
- p21-activated kinases (PAK) regulate cytoskeleton dynamics and cell cycle.
- PAK3 is a key member involved in neuronal functions.
- Alternative splicing is a mechanism for generating protein diversity.
Purpose of the Study:
- To characterize a novel mammalian PAK3 mRNA isoform (PAK3b) generated by alternative splicing.
- To investigate the functional and biochemical properties of PAK3b.
- To compare the signaling capabilities of PAK3b with the canonical PAK3a isoform.
Main Methods:
- RT-PCR and sequencing to identify and characterize the new mRNA.
- Kinase activity assays to measure enzymatic function.
- GTPase binding assays to assess protein-protein interactions.
Main Results:
- A new PAK3 isoform, PAK3b, was identified, featuring a 45-bp alternatively spliced exon encoding 15 amino acids.
- PAK3b exhibits high basal kinase activity, independent of GTPase stimulation, unlike PAK3a.
- The insertion in PAK3b disrupts the autoinhibitory domain, impairs Rac and Cdc42 binding, and alters GTPase specificity.
Conclusions:
- The novel PAK3b isoform possesses unique biochemical properties.
- PAK3b's altered activity and binding suggest distinct roles in neuronal signaling pathways.
- Alternative splicing of PAK3 contributes to functional diversity in the nervous system.