Phylogenetic and structural analysis of the Drosophila melanogaster p21-activated kinase DmPAK3

P21-activated kinases (PAKs) are a family of serine/threonine kinases whose diverse cellular functions in cytoskeletal reorganisation, cell motility, transformation and cell death are regulated both by the binding of the small RhoGTPases RAC and CDC42 and by RhoGTPase independent mechanisms. The genome of Drosophila melanogaster encodes three different PAK proteins: DmPAK1, DmMBT (DmPAK2) and DmPAK3. Although structurally related, DmPAK and DmMBT control different developmental processes and also differ in the regulation of their kinase activity through binding of RAC or CDC42. Here, we report the characterisation of DmPAK3. The phylogenetic analysis provides evidence that DmPAK3 and the related proteins from Drosophila pseudoobscura, Anopheles gambiae and Apis mellifera make up a distinct subgroup within the PAK protein family, which might be confined to insects. The structural differences of this PAK subgroup are also reflected by the selective binding of DmPAK3 to RAC-like RhoGTPases. Our biochemical analysis supports a model in which DmPAK3 can form homodimers where the N-terminal regulatory domain of one DmPAK3 protein can bind to and thereby inhibit the catalytic domain of the other DmPAK3 protein. Binding of activated RAC to the regulatory domain or mutation of the RAC-binding site in DmPAK3 relieves the inhibitory effect on kinase activity. Furthermore, our data indicate a function of DmPAK3 in reorganisation of the actin cytoskeleton.