p21-activated kinases (PAKs) are Cdc42 effectors found in metazoans, fungi and protozoa. They are subdivided into PAK1-like (group I) or PAK4-like (group II) kinases. Human PAK4 is widely expressed and its regulatory mechanism is unknown. We show that PAK4 is strongly inhibited by a newly identified autoinhibitory domain (AID) formed by amino acids 20 to 68, which is evolutionarily related to that of other PAKs. In contrast to group I kinases, PAK4 is constitutively phosphorylated on Ser 474 in the activation loop, but held in an inactive state until Cdc42 binding. Thus, group II PAKs are regulated through conformational changes in the AID rather than A-loop phosphorylation.

p21-activated kinases (PAKs) associate with a guanine nucleotide exchange factor, Pak-interacting exchange factor (PIX), which in turn binds the paxillin-associated adaptor GIT1 that targets the complex to focal adhesions. Here, a detailed structure-function analysis of GIT1 reveals how this multidomain adaptor also participates in activation of PAK. Kinase activation does not occur via Cdc42 or Rac1 GTPase binding to PAK. The ability of GIT1 to stimulate ␣PAK autophosphorylation requires the participation of the GIT N-terminal Arf-GAP domain but not Arf-GAP activity and involves phosphorylation of PAK at residues common to Cdc42-mediated activation. Thus, the activation of PAK at adhesion complexes involves a complex interplay between the kinase, Rho GTPases and protein partners that provide localization cues.

Paxillin, a major focal-adhesion complex component belongs to the subfamily of LIM domain proteins and participates in cell adhesion-mediated signal transduction. It is implicated in cell-motility responses upon activation of cell-surface receptors and can recruit, among others, the GIT1 [GRK (G-protein-coupled-receptor kinase)-interacting ARF (ADP-ribosylation factor) GAP (GTPase-activating protein)]-PIX [PAK (p21-activated kinase)-interacting exchange factor]-PAK1 complex. Several adhesion proteins including zyxin, Hic5 and Trip6 are also nuclear and can exert transcriptional effects. In the present study we show that endogenous paxillin shuttles between the cytoplasm and nucleus, and we have used a variety of tagged paxillin constructs to map the nuclear export signal. This region overlaps an important LD(4) motif that binds GIT1 and FAK1 (focal-adhesion kinase 1). We provide evidence that phosphorylation of Ser(272) within LD(4) blocks nuclear export, and we show that this modification also reduces GIT1, but not FAK1, binding; however, Ser(272) phosphorylation does not appear to be mediated by PAK1 as previously suggested. Expression of nuclear-localized paxillin LIM domains stimulate DNA synthesis and cell proliferation. By real-time PCR analysis we have established that overexpression of either full-length paxillin or a truncated nuclear form suppresses expression of the parental imprinted gene H19, and modulation of this locus probably affects the rate of NIH-3T3 cell proliferation.

Crystal structures of inactive PAK1(K299R) and the activation (A)-loop phospho-mimetic PAK1(T423E) have suggested that the kinase domain is in an active state regardless of activation loop status. Contrary to a large body of literature, we find that neither is PAK1(T423E) active in cells, nor does it exhibit significant activity in vitro. To explain these discrepancies all-atom molecular dynamics (MD) simulations of PAK1(phospho-T423) in complex with ATP and substrate were performed. These simulations point to a key interaction between PAK1 Lys308, at the end of the alphaC helix, and the pThr423 phosphate group, not seen in X-ray structures. The orthologous PAK4 Arg359 fulfills the same role in immobilizing the alphaC helix. These in silico predictions were validated by experimental mutagenesis of PAK1 and PAK4. The simulations explain why the PAK1 A-loop phospho-mimetic is inactive, but also point to a key functional interaction likely found in other protein kinases.

The regulation of Rac1 by HACE1-mediated ubiquitination and proteasomal degradation is emerging as an essential element in the maintenance of cell homeostasis. However, how the E3 ubiquitin ligase activity of HACE1 is regulated remains undetermined. Using a proteomic approach, we identified serine 385 as a target of group-I PAK kinases downstream Rac1 activation by CNF1 toxin from pathogenic E. coli. Moreover, cell treatment with VEGF also promotes Ser-385 phosphorylation of HACE1. We have established in vitro that HACE1 is a direct target of PAK1 kinase activity. Mechanistically, we found that the phospho-mimetic mutant HACE1(S385E), as opposed to HACE1(S385A), displays a lower capacity to ubiquitinate Rac1 in cells. Concomitantly, phosphorylation of Ser-385 plays a pivotal role in controlling the oligomerization state of HACE1. Finally, Ser-385 phosphorylated form of HACE1 localizes in the cytosol away from its target Rac1. Together, our data point to a feedback inhibition of HACE1 ubiquitination activity on Rac1 by group-I PAK kinases.