In this study, we show that phosphorylated 3-phosphoinositide-dependent kinase 1 (PDK1) phosphorylates p21-activated kinase 1 (PAK1) in the presence of sphingosine. We identify threonine 423, a conserved threonine in the activation loop of kinase subdomain VIII, as the PDK1 phosphorylation site on PAK1. Threonine 423 is a previously identified PAK1 autophosphorylation site that lies within a PAK consensus phosphorylation sequence. After pretreatment with phosphatases, autophosphorylation of PAK1 occurred at all major sites except threonine 423. A phosphothreonine 423-specific antibody detected phosphorylation of recombinant, catalytically inactive PAK1 after incubation with wild-type PAK1, indicating phosphorylation of threonine 423 occurs by an intermolecular mechanism. The biological significance of PDK1 phosphorylation of PAK1 at threonine 423 in vitro is supported by the observation that these two proteins interact in vivo and that PDK1-phosphorylated PAK1 has an increased activity toward substrate. An increase of phosphorylation of catalytically inactive PAK1 was observed in COS-7 cells expressing wild-type, but not catalytically inactive, PDK1 upon elevation of intracellular sphingosine levels. PDK1 phosphorylation of PAK1 was not blocked by pretreatment with wortmannin or when PDK1 was mutated to prevent phosphatidylinositol binding, indicating this process is independent of phosphatidylinositol 3-kinase activity. The data presented here provide evidence for a novel mechanism for PAK1 regulation and activation.

The ability of cells to recognize and respond with directed motility to chemoattractant agents is critical to normal physiological function. Neutrophils represent the prototypic chemotactic cell in that they respond to signals initiated through the binding of bacterial peptides and other chemokines to G protein-coupled receptors with speeds of up to 30 microm/min. It has been hypothesized that localized regulation of cytoskeletal dynamics by Rho GTPases is critical to orchestrating cell movement. Using a FRET-based biosensor approach, we investigated the dynamics of Rac GTPase activation during chemotaxis of live primary human neutrophils. Rac has been implicated in establishing and maintaining the leading edge of motile cells, and we show that Rac is dynamically activated at specific locations in the extending leading edge. However, we also demonstrate activated Rac in the retracting tail of motile neutrophils. Rac activation is both stimulus and adhesion dependent. Expression of a dominant-negative Rac mutant confirms that Rac is functionally required both for tail retraction and for formation of the leading edge during chemotaxis. These data establish that Rac GTPase is spatially and temporally regulated to coordinate leading-edge extension and tail retraction during a complex motile response, the chemotaxis of human neutrophils.

Genetic efficiency in higher organisms depends on mechanisms to create multiple functions from single genes. To investigate this question for an enzyme family, we chose aminoacyl tRNA synthetases (AARSs). They are exceptional in their progressive and accretive proliferation of noncatalytic domains as the Tree of Life is ascended. Here we report discovery of a large number of natural catalytic nulls (CNs) for each human AARS. Splicing events retain noncatalytic domains while ablating the catalytic domain (CD) to create CNs with diverse functions. Each synthetase is converted into several new signaling proteins with biological activities ‘orthogonal’ to that of the catalytic parent. We suggest that splice variants with non-enzymatic functions may be more general, as evidenced by recent findings of other catalytically inactive splice-variant enzymes.