PAK1 Kinase Activity Research Articles

Stimulation of NFκB Activity by Multiple Signaling Pathways Requires PAK1

The p21-activated kinase (PAK1) is a serine-threonine protein kinase that is activated by binding to the Rho family small G proteins Rac and Cdc42hs. Both Rac and Cdc42hs have been shown to regulate the activity of the transcription factor NFkappaB. Here we show that expression of active Ras, Raf-1, or Rac1 in fibroblasts stimulates NFkappaB in a PAK1-dependent manner and that expression of active PAK1 can stimulate NFkappaB on its own. Similarly, in macrophages activation of NFkappaB as well as transcription from the tumor necrosis factor alpha promoter depends on PAK1. In these cells lipopolysaccharide is a potent activator of PAK1 kinase activity. We also demonstrate that expression of active PAK1 stimulates the nuclear translocation of the p65 subunit of NFkappaB but does not activate the inhibitor of kappaB kinases alpha or beta. These data demonstrate that PAK1 is a crucial signaling molecule involved in NFkappaB activation by multiple stimuli.

Reciprocal Signaling between Heterotrimeric G Proteins and the p21-stimulated Protein Kinase

p21-activated protein kinase (PAK)-1 phosphorylated Galpha(z), a member of the Galpha(i) family that is found in the brain, platelets, and adrenal medulla. Phosphorylation approached 1 mol of phosphate/mol of Galpha(z) in vitro. In transfected cells, Galpha(z) was phosphorylated both by wild-type PAK1 when stimulated by the GTP-binding protein Rac1 and by constitutively active PAK1 mutants. In vitro, phosphorylation occurred only at Ser(16), one of two Ser residues that are the major substrate sites for protein kinase C (PKC). PAK1 did not phosphorylate other Galpha subunits (i1, i2, i3, o, s, or q). PAK1-phosphorylated Galpha(z) was resistant both to RGSZ1, a G(z)-selective GTPase-activating protein (GAP), and to RGS4, a relatively nonselective GAP for the G(i) and G(q) families of G proteins. Phosphorylation of Ser(27) by PKC did not alter sensitivity to either GAP. The previously described inhibition of G(z) GAPs by PKC is therefore mediated by phosphorylation of Ser(16). Phosphorylation of either Ser(16) by PAK1 or Ser(27) by PKC decreased the affinity of Galpha(z) for Gbetagamma; phosphorylation of both residues by PKC caused no further effect. PAK1 thus regulates Galpha(z) function by attenuating the inhibitory effects of both GAPs and Gbetagamma. In this context, the kinase activity of PAK1 toward several protein substrates was directly inhibited by Gbetagamma, suggesting that PAK1 acts as a Gbetagamma-regulated effector protein. This inhibition of mammalian PAK1 by Gbetagamma contrasts with the stimulation of the PAK homolog Ste20p in Saccharomyces cerevisiae by the Gbetagamma homolog Ste4p/Ste18p.

Pak1 kinase activity affects the character of cell morphology and movement

As described in the review by Bagrodia and Cerione1xBagrodia, S. and Cerione, R. Trends Cell Biol. 1999; 9: 350–355Abstract | Full Text | Full Text PDF | PubMed | Scopus (323)See all References1, inducible expression of different forms of Pak1 in NIH3T3 cells leads to changes in cell morphology and pattern of movement2xSells, M.A. et al. J. Cell Biol. 1999; 145: 837–849Crossref | PubMed | Scopus (291)See all References2. This figure shows wild-type cells (a), and cells overexpressing either an active form of Pak1 (b) or a kinase-dead form (c). The cells expressing constitutively active Pak1 have large, polarized lamellipodia at their leading edge and are more motile than the control cells (Fig. 1Fig. 1). Sells and colleagues also observed that these cells moved more rapidly in response to a collagen gradient when plated on a fibronectin surface. By contrast, the cells expressing the kinase-dead form of Pak1 had several lamellipodia projecting in different directions simultaneously. They were highly motile, but in a more random manner, and their ability to respond to the collagen gradient by directional movement was impaired. These movement differences correlated with changes in the organization of the actin cytoskeleton and are consistent with previous findings. Sells et al. also found that cells expressing the constitutively active form of Pak1 showed increased myosin light chain (MLC) phosphorylation and proposed that this is involved in the regulation of cell movement by Pak1. However, as described by Bagrodia and Cerione1xBagrodia, S. and Cerione, R. Trends Cell Biol. 1999; 9: 350–355Abstract | Full Text | Full Text PDF | PubMed | Scopus (323)See all References1, this does not agree with previous results in other cell types, where activated Pak reduced MLC phosphorylation. The influence of Pak, both direct and indirect, on MLC phosphorylation and the role of this modification in influencing cell movement both require further investigation.FIGURE 1Pak1 expression was induced in Pak1-overexpressing NIH 3T3 cells by removing tetracycline from the growth medium for 16 hours. The cells were then trypsinized and, after a 30-minute incubation on plates coated with bovine serum albumin, the cells were transferred to fibronectin-coated plates. Phase-contrast micrographs were taken 95 min later. The cells shown express: (a) empty vector (control cells), (b) Pak1L83,L86 (constitutively active) and (c) Pak1R299 (kinase dead). Bar, 50 μm.View Large Image | Download PowerPoint Slide

αPix Stimulates p21-activated Kinase Activity through Exchange Factor-dependent and -independent Mechanisms

Activation of p21-activated kinases (Paks) is achieved through binding of the GTPases Rac or Cdc42 to a conserved domain in the N-terminal regulatory region of Pak. Additional signaling components are also likely to be important in regulating Pak activation. Recently, a family of Pak-interacting guanine nucleotide exchange factors (Pix) have been identified and which are good candidates for regulating Pak activity. Using an active, truncated form of alphaPix (amino acids 155-545), we observe stimulation of Pak1 kinase activity when alphaPix155-545 is co-expressed with Cdc42 and wild-type Pak1 in COS-1 cells. This activation does not occur when we co-express a Pak1 mutant unable to bind alphaPix. The activation of wild-type Pak1 by alphaPix155-545 also requires that alphaPix155-545 retain functional exchange factor activity. However, the Pak1(H83,86L) mutant that does not bind Rac or Cdc42 is activated in the absence of GTPase by alphaPix155-545 and by a mutant of alphaPix155-545 that no longer has exchange factor activity. Pak1 activity stimulated in vitro using GTPgammaS-loaded Cdc42 was also enhanced by recombinant alphaPix155-545 in a binding-dependent manner. These data suggest that Pak activity can be modulated by physical interaction with alphaPix and that this specific effect involves both exchange factor-dependent and -independent mechanisms.

Differential Effects of PAK1-activating Mutations Reveal Activity-dependent and -independent Effects on Cytoskeletal Regulation

PAKs are serine/threonine protein kinases that are activated by binding to Rac or Cdc42hs. Different forms of activated PAK1 have been reported to either promote membrane ruffling and focal adhesion assembly or cause focal adhesion disassembly and stress fiber dissolution. To understand the basis for these distinct morphological effects, we have examined the mechanism of mutational activation of PAK1, and characterized the effects of different active PAK1 proteins on cytoskeletal structure in vivo. We find that PAK1 contains an autoinhibitory domain that overlaps with its small G protein binding domain and that two separate activating mutations within this regulatory region each decrease autoinhibitory activity. Because only one of these mutations affects Cdc42hs binding activity, this indicates that activation of PAK1 by these mutations results from interference with the function of the autoinhibitory domain and not with small G protein binding activity. When we examined the morphological effects of these different forms of PAK1 in vivo, we found that PAK1 kinase activity was associated with disassembly of focal adhesions and actin stress fibers and that this may require interaction with potential SH3 domain-containing proteins. Lamellipodia formation and membrane ruffling caused by active PAK1 expression, however, was independent of PAK1 catalytic activity and likely requires interaction among multiple proteins binding to the PAK1 regulatory domain.

The p35/Cdk5 kinase is a neuron-specific Rac effector that inhibits Pak1 activity.

Cyclin-dependent kinase 5 (Cdk5) and its neuron-specific regulator p35 are essential for neuronal migration and for the laminar configuration of the cerebral cortex. In addition, p35/Cdk5 kinase concentrates at the leading edges of axonal growth cones and regulates neurite outgrowth in cortical neurons in culture. The Rho family of small GTPases is implicated in a range of cellular functions, including cell migration and neurite outgrowth. Here we show that the p35/Cdk5 kinase co-localizes with Rac in neuronal growth cones. Furthermore, p35 associates directly with Rac in a GTP-dependent manner. Another Rac effector, Pak1 kinase, is also present in the Rac-p35/Cdk5 complexes and co-localizes with p35/Cdk5 and Rac at neuronal peripheries. The active p35/Cdk5 kinase causes Pak1 hyperphosphorylation in a Rac-dependent manner, which results in down-regulation of Pak1 kinase activity. Because the Rho family of GTPases and the Pak kinases are implicated in actin polymerization, the modification of Pak1, imposed by the p35/Cdk5 kinase, is likely to have an impact on the dynamics of the reorganization of the actin cytoskeleton in neurons, thus promoting neuronal migration and neurite outgrowth.

Structural Requirements for PAK Activation by Rac GTPases

The Rho family GTPases, Rac1 and Rac2, regulate a variety of cellular functions including cytoskeletal reorganization, the generation of reactive oxygen species, G1 cell cycle progression and, in concert with Ras, oncogenic transformation. Among the many putative protein targets identified for Rac (and/or Cdc42), the Ser/Thr kinase p21-activated kinase (PAK) is a prime candidate for mediating some of Rac's cellular effects. This report shows that Rac1 binds to and stimulates the kinase activity of PAK1 approximately 2- and 4-5-fold, respectively, better than Rac2. Mutational analysis was employed to determine the structural elements on Rac and PAK that are important for optimal binding and activation. The most notable difference between the highly homologous Rac isomers is the composition of their C-terminal polybasic domains. Mutation of these six basic residues in Rac1 to neutral amino acids dramatically decreased the ability of Rac1 to bind PAK1 and almost completely abolished its ability to stimulate PAK activity. Moreover, replacing the highly charged polybasic domain of Rac1 with the less charged domain of Rac2 (and vice versa) completely reversed the PAK binding/activation properties of the two Rac isomers. Thus, polybasic domain differences account for the disparate abilities of Rac1 and Rac2 to activate PAK. PAK proteins also contain a basic region, consisting of three contiguous lysine residues (Lys66-Lys67-Lys68), which lies outside of the previously identified Cdc42/Rac-binding domain. Mutation of these Lys residues to neutral residues decreased PAK binding to activated Rac1 and Rac2 (but not Cdc42) and greatly reduced PAK1 activation by Rac1, Rac2, and Cdc42 proteins in vivo. In contrast, mutation of lysines 66-68 to basic Arg residues did not decrease (and in some cases enhanced) the ability of Rac1, Rac2, and Cdc42 to bind and activate PAK1. Our studies suggest that the polybasic domain of Rac is a novel effector domain that may allow the two Rac isomers to activate different effector proteins. In addition, our results indicate that a basic region in PAK is required for PAK activation and that binding of Rac/Cdc42 to PAK is not sufficient for kinase activation.