Association of PI-3 kinase with PAK1 leads to actin phosphorylation and cytoskeletal reorganization

The family of p21-activated kinases (PAKs) have been implicated in the rearrangement of actin cytoskeleton by acting downstream of the small GTPases Rac and Cdc42. Here we report that even though Cdc42/Rac1 or Akt are not activated, phosphatidylinositol-3 (PI-3) kinase activation induces PAK1 kinase activity. Indeed, we demonstrate that PI-3 kinase associates with the N-terminal regulatory domain of PAK1 (amino acids 67-150) leading to PAK1 activation. The association of the PI-3 kinase with the Cdc42/Rac1 binding-deficient PAK1(H83,86L) confirms that the small GTPases are not involved in the PI-3 kinase-PAK1 interaction. Furthermore, PAK1 was activated in cells expressing the dominant-negative forms of Cdc42 or Rac1. Additionally, we show that PAK1 phosphorylates actin, resulting in the dissolution of stress fibers and redistribution of microfilaments. The phosphorylation of actin was inhibited by the kinase-dead PAK1(K299R) or the PAK1 autoinhibitory domain (PAK1(83-149)), indicating that PAK1 was responsible for actin phosphorylation. We conclude that the association of PI-3 kinase with PAK1 regulates PAK1 kinase activity through a Cdc42/Rac1-independent mechanism leading to actin phosphorylation and cytoskeletal reorganization.     

Phosphorylation of RhoGDI1 by p21-activated kinase 2 mediates basic fibroblast growth factor-stimulated neurite outgrowth in PC12 cells

We previously showed that p21-activated kinase 2 (PAK2), a major PAK isoform expressed in PC12 cells, mediates neurite outgrowth via Rac1 GTPase. RhoGDI1 forms a complex with Rac1, resulting in its inhibition. Rac1 activation requires dissociation from RhoGDI1. Here, we show that PAK2 mediates basic fibroblast growth factor (bFGF)-stimulated neurite outgrowth via phosphorylation of RhoGDI1. RhoGDI1 was shown to be associated with PAK2, with phosphorylation of Ser34 and Ser101 by active PAK2 evident in vitro and in vivo. A RhoGDI1 phosphomimetic mutant (S34E/S101E) was dissociated from Rac1/Cdc42, whereas the wild-type or a nonphosphorylatable mutant (S34A/S101A) formed a tight complex. Consistent with this, PC12 cells expressing the phosphomimetic mutant displayed Rac1/Cdc42 activation in response to bFGF stimulation. Neurite outgrowth was also enhanced in PC12 cells expressing the phosphomimetic mutant. These results suggest that PAK2-mediated RhoGDI1 phosphorylation stimulates dissociation of RhoGDI1-Rac1/Cdc42 complex accompanied by relief of inhibitory effect on Rac1/Cdc42, which promotes neuronal differentiation.     

Requirement of Rho-family GTPases in the invasion of Type 1-piliated uropathogenic Escherichia coli

Bladder infections caused by uropathogenic Escherichia coli (UPEC) depends on the ability of E. coli to express type 1 pili. The adhesive component of the pilus, FimH, mediates the invasion of E. coli into the bladder epithelium, a mechanism that facilitates the survival and persistence of E. coli in the bladder. The invasion mechanism requires actin polymerization, focal adhesion kinase phosphorylation and PI 3-kinase activation as well as the formation of FAK/PI 3-kinase and downstream vinculin/alpha-actinin complexes. In this study, we report a role for Rho-GTPase family members, namely RhoA, Cdc42 and Rac1, in the invasion process. Internalization of type 1-piliated E. coli (fimH+) and FimH-coated micro-spheres was inhibited by compactin, a pan-Rho-GTPase inhibitor and dominant negative isoforms of Rac1 and Cdc42. Expression of active Rac1 induced an internalization of E. coli that was insensitive to wortmannin and genistein. Expression of constitutively active Cdc42 induced the formation of FAK/PI 3-kinase and vinculin/alpha-actinin complexes whereas active Rac1 induced only a vinculin/alpha-actinin complex. Taken together, these data suggest that FimH-mediated invasion is dependent on GTP-binding protein activity that involves Cdc42 and PI 3-kinase activation probably upstream of Rac1.     

Escherichia coli K-1 interaction with human brain micro-vascular endothelial cells triggers phospholipase C-gamma1 activation downstream of phosphatidylinositol 3-kinase

Escherichia coli, the most common Gram-negative bacterium that causes meningitis in neonates, invades human brain microvascular endothelial cells (HBMEC) by rearranging host cell actin via the activation of phosphatidylinositol 3-kinase (PI3K) and PKC-alpha. Here, further, we show that phospholipase (PLC)-gamma1 is phosphorylated on tyrosine 783 and condenses at the HBMEC membrane beneath the E. coli entry site. Overexpression of a dominant negative (DN) form of PLC-gamma, the PLC-z fragment, in HBMEC inhibits PLC-gamma1 activation and significantly blocks E. coli invasion. PI3K activation is not affected in PLC-z/HBMEC upon infection, whereas PKC-alpha phosphorylation is completely abolished, indicating that PLC-gamma1 is downstream of PI3K. Concomitantly, the phosphorylation of PLC-gamma1 is blocked in HBMEC overexpressing a dominant negative form of the p85 subunit of PI3K but not in HBMEC overexpressing a dominant negative form of PKC-alpha. In addition, the recruitment of PLC-gamma1 to the cell membrane in both PLC-z/HBMEC and DN-p85/HBMEC is inhibited. Activation of PI3K is associated with the conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 1,4,5-trisphosphate (PIP3), which in turn recruits PLC-gamma1 to the cell membrane via its interaction with pleckstrin homology domain of PLC-gamma1. Utilizing the pleckstrin homology domains of PKC-delta and Btk proteins fused to green fluorescent protein (GFP), which specifically interact with PIP2 and PIP3, respectively, we show herein that E. coli invasion induces the breakdown of PIP2 at the plasma membrane near the site of E. coli interaction. PIP3, on the other hand, recruits the GFPBkt to the cell membrane beneath the sites of E. coli attachment. Our studies further show that E. coli invasion induces the release of Ca2+ from intracellular pools as well as the influx of Ca2+ from the extracellular medium. This elevation in Ca2+ levels is completely blocked both in PLC-z/HBMEC and DN-p85/HBMEC, but not in DN-PKC/HBMEC. Taken together, these results suggest that E. coli infection of HBMEC induces PLC-gamma1 activation in a PI3K-dependent manner to increase Ca2+ levels in HBMEC. This is the first report demonstrating the recruitment of activated PLC-gamma1 to the sites of bacterial entry.     

Gi-coupled receptors mediate phosphorylation of CPI-17 and MLC20 via preferential activation of the PI3K/ILK pathway

Sustained smooth-muscle contraction or its experimental counterpart, Ca2+ sensitization, by G(q/13)-coupled receptor agonists is mediated via RhoA-dependent inhibition of MLC (myosin light chain) phosphatase and MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation by a Ca2+-independent MLCK (MLC kinase). The present study identified the corresponding pathways initiated by G(i)-coupled receptors. Somatostatin acting via G(i)1-coupled sstr3 receptor, DPDPE ([D-Pen2,D-Pen5]enkephalin; where Pen is penicillamine) acting via G(i)2-coupled delta-opioid receptors, and cyclopentyl adenosine acting via G(i)3-coupled adenosine A1 receptors preferentially activated PI3K (phosphoinositide 3-kinase) and ILK (integrin-linked kinase), whereas ACh (acetylcholine) acting via G(i)3-coupled M2 receptors preferentially activated PI3K, Cdc42 (cell division cycle 42)/Rac1, PAK1 (p21-activated kinase 1) and p38 MAPK (mitogen-activated protein kinase). Only agonists that activated ILK induced sustained CPI-17 (protein kinase C potentiated inhibitor 17 kDa protein) phosphorylation at Thr38, MLC20 phosphorylation at Ser19, and contraction, consistent with recent evidence that ILK can act as a Ca2+-independent MLCK capable of phosphorylating the MLC phosphatase inhibitor, CPI-17, at Thr38. ILK activity, and CPI-17 and MLC20 phosphorylation were inhibited by LY294002 and in muscle cells expressing ILK(R211A) or treated with siRNA (small interfering RNA) for ILK. ACh acting via M2 receptors activated ILK, and induced CPI-17 and MLC20 phosphorylation and muscle contraction, but only after inhibition of p38 MAPK; all these responses were inhibited in cells expressing ILK(R211A). Conversely, ACh activated PAK1, a step upstream of p38 MAPK, whereas the three other agonists did so only in cells transfected with ILK(R211A) or siRNA for ILK. The results demonstrate reciprocal inhibition between two pathways downstream of PI3K, with ILK inhibiting PAK1, and p38 MAPK inhibiting ILK. Sustained contraction via G(i)-coupled receptors is dependent on CPI-17 and MLC20 phosphorylation by ILK.     

Actopaxin (α-Parvin) Phosphorylation Is Required for Matrix Degradation and Cancer Cell Invasion

Dysregulation of cell adhesion and motility is known to be an important factor in the development of tumor malignancy. Actopaxin (α-parvin) is a paxillin, integrin-linked kinase, and F-actin binding focal adhesion protein with several serine phosphorylation sites in the amino terminus that contribute to the regulation of cell spreading and migration. Here, phosphorylation of actopaxin is shown to contribute to the regulation of matrix degradation and cell invasion. Osteosarcoma cells stably expressing wild type (WT), nonphosphorylatable (Quint), and phosphomimetic (S4D/S8D) actopaxin demonstrate that actopaxin phosphorylation is necessary for efficient Src and matrix metalloproteinase-driven degradation of extracellular matrix. Rac1 was found to be required for actopaxin-induced matrix degradation whereas inhibition of myosin contractility promoted degradation in the phosphomutant-expressing Quint cells, indicating that a balance of Rho GTPase signaling and regulation of cellular tension are important for the process. Furthermore, actopaxin forms a complex with the Rac1/Cdc42 GEF β-PIX and Rac1/Cdc42 effector PAK1, to regulate actopaxin-dependent matrix degradation. Actopaxin phosphorylation is elevated in the invasive breast cancer cell line MDA-MB-231 compared with normal breast epithelial MCF10A cells. Expression of the nonphosphorylatable Quint actopaxin in MDA-MB-231 cells inhibits cell invasion whereas overexpression of WT actopaxin promotes invasion in MCF10A cells. Taken together, this study demonstrates a new role for actopaxin phosphorylation in matrix degradation and cell invasion via regulation of Rho GTPase signaling.     

Rac-induced increase of phosphorylation of myosin regulatory light chain in HeLa cells

The pathways by which activation of the small GTP-binding protein Rac causes cytoskeletal changes are not fully understood but are likely to involve both assembly of new actin filaments and reorganization of actin filaments driven by the actin-dependent ATPase activity of myosin II. Here we show that expression of active RacQ61 in growing HeLa cells, in addition to inducing ruffling, substantially enhances the level of phosphorylation of serine-19 of the myosin II regulatory light chain (MLC), which would increase actomyosin II ATPase and motor activities. Phosphorylated myosin was localized to RacQ61-induced ruffles and stress fibers. RacQ61-induced phosphorylation of MLC was reduced by a maximum of about 38% by an inhibitor (Tat-PAK) of p21-activated kinase (PAK), about 35% by an inhibitor (Y-27632) of Rho kinase, 51% by Tat-PAK plus Y-27632, and 10% by an inhibitor (ML7) of myosin light chain kinase. Staurosporine, a non-specific inhibitor of serine/threonine kinases, reduced RacQ61-induced phosphorylation of MLC by about 58%, at the maximum concentration that did not kill cells. Since Rac activates PAK and PAK can phosphorylate MLC, these data strongly suggest that PAK is responsible for a significant fraction of RacQ61-induced MLC phosphorylation. To our knowledge, this is the first evidence that active Rac causes phosphorylation of MLC in cells, thus implicating activation of the ATPase activity of actomyosin II as one of the ways by which Rac may induce cytoskeletal changes.     

Serine-71 phosphorylation of rac1 modulates downstream signaling

The Rho GTPases Rac1 and Cdc42 regulate a variety of cellular functions by signaling to different signal pathways. It is believed that the presence of a specific effector at the location of GTPase activation determines the route of downstream signaling. We previously reported about EGF-induced Ser-71 phosphorylation of Rac1/Cdc42. By using the phosphomimetic S71E-mutants of Rac1 and Cdc42 we investigated the impact of Ser-71 phosphorylation on binding to selected effector proteins. Binding of the constitutively active (Q61L) variants of Rac1 and Cdc42 to their specific interaction partners Sra-1 and N-WASP, respectively, as well as to their common effector protein PAK was abrogated when Ser-71 was exchanged to glutamate as phosphomimetic substitution. Interaction with their common effector proteins IQGAP1/2/3 or MRCK alpha was, however, hardly affected. This ambivalent behaviour was obvious in functional assays. In contrast to Rac1 Q61L, phosphomimetic Rac1 Q61L/S71E was not able to induce increased membrane ruffling. Instead, Rac1 Q61L/S71E allowed filopodia formation, which is in accordance with abrogation of the dominant Sra-1/Wave signalling pathway. In addition, in contrast to Rac1 transfected cells Rac1 S71E failed to activate PAK1/2. On the other hand, Rac1 Q61L/S71E was as effective in activation of NF-kappaB as Rac1 Q61L, illustrating positive signal transduction of phosphorylated Rac1. Together, these data suggest that phosphorylation of Rac1 and Cdc42 at serine-71 represents a reversible mechanism to shift specificity of GTPase/effector coupling, and to preferentially address selected downstream pathways.     

Activation of p21-activated kinase 1 is required for lysophosphatidic acid-induced focal adhesion kinase phosphorylation and cell motility in human melanoma A2058 cells.

Lysophosphatidic acid (LPA), one of the naturally occurring phospholipids, stimulates cell motility through the activation of Rho family members, but the signaling mechanisms remain to be elucidated. In the present study, we investigated the roles of p21-activated kinase 1 (PAK1) on LPA-induced focal adhesion kinase (FAK) phosphorylation and cell motility. Treatment of human melanoma cells A2058 with LPA increased phosphorylation and activation of PAK1, which was blocked by treatment with pertussis toxin and by inhibition of phosphoinositide 3-kinase (PI3K) with an inhibitor LY294002 or by overexpression of catalytically inactive mutant of PI3Kgamma, indicating that LPA-induced PAK1 activation was mediated via a Gi protein and the PI3Kgamma signaling pathway. In addition, we demonstrated that Rac1/Cdc42 signals acted as upstream effector molecules of LPA-induced PAK activation. However, Rho-associated kinase, MAP kinase kinase 1/2 or phospholipase C might not be involved in LPA-induced PAK1 activation or cell motility stimulation. Furthermore, PAK1 was necessary for FAK phosphorylation by LPA, which might cause cell migration, as transfection of the kinase deficient mutant of PAK1 or PAK auto-inhibitory domain significantly abrogated LPA-induced FAK phosphorylation. Taken together, these findings strongly indicated that PAK1 activation was necessary for LPA-induced cell motility and FAK phosphorylation that might be mediated by sequential activation of Gi protein, PI3Kgamma and Rac1/Cdc42.     

67-kDa laminin receptor promotes internalization of cytotoxic necrotizing factor 1-expressing Escherichia coli K1 into human brain microvascular endothelial cells

Escherichia coli K1 is the most common Gram-negative organism causing meningitis, and its invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for penetration into the central nervous system. We have reported previously that cytotoxic necrotizing factor 1 (CNF1) contributes to E. coli K1 invasion of HBMEC and interacts with 37-kDa laminin receptor precursor (37LRP) of HBMEC, which is a precursor of 67-kDa laminin receptor (67LR). In the present study, we examined the role of 67LR in the CNF1-expressing E. coli K1 invasion of HBMEC. Immunofluorescence microscopy and ligand overlay assays showed that 67LR is present on the HBMEC membrane and interacts with CNF1 protein as well as the CDPGYIGSR laminin peptide. 67LR was up-regulated and clustered at the sites of E. coli K1 on HBMEC in a CNF1-dependent manner. Pretreatment of CNF1+ E. coli K1 with recombinant 37-kDa laminin receptor precursor reduced the invasion rate to the level of Deltacnf1 mutant, and the invasion rate of CNF1+ E. coli K1 was enhanced in 67LR-overexpressing HBMEC, indicating 67LR is involved in the CNF1+ E. coli K1 invasion of HBMEC. Coimmunoprecipitation analysis showed that, upon incubation with CNF1+ E. coli K1 but not with Deltacnf1 mutant, focal adhesion kinase and paxillin were recruited and associated with 67LR. When immobilized onto polystyrene beads, CNF1 was sufficient to induce internalization of coupled beads into HBMEC through interaction with 67LR. Taken together, this is the first demonstration that E. coli K1 invasion of HBMEC occurs through the ligand-receptor (CNF1-67LR) interaction, and 67LR promotes CNF1-expressing E. coli K1 internalization of HBMEC.     

Phosphatidylinositol 3-kinase activation and interaction with focal adhesion kinase in Escherichia coli K1 invasion of human brain microvascular endothelial cells

Invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for successful crossing of the blood-brain barrier by Escherichia coli K1. We have previously demonstrated the requirement of cytoskeletal rearrangements and activation of focal adhesion kinase (FAK) in E. coli K1 invasion of human BMEC (HBMEC). The current study investigated the role of phosphatidylinositol 3-kinase (PI3K) activation and PI3K interaction with FAK in E. coli invasion of HBMEC. PI3K inhibitor LY294002 blocked E. coli K1 invasion of HBMEC in a dose-dependent manner, whereas an inactive analogue LY303511 had no such effect. In HBMEC, E. coli K1 increased phosphorylation of Akt, a downstream effector of PI3K, which was completely blocked by LY294002. In contrast, non-invasive E. coli failed to activate PI3K. Overexpression of PI3K mutants Deltap85 and catalytically inactive p110 in HBMEC significantly inhibited both PI3K/Akt activation and E. coli K1 invasion of HBMEC. Stimulation of HBMEC with E. coli K1 increased PI3K association with FAK. Furthermore, PI3K/Akt activation was blocked in HBMEC-overexpressing FAK dominant-negative mutants (FRNK and Phe397FAK). These results demonstrated the involvement of PI3K signaling in E. coli K1 invasion of HBMEC and identified a novel role for PI3K interaction with FAK in the pathogenesis of E. coli meningitis.     

Rac1-dependent recruitment of PAK2 to G2 phase centrosomes and their roles in the regulation of mitotic entry

During mitotic entry, the centrosomes provide a scaffold for initial activation of the CyclinB/Cdk1 complex, the mitotic kinase Aurora A, and the Aurora A-activating kinase p21-activated kinase (PAK). The activation of PAK at the centrosomes is yet regarded to happen independently of the Rho-GTPases Rac/Cdc42. In this study, Rac1 (but not RhoA or Cdc42) is presented to associate with the centrosomes from early G₂ phase until prometaphase in a cell cycle-dependent fashion, as evidenced by western blot analysis of prepared centrosomes and by immunolabeling. PAK associates with the G₂/M-phase centrosomes in a Rac1-dependent fashion. Furthermore, specific inhibition of Rac1 by C. difficile toxinB-catalyzed glucosylation or by knockout results in inhibited activation of PAK1/2, Aurora A, and the CyclinB/Cdk1 complex in late G₂ phase/prophase and delayed mitotic entry. Inhibition of PAK activation at late G₂-phase centrosomes caused by Rac1 inactivation coincides with impeded activation of Aurora A and the CyclinB/Cdk1 complex and delayed mitotic entry.     

GIT1 activates p21-activated kinase through a mechanism independent of p21 binding

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 alphaPAK 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.     

A Conserved Negative Regulatory Region in αPAK: Inhibition of PAK Kinases Reveals Their Morphological Roles Downstream of Cdc42 and Rac1

αPAK in a constitutively active form can exert morphological effects (E. Manser, H.-Y. Huang, T.-H. Loo, X.-Q. Chen, J.-M. Dong, T. Leung, and L. Lim, Mol. Cell. Biol. 17:1129–1143, 1997) resembling those of Cdc42^G12V. PAK family kinases, conserved from yeasts to humans, are directly activated by Cdc42 or Rac1 through interaction with a conserved N-terminal motif (corresponding to residues 71 to 137 in αPAK). αPAK mutants with substitutions in this motif that resulted in severely reduced Cdc42 binding can be recruited normally to Cdc42^G12V-driven focal complexes. Mutation of residues in the C-terminal portion of the motif (residues 101 to 137), though not affecting Cdc42 binding, produced a constitutively active kinase, suggesting this to be a negative regulatory region. Indeed, a 67-residue polypeptide encoding αPAK^83-149 potently inhibited GTPγS-bound Cdc42-mediated kinase activation of both αPAK and βPAK. Coexpression of this PAK inhibitor with Cdc42^G12V prevented the formation of peripheral actin microspikes and associated loss of stress fibers normally induced by the p21. Coexpression of PAK inhibitor with Rac1^G12V also prevented loss of stress fibers but not ruffling induced by the p21. Coexpression of αPAK^83-149 completely blocked the phenotypic effects of hyperactive αPAK^L107F in promoting dissolution of focal adhesions and actin stress fibers. These results, coupled with previous observations with constitutively active PAK, demonstrate that these kinases play an important role downstream of Cdc42 and Rac1 in cytoskeletal reorganization.