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1.  Matrix-specific protein kinase A signaling regulates p21 activated kinase activation by flow in endothelial cells 
Circulation research  2010;106(8):1394-1403.
Rationale
Atherosclerosis is initiated by blood flow patterns that activate inflammatory pathways in endothelial cells. Activation of inflammatory signaling by fluid shear stress is highly dependent on the composition of the subendothelial extracellular matrix. The basement membrane proteins laminin and collagen found in normal vessels suppress flow-induced p21 activated kinase (PAK) and NF-κB activation. By contrast, the provisional matrix proteins fibronectin and fibrinogen found in wounded or inflamed vessels support flow-induced PAK and NF-κB activation. PAK mediates both flow-induced permeability and matrix-specific activation of NF-κB.
Objective
To elucidate the mechanisms regulating matrix-specific PAK activation.
Methods and Results
We now show that matrix composition does not affect the upstream pathway by which flow activates PAK (integrin activation, Rac). Instead basement membrane proteins enhance flow-induced protein kinase A (PKA) activation, which suppresses PAK. Inhibiting PKA restored flow-induced PAK and NF-κB activation in cells on basement membrane proteins, whereas stimulating PKA inhibited flow-induced activation of inflammatory signaling in cells on fibronectin. PKA suppressed inflammatory signaling through PAK inhibition. Activating PKA by injection of the PGI2 analog iloprost reduced PAK activation and inflammatory gene expression at sites of disturbed flow in vivo, whereas inhibiting PKA by PKI injection enhanced PAK activation and inflammatory gene expression. Inhibiting PAK prevented the enhancement of inflammatory gene expression by PKI.
Conclusions
Basement membrane proteins inhibit inflammatory signaling in endothelial cells via PKA-dependent inhibition of PAK.
doi:10.1161/CIRCRESAHA.109.210286
PMCID: PMC2862370  PMID: 20224042
Shear stress; extracellular matrix; protein kinase A; p21 activated kinase; NF-κB
2.  Matrix-specific p21-activated kinase activation regulates vascular permeability in atherogenesis 
The Journal of Cell Biology  2007;176(5):719-727.
Elevated permeability of the endothelium is thought to be crucial in atherogenesis because it allows circulating lipoproteins to access subendothelial monocytes. Both local hemodynamics and cytokines may govern endothelial permeability in atherosclerotic plaque. We recently found that p21-activated kinase (PAK) regulates endothelial permeability. We now report that onset of fluid flow, atherogenic flow profiles, oxidized LDL, and proatherosclerotic cytokines all stimulate PAK phosphorylation and recruitment to cell–cell junctions. Activation of PAK is higher in cells plated on fibronectin (FN) compared to basement membrane proteins in all cases. In vivo, PAK is activated in atherosclerosis-prone regions of arteries and correlates with FN in the subendothelium. Inhibiting PAK in vivo reduces permeability in atherosclerosis-prone regions. Matrix-specific PAK activation therefore mediates elevated vascular permeability in atherogenesis.
doi:10.1083/jcb.200609008
PMCID: PMC2064028  PMID: 17312022
3.  PAK signaling regulates oxidant-dependent NF-κB activation by flow 
Circulation research  2008;103(6):671-679.
Disturbed blood flow promotes atherosclerosis mainly by inducing inflammatory gene expression in endothelial cells. Flow stimulates the proinflammatory transcription factor NF-κB through integrin- and Rac-dependent production of reactive oxygen species (ROS). Previous work demonstrated that NF-κB activation by flow is matrix-specific, occurring in cells on fibronectin but not collagen. Activation of p21-activated kinase (PAK) followed the same matrix-dependent pattern. We now show that inhibiting PAK in cells on fibronectin blocked NF-κB activation by both laminar and oscillatory flow in vitro and at sites of disturbed flow in vivo. Constitutively active PAK rescued flow-induced NF-κB activation in cells on collagen. Surprisingly, PAK was not required for flow-induced ROS production. Instead, PAK modulated the ability of H2O2 to activate the NF-κB pathway. These data demonstrate that PAK controls NF-κB activation by modulating cells’ sensitivity to ROS.
doi:10.1161/CIRCRESAHA.108.182097
PMCID: PMC2697905  PMID: 18669917
4.  The subendothelial extracellular matrix modulates JNK activation by flow 
Circulation research  2009;104(8):995-1003.
Atherosclerosis begins as local inflammation of artery walls at sites of disturbed flow. The c-Jun NH2-terminal kinase (JNK) is thought to be one of the major regulators of flow-dependent inflammatory gene expression in endothelial cells in atherosclerosis. We now show that JNK activation by both onset of laminar flow and long-term oscillatory flow is matrix-specific, with enhanced activation on fibronectin compared to basement membrane protein or collagen. Flow-induced JNK activation on fibronectin requires new integrin ligation, and requires both the MAP kinase kinase MKK4 and p21-activated kinase (PAK). In vivo, JNK activation at sites of early atherogenesis correlates with the deposition of fibronectin. Inhibiting PAK reduces JNK activation in atheroprone regions of the vasculature in vivo. These results identify JNK as a matrix-specific, flow-activated inflammatory event. Together with other studies, these data elucidate a network of matrix-specific pathways that determine inflammatory events in response to fluid shear stress.
doi:10.1161/CIRCRESAHA.108.186486
PMCID: PMC2702158  PMID: 19286608
shear stress; atherosclerosis; JNK
5.  Endothelial Cell PECAM-1 Promotes Atherosclerotic Lesions in Areas of Disturbed Flow in ApoE-Deficient Mice 
Objective
Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) has recently been shown to form an essential element of a mechanosensory complex that mediates endothelial responses to fluid shear stress. The aim of this study was to determine the in vivo role of PECAM-1 in atherosclerosis.
Methods and Results
We crossed C57BL/6 Pecam1−/− mice with apolipoprotein E–deficient (Apoe−/−) mice. On a Western diet, Pecam1−/−Apoe−/− mice showed reduced atherosclerotic lesion size compared to Apoe−/− mice. Striking differences were observed in the lesser curvature of the aortic arch, an area of disturbed flow, but not in the descending thoracic or abdominal aorta. Vascular cell adhesion molecule-1 (VCAM-1) expression, macrophage infiltration, and endothelial nuclear NF-κB were all reduced in Pecam1−/−Apoe−/− mice. Bone marrow transplantation suggested that endothelial PECAM-1 is the main determinant of atherosclerosis in the aortic arch, but that hematopoietic PECAM-1 promotes lesions in the abdominal aorta. In vitro data show that siRNA-based knockdown of PECAM-1 attenuates endothelial NF-κB activity and VCAM-1 expression under conditions of atheroprone flow.
Conclusion
These results indicate that endothelial PECAM-1 contributes to atherosclerotic lesion formation in regions of disturbed flow by regulating NF-κB–mediated gene expression.
doi:10.1161/ATVBAHA.108.164707
PMCID: PMC2651147  PMID: 18688018
atherosclerosis; shear stress sensing; adhesion molecules; endothelium; macrophages
6.  Altered nitric oxide production mediates matrix-specific PAK2 and NF-κB activation by flow 
Molecular Biology of the Cell  2013;24(3):398-408.
PAK2 mediates shear stress–induced NF-κB activation. Basement membrane proteins limit the proinflammatory response to shear by blocking the interaction of PAK2 with the adaptor protein Nck. This uncoupling response requires protein kinase A–dependent nitric oxide production and subsequent PAK2 phosphorylation on Ser-20 in the Nck-binding domain.
Shear stress generated by distinct blood flow patterns modulates endothelial cell phenotype to spatially restrict atherosclerotic plaque development. Signaling through p21-activated kinase (PAK) mediates several of the deleterious effects of shear stress, including enhanced NF-κB activation and proinflammatory gene expression. Whereas shear stress activates PAK in endothelial cells on a fibronectin matrix, basement membrane proteins limit shear-induced PAK activation and inflammation through a protein kinase A–dependent pathway; however, the mechanisms underlying this regulation were unknown. We show that basement membrane proteins limit membrane recruitment of PAK2, the dominant isoform in endothelial cells, by blocking its interaction with the adaptor protein Nck. This uncoupling response requires protein kinase A–dependent nitric oxide production and subsequent PAK2 phosphorylation on Ser-20 in the Nck-binding domain. Of importance, shear stress does not stimulate nitric oxide production in endothelial cells on fibronectin, resulting in enhanced PAK activation, NF-κB phosphorylation, ICAM-1 expression, and monocyte adhesion. These data demonstrate that differential flow–induced nitric oxide production regulates matrix-specific PAK signaling and describe a novel mechanism of nitric oxide–dependent NF-κB inhibition.
doi:10.1091/mbc.E12-07-0513
PMCID: PMC3564533  PMID: 23171552
7.  Increased endothelial expression of Toll-like receptor 2 at sites of disturbed blood flow exacerbates early atherogenic events 
Toll-like receptors (TLRs) are pattern recognition receptors of innate immunity. TLRs initiate inflammatory pathways that may exacerbate chronic inflammatory diseases like atherosclerosis. En face laser scanning confocal microscopy (LSCM) of isolated aortic segments revealed the distribution of intimal TLR2 expression and the atheroprotective outcomes resulting from a TLR2 deficiency. TLR2 expression was restricted to endothelial cells in regions of disturbed blood flow, such as the lesser curvature region, in atherosclerosis-prone, low-density lipoprotein receptor–deficient (LDLr−/−) mice. Diet-induced hyperlipidemia in LDLr−/− mice increased this regional endothelial TLR2 expression. Bone marrow (BM) reconstitution of LDLr−/− and LDLr−/−TLR2−/− mice created chimeric mice with green fluorescent protein (GFP) expression in BM-derived cells (BMGFP+). Lesser curvature BMGFP+ leukocyte accumulation, lipid accumulation, foam cell generation and endothelial cell injury were all increased by hyperlipidemia, whereas hyperlipidemic double mutant BMGFP+LDLr−/−TLR2−/− mice had reduced BMGFP+ leukocyte accumulation, lipid accumulation, foam cells, and endothelial cell injury. This is the first report of in vivo site-specific expression of endothelial cell TLR2. Expression of this receptor on endothelial cells contributed to early atherosclerotic processes in lesion-prone areas of the mouse aorta.
doi:10.1084/jem.20071096
PMCID: PMC2271019  PMID: 18250194
8.  Molecular Mechanisms Responsible for the Atheroprotective Effects of Laminar Shear Stress 
Antioxidants & Redox Signaling  2009;11(7):1669-1682.
Abstract
The endothelium lining the inner surface of blood vessels of the cardiovascular system is constantly exposed to hemodynamic shear stress. The interaction between endothelial cells and hemodynamic shear stress has critical implications for atherosclerosis. Regions of arterial narrowing, curvatures, and bifurcations are especially susceptible to atherosclerotic lesion formation. In such areas, endothelial cells experience low, or oscillatory, shear stress. Corresponding changes in endothelial cell structure and function make them susceptible to the initiation and development of atherosclerosis. In contrast, blood flow with high laminar shear stress activates signal transductions as well as gene and protein expressions that play important roles in vascular homeostasis. In response to laminar shear stress, the release of vasoactive substances such as nitric oxide and prostacyclin decreases permeability to plasma lipoproteins as well as the adhesion of leukocytes, and inhibits smooth muscle cell proliferation and migration. In summary, different flow patterns directly determine endothelial cell morphology, metabolism, and inflammatory phenotype through signal transduction and gene and protein expression. Thus, high laminar shear stress plays a key role in the prevention of atherosclerosis through its regulation of vascular tone and long-term maintenance of the integrity and function of endothelial cells. Antioxid. Redox Signal. 11, 1669–1682.
doi:10.1089/ars.2009.2487
PMCID: PMC2842586  PMID: 19309258
9.  Hemodynamic shear stress and the endothelium in cardiovascular pathophysiology 
SUMMARY
Endothelium lining the cardiovascular system is highly sensitive to hemodynamic shear stresses that act at the vessel luminal surface in the direction of blood flow. Physiological variations of shear stress regulate acute changes in vascular diameter and when sustained induce slow, adaptive, structural-wall remodeling. Both processes are endothelium-dependent and are systemically and regionally compromised by hyperlipidemia, hypertension, diabetes and inflammatory disorders. Shear stress spans a range of spatiotemporal scales and contributes to regional and focal heterogeneity of endothelial gene expression, which is important in vascular pathology. Regions of flow disturbances near arterial branches, bifurcations and curvatures result in complex spatiotemporal shear stresses and their characteristics can predict atherosclerosis susceptibility. Changes in local artery geometry during atherogenesis further modify shear stress characteristics at the endothelium. Intravascular devices can also influence flow-mediated endothelial responses. Endothelial flow-induced responses include a cell-signaling repertoire, collectively known as mechanotransduction, that ranges from instantaneous ion fluxes and biochemical pathways to gene and protein expression. A spatially decentralized mechanism of endothelial mechanotransduction is dominant, in which deformation at the cell surface induced by shear stress is transmitted as cytoskeletal tension changes to sites that are mechanically coupled to the cytoskeleton. A single shear stress mechanotransducer is unlikely to exist; rather, mechanotransduction occurs at multiple subcellular locations.
doi:10.1038/ncpcardio1397
PMCID: PMC2851404  PMID: 19029993
atherosclerosis; endothelial mechanotransduction; hemodynamics
10.  Functional Deficits in Pak5, Pak6 and Pak5/Pak6 Knockout Mice 
PLoS ONE  2013;8(4):e61321.
The p21-activated kinases are effector proteins for Rho-family GTPases. PAK4, PAK5, and PAK6 are the group II PAKs associated with neurite outgrowth, filopodia formation, and cell survival. Pak4 knockout mice are embryonic lethal, while Pak5, Pak6, and Pak5/Pak6 double knockout mice are viable and fertile. Our previous work found that the double knockout mice exhibit locomotor changes and learning and memory deficits. We also found some differences with Pak5 and Pak6 single knockout mice and the present work further explores the potential differences of the Pak5 knockout and Pak6 knockout mice in comparison with wild type mice. The Pak6 knockout mice were found to weigh significantly more than the other genotypes. The double knockout mice were found to be less active than the other genotypes. The Pak5 knockout mice and the double knockout mice performed worse on the rotorod test. All the knockout genotypes were found to be less aggressive in the resident intruder paradigm. The double knockout mice were, once again, found to perform worse in the active avoidance assay. These results indicate, that although some behavioral differences are seen in the Pak5 and Pak6 single knockout mice, the double knockout mice exhibit the greatest changes in locomotion and learning and memory.
doi:10.1371/journal.pone.0061321
PMCID: PMC3620390  PMID: 23593460
11.  Low-grade chronic inflammation in regions of the normal mouse arterial intima predisposed to atherosclerosis 
The Journal of Experimental Medicine  2006;203(9):2073-2083.
Atherosclerotic lesions develop in regions of arterial curvature and branch points, which are exposed to disturbed blood flow and have unique gene expression patterns. The cellular and molecular basis for atherosclerosis susceptibility in these regions is not completely understood. In the intima of atherosclerosis-predisposed regions of the wild-type C57BL/6 mouse aorta, we quantified increased expression of several proinflammatory genes that have been implicated in atherogenesis, including vascular cell adhesion molecule–1 (VCAM-1) and a relative abundance of dendritic cells, but only occasional T cells. In contrast, very few intimal leukocytes were detected in regions resistant to atherosclerosis; however, abundant macrophages, including T cells, were found throughout the adventitia (Adv). Considerably lower numbers of intimal CD68+ leukocytes were found in inbred atherosclerosis-resistant C3H and BALB/c mouse strains relative to C57BL/6 and 129; however, leukocyte distribution throughout the Adv of all strains was similar. The predominant mechanism for the accumulation of intimal CD68+ cells was continued recruitment of bone marrow–derived blood monocytes, suggestive of low-grade chronic inflammation. Local proliferation of intimal leukocytes was low. Intimal CD68+ leukocytes were reduced in VCAM-1–deficient mice, suggesting that mechanisms of leukocyte accumulation in the intima of normal aorta are analogous to those in atherosclerosis.
doi:10.1084/jem.20060245
PMCID: PMC2118407  PMID: 16894012
12.  Inhibition of p21 Activated Kinase (PAK) Reduces Airway Responsiveness In Vivo and In Vitro in Murine and Human Airways 
PLoS ONE  2012;7(8):e42601.
The p21-activated protein kinases (Paks) have been implicated in the regulation of smooth muscle contractility, but the physiologic effects of Pak activation on airway reactivity in vivo are unknown. A mouse model with a genetic deletion of Pak1 (Pak1−/−) was used to determine the role of Pak in the response of the airways in vivo to challenge with inhaled or intravenous acetylcholine (ACh). Pulmonary resistance was measured in anesthetized mechanically ventilated Pak1−/− and wild type mice. Pak1−/− mice exhibited lower airway reactivity to ACh compared with wild type mice. Tracheal segments dissected from Pak1−/− mice and studied in vitro also exhibited reduced responsiveness to ACh compared with tracheas from wild type mice. Morphometric assessment and pulmonary function analysis revealed no differences in the structure of the airways or lung parenchyma, suggesting that that the reduced airway responsiveness did not result from structural abnormalities in the lungs or airways due to Pak1 deletion. Inhalation of the small molecule synthetic Pak1 inhibitor, IPA3, also significantly reduced in vivo airway responsiveness to ACh and 5-hydroxytryptamine (5-Ht) in wild type mice. IPA3 inhibited the contractility of isolated human bronchial tissues to ACh, confirming that this inhibitor is also effective in human airway smooth muscle tissue. The results demonstrate that Pak is a critical component of the contractile activation process in airway smooth muscle, and suggest that Pak inhibition could provide a novel strategy for reducing airway hyperresponsiveness.
doi:10.1371/journal.pone.0042601
PMCID: PMC3416806  PMID: 22900031
13.  Activation of Syk Protein Tyrosine Kinase in Response to Osmotic Stress Requires Interaction with p21-Activated Protein Kinase Pak2/γ-PAK 
Molecular and Cellular Biology  2004;24(1):71-83.
The p21-activated serine/threonine protein kinase Pak2/γ-PAK and the nonreceptor type of protein tyrosine kinase Syk are known to be activated when the cells are exposed to osmotic stress. The purpose of the present study was to examine whether Pak2 and Syk functionally cooperate in cellular signaling. Cotransfection studies revealed that Pak2 associates with Syk in COS cells. The constitutively active form of Cdc42 increases the association of Pak2 with Syk. Pak2 coexpressed with an inactive form of Cdc42 or kinase-inactive Pak2 interacts to a lesser extent with Syk, suggesting that Pak2-Syk association is enhanced by Pak2 activation. Interaction with Pak2 enhances the intrinsic kinase activity of Syk. This is supported by in vitro studies showing that Pak2 phosphorylates and activates Syk. Treatment of cells with sorbitol to induce hyperosmolarity results in the translocation of Pak2 and Syk to the region surrounding the nucleus and in dramatic enhancement of their association. Furthermore, cotransfection of Pak2 and Syk leads to the activation of c-Jun N-terminal kinase (JNK) under hyperosmotic conditions. Pak2 short interfering RNA suppresses sorbitol-mediated activation of endogenous Syk and JNK, thus identifying a novel pathway for JNK activation by Cdc42. These results demonstrate that Pak2 and Syk positively cooperate to regulate cellular responses to stress.
doi:10.1128/MCB.24.1.71-83.2004
PMCID: PMC303346  PMID: 14673144
14.  Essential role of CIB1 in regulating PAK1 activation and cell migration 
The Journal of Cell Biology  2005;170(3):465-476.
p21-activated kinases (PAKs) regulate many cellular processes, including cytoskeletal rearrangement and cell migration. In this study, we report a direct and specific interaction of PAK1 with a 22-kD Ca2+-binding protein, CIB1, which results in PAK1 activation both in vitro and in vivo. CIB1 binds to PAK1 within discrete regions surrounding the inhibitory switch domain in a calcium-dependent manner, providing a potential mechanism of CIB1-induced PAK1 activation. CIB1 overexpression significantly decreases cell migration on fibronectin as a result of a PAK1-and LIM kinase–dependent increase in cofilin phosphorylation. Conversely, the RNA interference–mediated depletion of CIB1 increases cell migration and reduces normal adhesion-induced PAK1 activation and cofilin phosphorylation. Together, these results demonstrate that endogenous CIB1 is required for regulated adhesion-induced PAK1 activation and preferentially induces a PAK1-dependent pathway that can negatively regulate cell migration. These results point to CIB1 as a key regulator of PAK1 activation and signaling.
doi:10.1083/jcb.200502090
PMCID: PMC2171473  PMID: 16061695
15.  Do PAKs make good drug targets? 
p21-activated kinases (PAKs) act downstream of Rho-family GTPase and are linked to steps in both cancer initiation and progression. There are six mammalian PAK isoforms that are divided into two groups, and for different reasons both groups are attractive targets for cancer therapy. We describe the background and recent development of a PAK inhibitor, PF-3758309, which exhibits relatively good selectivity and high potency for PAKs. Experiments using PF-3758309 confirm that inhibiting PAK is a beneficial strategy to combat some tumors, and this activity is likely related to modulation of both cell proliferation and survival. The genetic loss of NF2 (neurofibromatosis type 2) leading to increased cell proliferation through a Ras-Rac-PAK pathway may represent a good test system to analyze this new PAK inhibitor.
doi:10.3410/B2-70
PMCID: PMC2989626  PMID: 21173843
16.  Endothelial Protein Kinase C Isoform Identity and Differential Activity of PKCζ in an Athero-Susceptible Region of Porcine Aorta 
Circulation research  2005;97(5):443-449.
Endothelial protein kinase C (PKC) signaling was investigated in different regions of normal porcine aorta. The locations map to differential atherosclerotic susceptibility and correlate with sites of disturbed (DF) or undisturbed (UF) local flow profiles. Endothelial lysates were isolated from the inner curvature of the aortic arch (DF; athero-susceptible) and a nearby UF region of the descending thoracic aorta (UF; athero-protected), and in some experiments a distant athero-protected UF site, the common carotid artery. Total endothelial PKC activity in the DF regions was 145% to 240% of that in both UF locations (P<0.05), whereas the UF regions were not significantly different from each other. PKC protein isoforms α, β, ε, ι, λ, and ζ were expressed in similar proportions in both aortic regions, suggesting that differences of kinase activity were not directly attributable to expression levels. Inhibition of members of the “conventional” and “novel” PKC families had no differential effect on regional kinase activity. However, inhibition of PKCζ, a member of the “atypical” PKC family, reduced the DF lysate kinase activity to that of UF levels (NS P=0.35). Differential phosphorylation of PKCζ Thr410 and Thr560, along with increased levels of PKCζ degradation products in UF endothelial lysates, suggested posttranslational modification of PKCζ as the basis for site-specific differences in vivo. Steady-state regional heterogeneity of an important family of regulatory proteins in intact arterial endothelium in vivo may link localized athero-susceptibility and the associated hemodynamic environment.
doi:10.1161/01.RES.0000179767.37838.60
PMCID: PMC3057121  PMID: 16051884
endothelial phenotypic heterogeneity; site-specific PKC activity; flow disturbance; hemodynamics
17.  Substrate and Inhibitor Specificity of the Type II p21-Activated Kinase, PAK6 
PLoS ONE  2013;8(10):e77818.
The p21-activated kinases (PAKs) are important effectors of Rho-family small GTPases. The PAK family consists of two groups, type I and type II, which have different modes of regulation and signaling. PAK6, a type II PAK, influences behavior and locomotor function in mice and has an ascribed role in androgen receptor signaling. Here we show that PAK6 has a peptide substrate specificity very similar to the other type II PAKs, PAK4 and PAK5 (PAK7). We find that PAK6 catalytic activity is inhibited by a peptide corresponding to its N-terminal pseudosubstrate. Introduction of a melanoma-associated mutation, P52L, into this peptide reduces pseudosubstrate autoinhibition of PAK6, and increases phosphorylation of its substrate PACSIN1 (Syndapin I) in cells. Finally we determine two co-crystal structures of PAK6 catalytic domain in complex with ATP-competitive inhibitors. We determined the 1.4 Å co-crystal structure of PAK6 with the type II PAK inhibitor PF-3758309, and the 1.95 Å co-crystal structure of PAK6 with sunitinib. These findings provide new insights into the structure-function relationships of PAK6 and may facilitate development of PAK6 targeted therapies.
doi:10.1371/journal.pone.0077818
PMCID: PMC3810134  PMID: 24204982
18.  PAK4–6 in cancer and neuronal development 
Cellular Logistics  2012;2(2):95-104.
PAKs 4, 5 and 6 are members of the group B family of p21-activated kinases. Among this group, PAK4 has been most extensively studied. While it has essential roles in embryonic development, in adults high levels of PAK4 are frequently associated with cancer. PAK4 is overexpressed in a variety of cancers, and the Pak4 gene is amplified in some cancers. PAK4 overexpression is sufficient to cause oncogenic transformation in cells and in mouse models. The tight connection between PAK4 and cancer make it a promising diagnostic tool as well as a potential drug target. The group B PAKs also have important developmental functions. PAK4 is important for many early developmental processes, while PAK5 and PAK6 play roles in learning and memory in mice. This chapter provides an overview of the roles of the group B PAKs in cancer as well as development, and includes a discussion of PAK mediated signaling pathways and cellular functions.
doi:10.4161/cl.21171
PMCID: PMC3485745  PMID: 23125951
PAK4; group B PAKs; signal transduction; development; cancer
19.  Induction of Vascular Permeability: βPIX and GIT1 Scaffold the Activation of Extracellular Signal-regulated Kinase by PAK 
Molecular Biology of the Cell  2007;18(6):2346-2355.
Increased permeability of blood vessels is an important component of inflammation, but in some circumstances it contributes to tissue injury and organ failure. Previous work showed that p21-activated kinase (PAK) is a critical regulator of endothelial cell–cell junctions through effects on myosin light chain phosphorylation and cell contractility. We now show that blocking PAK function inhibits fluid leak in a mouse model of acute lung injury. In cultured endothelial cells, induction of myosin light chain phosphorylation by PAK is mediated by mitogen-activated protein kinase kinase and extracellular signal-regulated kinase (Erk). Erk in lipopolysaccharide (LPS)-treated mouse lung is activated in a PAK-dependent manner in several cell types, most prominently vascular endothelium. Activation of Erk requires the integrity of the complex between PAK, PIX, and GIT1. Several means of disrupting this complex inhibit stimulation of vascular permeability in vitro. A cell-permeant peptide that blocks binding of PAK to PIX inhibits LPS-induced fluid leak in the mouse lung injury model. We conclude that the PAK–PIX–GIT1 complex is critical for Erk-dependent myosin phosphorylation and vascular permeability.
doi:10.1091/mbc.E06-07-0584
PMCID: PMC1877103  PMID: 17429073
20.  Blocking p21-activated Kinase Reduces Lipopolysaccharide-induced Acute Lung Injury by Preventing Polymorphonuclear Leukocyte Infiltration 
Rationale: Excessive recruitment of polymorphonuclear leukocytes (PMNs) to the lung promotes acute lung injury (ALI). Chemokine receptors and adhesion molecules initiate leukocyte–endothelial interactions, but mediators of PMN migration through the alveolo-capillary membrane remain to be identified. p21-Activated kinase (PAK) is an effector of small GTPases and has been implicated in cell migration.
Objectives: To test the role of PAK in ALI.
Methods: An inhibitory PAK peptide was used to determine the role of PAK in cytoskeletal actin polymerization, cell adhesion, and oxidative burst. PMN migration was investigated in vitro and in a murine model of lipopolysaccharide-induced lung injury.
Measurements and Main Results: PMN migration into lung interstitium and alveolar space was suppressed by an inhibitory PAK peptide. Neutrophils that had taken up the inhibitory PAK peptide were unable to enter the alveolar space. CXCL2/3, an important PMN chemoattractant in murine lung injury, induced PAK phosphorylation in PMNs. Blocking PAK function inhibited chemotaxis, chemokine-induced cytoskeletal actin polymerization, and adhesion-induced oxidative burst.
Conclusions: We conclude that neutrophil PAK is a critical mediator of PMN migration and may be an attractive target in ALI.
doi:10.1164/rccm.200612-1822OC
PMCID: PMC1899271  PMID: 17322107
acute respiratory distress syndrome; polymorphonuclear leukocytes; inflammation; migration
21.  3D structure analysis of PAKs 
Cellular Logistics  2012;2(2):69-77.
The p21-activated kinase (PAK) family plays a versatile role in cell signaling by forming a hub of interactions. PAKs bind the GTPases like RAC and CDC42. Their proline-rich motifs bind SH3 adaptor proteins such as PIX and NCK. PAKs display nuclear localization signal sites and a potential Integrin binding site. No fully complete structure of the PAKs has been published; partial 3D structures of the PAK family kinases include portions of the auto-inhibited PAK1, GTPase bound to small peptides from PAKs, and the kinase domains from PAK1 and PAK4–6 (with small ligands in a few cases). This review focuses on exploring the intermolecular interaction regions in these 3D structures and we offer insights on the missing regions in crystal structure of the auto-inhibited PAK1. Understanding and modulation of PAK intermolecular interactions can pave the way for PAK blockers and biosensors.
doi:10.4161/cl.21883
PMCID: PMC3490965  PMID: 23162739
PAK; 3D structure; ligand binding site; affinity reagent; small-molecule inhibitor
22.  The Pak4 Protein Kinase Plays a Key Role in Cell Survival and Tumorigenesis in Athymic Mice 
Pak4 is a member of the B group of p21-activated (Pak) kinases, originally identified as an effector protein for Cdc42. Although Pak4 is expressed at low levels in most adult tissues, it is highly overexpressed in tumor cell lines. Here, we show that Pak4 is also overexpressed in primary tumors, including colon, esophageal, and mammary tumors. Overexpression of Pak4 also leads to tumor formation in athymic mice, whereas deletion of Pak4 inhibits tumorigenesis. Although a constitutively active Pak4 mutant was previously shown to promote oncogenic transformation in cultured cells, our results are the first to show that Pak4 also promotes tumorigenesis in experimental animals. Furthermore, these results show for the first time that not only constitutively active Pak4, but also wild-type Pak4, is transforming, when experimental animals are used. These results are highly significant because wild-type Pak4, rather than activated Pak4, is overexpressed in tumor cells. Our results suggest that overexpression or activation of Pak4 is a key step in oncogenic transformation, due to its ability to promote cell survival and subsequent uncontrolled proliferation. The finding that Pak4 is up-regulated in so many types of cancers indicates that Pak4 may play a vital role in a wide range of different types of cancer. This makes it an attractive candidate for drug therapy for different types of cancer.
doi:10.1158/1541-7786.MCR-08-0087
PMCID: PMC2822623  PMID: 18644984
23.  Noninvasive Ultrasound Molecular Imaging of the Effect of Statins on Endothelial Inflammatory Phenotype in Early Atherosclerosis 
PLoS ONE  2013;8(3):e58761.
Background/Objectives
Inflammatory changes on the endothelium are responsible for leukocyte recruitment to plaques in atherosclerosis. Noninvasive assessment of treatment-effects on endothelial inflammation may be of use for managing medical therapy and developing novel therapies. We hypothesized that molecular imaging of vascular cell adhesion molecule-1 (VCAM-1) with contrast enhanced ultrasound (CEU) could assess treatment effects on endothelial phenotype in early atherosclerosis.
Methods
Mice with atherosclerosis produced by gene deletion of the LDL-receptor and Apobec-1-editing protein were studied. At 12 weeks of age, mice received 8 weeks of regular chow or atorvastatin-enriched chow (10 mg/kg/day). At 20 weeks, CEU molecular imaging for aortic endothelial VCAM-1 expression was performed with VCAM-1-targeted (MBVCAM) and control microbubbles (MBCtr). Aortic wall thickness was assessed with high frequency ultrasound. Histology, immunohistology and Western blot were used to assess plaque burden and VCAM-1 expression.
Results
Plaque burden was reduced on histology, and VCAM-1 was reduced on Western blot by atorvastatin, which corresponded to less endothelial expression of VCAM-1 on immunohistology. High frequency ultrasound did not detect differences in aortic wall thickness between groups. In contrast, CEU molecular imaging demonstrated selective signal enhancement for MBVCAM in non-treated animals (MBVCAM 2±0.3 vs MBCtr 0.7±0.2, p<0.01), but not in statin-treated animals (MBVCAM 0.8±0.2 vs MBCtr 1.0±0.2, p = ns; p<0.01 for the effect of statin on MBVCAM signal).
Conclusions
Non-invasive CEU molecular imaging detects the effects of anti-inflammatory treatment on endothelial inflammation in early atherosclerosis. This easily accessible, low-cost technique may be useful in assessing treatment effects in preclinical research and in patients.
doi:10.1371/journal.pone.0058761
PMCID: PMC3598944  PMID: 23554922
24.  Hemodynamic activation of β-catenin and TCF signaling in vascular endothelium regulates fibronectin expression 
β-catenin/TCF signaling regulates a varied set of cellular functions including development and remodeling. Fibronectin is a TCF-regulated gene that is highly expressed in arterial endothelium during atherosclerosis development and contributes to the pathophysiology of the disease. However, the activation of endothelial β-catenin/TCF signaling and its role in fibronectin expression in atherosclerosis are not currently known.
Objective
To assess the activity of β-catenin/TCF signaling in atherosclerosis development and its regulation of fibronectin in vascular endothelium.
Methods and Results
Histological staining identified preferential nuclear localization of β-catenin in the endothelium of atheroprone aorta prior to and during lesion development. Transgenic reporter studies revealed that increased levels of TCF transcriptional activity in endothelium correlated anatomically with β-catenin nuclear localization and fibronectin deposition. Exposure of endothelial cells to human-derived atheroprone shear stress induced nuclear localization of β-catenin, transcriptional activation of TCF, and expression of fibronectin. Activation of fibronectin expression required β-catenin, TCF and the transcriptional co-activator CBP. Finally, we identified PECAM-1as a critical regulator of constitutive β-catenin and GSK-3β activities.
Conclusions
This data uncovers novel constitutive activation of the endothelial β-catenin/TCF signaling pathway in atherosclerosis and regulation of fibronectin through hemodynamic shear stress.
doi:10.1161/ATVBAHA.111.227827
PMCID: PMC3134525  PMID: 21527747
β-catenin; TCF/LEF; endothelium; atherosclerosis; hemodynamics; fibronectin
25.  PAK1-mediated activation of ERK1/2 regulates lamellipodial dynamics 
Journal of cell science  2008;121(Pt 22):3729-3736.
Summary
PAK1 is a member of the p21-activated kinase (PAK) family of serine/threonine kinases that are activated by the Rho GTPases Rac and Cdc42 and are implicated in regulating morphological polarity, cell migration and adhesion. Here we investigate the function of PAK1 in cell motility using macrophages derived from PAK1-null mice. We show that CSF-1, a macrophage chemoattractant, transiently stimulates PAK1 and MAPK activation, and that MAPK activation is reduced in PAK1−/− macrophages. PAK1 regulates the dynamics of lamellipodium extension as cells spread in response to adhesion but is not essential for macrophage migration or chemotaxis towards CSF-1. Following adhesion, PAK1−/− macrophages spread more rapidly than wild-type macrophages and have more but less stable lamellipodia. ERK1/2 activity was reduced in PAK1−/− macrophages during adhesion, and inhibition of ERK1/2 activation in wild-type macrophages was sufficient to increase the spread area and mimic the lamellipodial dynamics of PAK1−/− macrophages. Together, these data indicate that PAK1 signals via ERK1/2 to regulate lamellipodial stability.
doi:10.1242/jcs.027680
PMCID: PMC2702775  PMID: 18940914
PAK1; ERK1/2; cell adhesion; cell migration; macrophages; Rho GTPases

Results 1-25 (700845)