Search

Variants in RAC3, encoding a small GTPase RAC3 which is critical for the regulation of actin cytoskeleton and intracellular signal transduction, are associated with a rare neurodevelopmental disorder with structural brain anomalies and facial dysmorphism. We investigated a cohort of 10 unrelated participants presenting with global psychomotor delay, hypotonia, behavioural disturbances, stereotyped movements, dysmorphic features, seizures and musculoskeletal abnormalities. MRI of brain revealed a complex pattern of variable brain malformations, including callosal abnormalities, white matter thinning, grey matter heterotopia, polymicrogyria/dysgyria, brainstem anomalies and cerebellar dysplasia. These patients harboured eight distinct de novo RAC3 variants, including six novel variants (NM_005052.3): c.34G > C p.G12R, c.179G > A p.G60D, c.186_188delGGA p.E62del, c.187G > A p.D63N, c.191A > G p.Y64C and c.348G > C p.K116N. We then examined the pathophysiological significance of these novel and previously reported pathogenic variants p.P29L, p.P34R, p.A59G, p.Q61L and p.E62K. In vitro analyses revealed that all tested RAC3 variants were biochemically and biologically active to variable extent, and exhibited a spectrum of different affinities to downstream effectors including p21-activated kinase 1. We then focused on the four variants p.Q61L, p.E62del, p.D63N and p.Y64C in the Switch II region, which is essential for the biochemical activity of small GTPases and also a variation hot spot common to other Rho family genes, RAC1 and CDC42. Acute expression of the four variants in embryonic mouse brain using in utero electroporation caused defects in cortical neuron morphology and migration ending up with cluster formation during corticogenesis. Notably, defective migration by p.E62del, p.D63N and p.Y64C were rescued by a dominant negative version of p21-activated kinase 1. Our results indicate that RAC3 variants result in morphological and functional defects in cortical neurons during brain development through variant-specific mechanisms, eventually leading to heterogeneous neurodevelopmental phenotypes. Scala et al. identify six de novo variants in RAC3, which encodes a small GTPase, in 10 unrelated subjects with neurodevelopmental phenotypes. In vivo and in vitro analyses in mice reveal that RAC3 variants cause morpho-functional defects in cortical neurons through variant-specific mechanisms, disrupting corticogenesis.

The collective migration of vascular endothelial cells plays important roles in homeostasis and angiogenesis. Oxygen concentration in vivo, which is lower than in the atmosphere and changes due to diseases, is a key factor affecting the cellular dynamics of vascular endothelial cells. We previously reported that hypoxic conditions promote the internalization of vascular endothelial (VE)-cadherin, a specific cell-cell adhesion molecule, and increase the velocity of the collective migration of vascular endothelial cells. However, the mechanism through which cells regulate collective migration as affected by oxygen tension is not fully understood. Here, we investigated oxygen-dependent collective migration, focusing on intracellular protein p21-activated kinase (PAK) and hypoxia-inducing factor (HIF)-1α. A monolayer of human umbilical vein vascular endothelial cells (HUVECs) was formed in a microfluidic device with controllability of oxygen tension. The HUVECs were then exposed to various oxygen conditions in a range from 0.8% to 21% O2, with or without PAK inhibition or chemical stabilization of HIF-1α. Collective cell migration was measured by particle image velocimetry with time-lapse phase-contrast microscopic images. Localizations of VE-cadherin and HIF-1α were quantified by immunofluorescent staining. The collective migration of HUVECs varied in an oxygen-dependent fashion; the migration speed was increased by hypoxic exposure down to 1% O2, while it decreased under an extremely low oxygen tension of less than 1% O2. PAK inhibition suppressed the hypoxia-induced increase of the migration speed by preventing VE-cadherin internalization into HUVECs. A decrease in the migration speed was also obtained by chemical stabilization of HIF-1α, suggesting that excessive accumulation of HIF-1α diminishes collective cell migration. These results indicate that the oxygen-dependent variation of the migration speed of vascular endothelial cells is mediated by the regulation of VE-cadherin through the PAK pathway, as well as other mechanisms via HIF-1α, especially under extreme hypoxic conditions.

The “block and lock” strategy is one approach that might elicit a sterilizing cure for HIV-1 infection. The “block” refers to a compound’s ability to inhibit latent HIV-1 proviral transcription, while the “lock” refers to its capacity to induce permanent proviral silencing. We identified PF-3758309, a pan-isoform inhibitor of p21-activated kinases (PAKs), as a potent inhibitor of HIV-1 latency reversal (Vargas et al., Antimicrob Agents Chemother. 2019;63(2):e01744-18). The goal of this study was to define the mechanism(s) involved. We found that both 24ST1NLESG cells (a cell line model of HIV-1 latency) and purified CD4+ naïve and central memory T cells express high levels of PAK2, and lower levels of PAK1 and PAK4. Knockdown of PAK1 or PAK2, but not PAK4, in 24ST1NLESG cells resulted in a modest, but statistically significant decrease in the magnitude of HIV-1 latency reversal. Overexpression of PAK1 significantly increased the magnitude of latency reversal. A phospho-protein array analysis revealed that PF-3758309 down-regulates the NF-κB signaling pathway, which provides the most likely mechanism by which PF-3758309 inhibits latency reversal. Finally, we used cellular thermal shift assays combined with liquid chromatography and mass spectrometry to ascertain whether PF-3758309 off-target binding contributed to its activity. In 24ST1NLESG cells and in peripheral blood mononuclear cells, PF-3758309 bound to mitogen-activated protein kinase 1 and protein kinase A; however, knockdown of either of these kinases did not impact HIV-1 latency reversal. Collectively, our study suggests that PAK1 and PAK2 play a key role in the maintenance of HIV-1 latency.IMPORTANCEThe persistence of latent, replication-competent HIV-1 proviruses in resting CD4+ T cells, and other cellular reservoirs, represents a major barrier to a cure. The “block and lock” strategy is one approach proposed to elicit a sterilizing cure for HIV-1 infection. In this study, we define the mechanism by which PF-3758309, a pan-isoform p21-activated kinase (PAK) inhibitor, blocks the reversal of HIV-1 latency. Our data show that PAK1 and PAK2 play a role in the maintenance of HIV-1 latency, and further suggest that PAK inhibitors, such as PF-3758309, could form part of “block and lock” therapeutic strategies.

International audience; Purpose Gliomas are the most lethal adult primary brain cancers. Recent advances in their molecular characterization have contributed to a better understanding of their pathophysiology, but there is still a need to identify key genes controlling glioma cell proliferation and differentiation. The p21-activated kinases PAK1 and PAK2 play essential roles in cell division and brain development and are well-known oncogenes. In contrast, the role of PAK3 in cancer is poorly understood. It is known, however that this gene is involved in brain ontogenesis and has been identified as a gene of the proneural subtype signature in glioblastomas. Methods To better understand the role of PAK kinases in the pathophysiology of gliomas, we conducted expression analyses by querying multiple gene expression databases and analyzing primary human glioma samples. We next studied PAK3 expression upon differentiation in patient-derived cell lines (PDCLs) and the effects of PAK3 inhibition by lentiviral-mediated shRNA on glioma cell proliferation, differentiation and tumor growth. Results We show that contrary to PAK1 and PAK2, high PAK3 expression positively correlates with a longer survival of glioma patients. We also found that PAK3 displays differential expression patterns between glioma subgroups with a higher expression in 1p/19q-codeleted oligodendrogliomas, and is highly expressed in tumors and PDCLs of the proneural subtype. In PDCLs, high PAK3 expression negatively correlated with proliferation and positively correlated with neuronal differentiation. Inhibition of PAK3 expression increased PDCL proliferation and glioma tumor growth in nude mice. Conclusions Our results indicate that PAK3 plays a unique role among PAKs in glioma development and may represent a potential therapeutic target.

Neurodegenerative mechanisms due to mutations in spastin currently center on neuronal defects, primarily in microtubule and endomembrane regulation. Spastin loss in Drosophila larvae compromises neuronal microtubule distribution, alters synaptic bouton morphology, and weakens synaptic transmission at glutamatergic neuromuscular junction (NMJ) synapses. Pak3, a p21-activated kinase that promotes actin polymerization and filopodial projections, is required for these spastin mutant defects; animals lacking both genes have normal NMJs. Here we show that Pak3 is expressed in central and peripheral glial populations, and reduction of Pak3 specifically in subperineurial glial cells is sufficient to suppress the phenotypes associated with spastin loss. Subperineurial glia in the periphery ensheathe motor neuron axons and have been shown to extend actin-based projections that regulate synaptic terminals during normal NMJ development. We find that these subperineurial glial projections are Pak3-dependent and nearly twice as frequent in spastin mutants, while in Pak3, spastin double mutants, neither glial projections nor synaptic defects are observed. Spastin deficiency thus increases Pak3-dependent subperineurial glia activity, which is in turn required for neuronal defects. Our results demonstrate a central role for Pak3-mediated, altered glial behavior in the neuronal defects due to spastin loss, and suggest that a similar reactive glia-mediated mechanism may underlie human AD-HSP pathogenesis.

Aim Muscle contraction stimulates skeletal muscle glucose transport. Since it occurs independently of insulin, it is an important alternative pathway to increase glucose transport in insulin‐resistant states, but the intracellular signaling mechanisms are not fully understood. Muscle contraction activates group I p21‐activated kinases (PAKs) in mouse and human skeletal muscle. PAK1 and PAK2 are downstream targets of Rac1, which is a key regulator of contraction‐stimulated glucose transport. Thus, PAK1 and PAK2 could be downstream effectors of Rac1 in contraction‐stimulated glucose transport. The current study aimed to test the hypothesis that PAK1 and/or PAK2 regulate contraction‐induced glucose transport. Methods Glucose transport was measured in isolated soleus and extensor digitorum longus (EDL) mouse skeletal muscle incubated either in the presence or absence of a pharmacological inhibitor (IPA‐3) of group I PAKs or originating from whole‐body PAK1 knockout, muscle‐specific PAK2 knockout or double whole‐body PAK1 and muscle‐specific PAK2 knockout mice. Results IPA‐3 attenuated (−22%) the increase in glucose transport in response to electrically stimulated contractions in soleus and EDL muscle. PAK1 was dispensable for contraction‐stimulated glucose transport in both soleus and EDL muscle. Lack of PAK2, either alone (−13%) or in combination with PAK1 (−14%), partly reduced contraction‐stimulated glucose transport compared to control littermates in EDL, but not soleus muscle. Conclusion Contraction‐stimulated glucose transport in isolated glycolytic mouse EDL muscle is partly dependent on PAK2, but not PAK1., In this study, we show that contraction‐stimulated glucose transport in isolated mouse skeletal muscle is unaffected by whole‐body knockout of PAK1. On the contrary, muscle‐specific knockout of PAK2 either alone or in combination with whole‐body PAK1 knockout partially reduces contraction‐stimulated glucose transport in glycolytic EDL muscle. These data suggest that contraction‐stimulated glucose transport in isolated glycolytic mouse EDL muscle is partly dependent on PAK2, but not PAK1.

Microbial infections are most often countered by inflammatory responses that are initiated through the recognition of conserved microbial products by innate immune receptors and result in pathogen expulsion1-6. However, inflammation can also lead to pathology. Tissues such as the intestinal epithelium, which are exposed to microbial products, are therefore subject to stringent negative regulatory mechanisms to prevent signalling through innate immune receptors6-11. This presents a challenge to the enteric pathogen Salmonella Typhimurium, which requires intestinal inflammation to compete against the resident microbiota and to acquire the nutrients and electron acceptors that sustain its replication12,13. We show here that S. Typhimurium stimulates pro-inflammatory signalling by a unique mechanism initiated by effector proteins that are delivered by its type III protein secretion system. These effectors activate Cdc42 and the p21-activated kinase 1 (PAK1) leading to the recruitment of TNF receptor-associated factor 6 (TRAF6) and mitogen-activated protein kinase kinase kinase 7 (TAK1), and the stimulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inflammatory signalling. The removal of Cdc42, PAK1, TRAF6 or TAK1 prevented S. Typhimurium from stimulating NF-κB signalling in cultured cells. In addition, oral administration of a highly specific PAK inhibitor blocked Salmonella-induced intestinal inflammation and bacterial replication in the mouse intestine, although it resulted in a significant increase in the bacterial loads in systemic tissues. Thus, S. Typhimurium stimulates inflammatory signalling in the intestinal tract by engaging critical downstream signalling components of innate immune receptors. These findings illustrate the unique balance that emerges from host-pathogen co-evolution, in that pathogen-initiated responses that help pathogen replication are also important to prevent pathogen spread to deeper tissues.

The overexpression and hyperactivity of p21-activated serine/threonine kinases (PAKs) is known to facilitate tumorigenesis; however, the contribution of cancer-associated PAK mutations to tumor initiation and progression remains unclear. Here, we identify p21-activated serine/threonine kinase 5 (PAK5) as the most frequently altered PAK family member in human melanoma. More than 60% of melanoma-associated PAK5 gene alterations are missense mutations, and distribution of these variants throughout the protein coding sequence make it difficult to distinguish oncogenic drivers from passengers. To address this issue, we stably introduced the five most common melanoma-associated PAK5 missense mutations into human immortalized primary melanocytes (hMELTs). While expression of these mutants did not promote single-cell migration or induce temozolomide resistance, a subset of variants drove aberrant melanocyte proliferation. These mitogenic mutants, PAK5 S364L and D421N, clustered within an unstructured, serine-rich domain of the protein and inappropriately activated ERK and PKA through kinase-independent and -dependent mechanisms, respectively. Together, our findings establish the ability of mutant PAK5 to enhance PKA and MAPK signaling in melanocytes and localize the engagement of mitogenic pathways to a serine-rich region of PAK5.

P21-activated kinase 3 (PAK3) gene mutations are linked to several neurodevelopmental disorders, but the underlying mechanisms remain unclear. In this study, we used a tetracycline-inducible system to control the expression of a mutant PAK3 (mPAK3) protein in immediate early gene, namely cFos, positive cells to disrupt PAK signaling, specifically in cells activated by social interaction in transgenic mice. We show that the expression of mPAK3-GFP proteins was in cFos-expressing excitatory and inhibitory neurons in various brain regions, such as the cortex and hippocampus, commonly activated during learning and memory. Basal expression of mPAK3-GFP proteins in cFos-positive cells resulted in social recognition memory deficits in the three-chamber social interaction test, without affecting locomotor activity or other forms of memory. The social memory deficit was rescued by doxycycline to halt the mPAK3-GFP transgene expression. In addition, we show that the expression of mPAK3-GFP proteins in a subset of cFos-positive cells, induced by an antecedent short social interaction, termed social pairing, was sufficient to impair social recognition memory. These results indicate that normal PAK signaling in cFos-positive cells activated during social interaction is critical for social memory.

Activating mutations in the RAC1 gene have recently been discovered as driver events in malignant melanoma. Expression of this gene is associated with melanocyte proliferation, and melanoma cells bearing this mutation are insensitive to BRAF inhibitors such as vemurafenib and dabrafenib, and also may evade immune surveillance due to enhanced expression of PD-L1. Activating mutations in RAC1 are of special interest, as small molecule inhibitors for the RAC effector p21-activated kinase (PAK) are in late-stage clinical development and might impede oncogenic signaling from mutant RAC1. In this work, we explore the effects of PAK inhibition on RAC1P29S signaling in zebrafish embryonic development, in the proliferation, survival, and motility of RAC1P29S-mutant human melanoma cells, and on tumor formation and progression from such cells in mice. We report that RAC1P29S evokes a Rasopathy-like phenotype on zebrafish development that can be blocked by inhibitors of PAK or MEK. We also found and that RAC1 mutant human melanoma cells are resistant to clinical inhibitors of BRAF but are uniquely sensitive to PAK inhibitors. These data suggest that suppressing the PAK pathway might be of therapeutic benefit in this type of melanoma.

P21‐activated kinase‐1 (PAK‐1) is a serine/threonine kinase involved in multiple signaling pathways that mediate cellular functions such as cytoskeletal motility, cell proliferation, and survival. PAK‐1 expression is altered in various cancers, including prostate and breast. Our recent studies showed that prostate cancer cells expressing higher levels of PAK‐1 were resistant to the cytotoxic effects of the PAK‐1 inhibitor, inhibitor targeting PAK‐1 activation‐3 (IPA‐3), compared to those with lower expression. This study expanded these findings to other cancers (breast and melanoma) by testing the hypothesis that genetic and pharmacological inhibition of PAK‐1 alters cell growth, migration, and invasion in prostate, breast, and skin cancer cell lines. We also tested the specificity of IPA‐3 for PAK‐1 and the hypothesis that gene silencing of PAK‐1 altered the efficacy of sterically stabilized liposomes (SSL) containing IPA‐3 (SSL‐IPA‐3). PAK‐1 expression was identified in four different breast cancer cell lines, and in a melanoma cell line. The expression of PAK‐1 correlated to the IC50 of IPA‐3 as measured by MTT staining. PAK‐1 inhibition using shRNA correlated with decreased cell migration and invasion in prostate cancer DU‐145 and breast cancer MCF‐7 cells. Decreased migration and invasion also correlated to decreased expression of E‐cadherin and alterations in C‐X‐C Chemokine Receptor type 4 and Homing Cell Adhesion Molecule expression. PAK‐1 inhibition increased the cytotoxicity of IPA‐3, and the cytotoxicity of SSL‐IPA‐3 to levels comparable to that of free drug. These data demonstrate that both pharmacological and molecular inhibition of PAK‐1 decreased growth in prostate, breast, and melanoma cancer cell lines, and increased the toxicity of IPA‐3 and its liposomal formulation. These data also show the specificity of IPA‐3 for PAK‐1, are some of the first data suggesting that IPA‐3 is a therapeutic treatment for breast cancer and melanoma, and demonstrate the efficacy of liposome‐encapsulated IPA‐3 in breast cancer cells.

Normal cells exposed to cancer-causing events respond by triggering cellular senescence, a stress response which halts cell proliferation and constitutes a protective anti-cancer barrier. We have uncovered a previously unknown signaling pathway implicating p21-activated kinase 4 (PAK4) in the control of senescence in breast cancer, via the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) subunit RELB and the CCAAT-enhancer-binding protein beta (C/EBPβ).

A series of novel 2,4-diaminoquinazoline derivatives were designed, synthesized, and evaluated as p21-activated kinase 4 (PAK4) inhibitors. All compounds showed significant inhibitory activity against PAK4 (half-maximal inhibitory concentration IC50 < 1 μM). Among them, compounds 8d and 9c demonstrated the most potent inhibitory activity against PAK4 (IC50 = 0.060 μM and 0.068 μM, respectively). Furthermore, we observed that compounds 8d and 9c displayed potent antiproliferative activity against the A549 cell line and inhibited cell cycle distribution, migration, and invasion of this cell line. In addition, molecular docking analysis was performed to predict the possible binding mode of compound 8d. This series of compounds has the potential for further development as PAK4 inhibitors for anticancer activity.

Migrating cells sense variations of stiffness in connective tissue matrices but how cells detect and respond to stiffness orientation is not defined. We examined cell extension formation on collagen with underlying support (vertical stiffness gradient) or on collagen laterally supported by nylon (lateral stiffness gradient). At 6h after plating, cells plated on laterally-supported collagen exhibited >2-fold more abundant and ~2-fold longer cell extensions than cells plated on collagen with underlying support. We examined whether p21-activated kinase 1 (PAK1) influences extension formation that is dependent on the orientation of support. At 6h after plating on collagen with underlying support, wild-type cell extensions were 40% shorter than PAK1 knockdown cells. In contrast, on laterally-supported collagen, wild-type cell extensions were 2-fold longer than PAK1 knockdown cells. In cells plated on laterally-supported collagen, there were ~2-fold reductions of collagen fiber alignment and compaction in PAK1 knockdown cells compared with wild-type cells. PAK1 knockdown did not affect collagen fiber alignment or compaction by cells plated on collagen with underlying support. Wild-type cells with lateral support of collagen exhibited 3-fold increases of phospho-myosin staining at 6h, which was 2-fold lower in PAK1 knockdown cells. In contrast, cells on collagen with underlying support showed no increase of phospho-myosin staining at any times. PAK1 knockdown did not affect α2 or β1 integrin expression or function. We conclude that PAK1 is involved in the ability of cells to sense the orientation of stiffness in collagen substrates and generate contractile forces that affect cell extension formation.

p21-activated kinases (PAKs) are Cdc42/Rac–activated serine-threonine protein kinases that regulate of several key cancer-relevant signaling pathways, such as the Mek/Erk, PI3K/Akt, and Wnt/b-catenin signaling pathways. Pak1 is frequently overexpressed and/or hyperactivated in different human cancers, including human breast, ovary, prostate, and brain cancer, due to amplification of the PAK1 gene in an 11q13 amplicon. Genetic or pharmacological inactivation of Pak1 has been shown to reduce proliferation of different cancer cells in vitro and reduce tumor progression in vivo. In this work, we examined the roles of Pak1 in cellular and animal models of PAK1-amplified ovarian cancer. We found that inhibition of Pak1 leads to decreased proliferation and migration in PAK1 amplified/overexpressed ovarian cancer cells, and has no effect in cell that lack such amplification/overexpression. Further, we observed that loss of Pak1 function causes 11q13 amplified ovarian cancer cells to arrest in the G2/M phase of the cell cycle. This arrest correlates with activation of p53 and p21Cip and decreased expression of cyclin B1. These findings suggest that small molecule inhibitors of Pak1 may play a therapeutic role in the ~25% of ovarian cancers characterized by PAK1 gene amplification.

// Hoi-Yee Chow 1, * , Biao Dong 2, * , Sergio G. Duron 3 , David A. Campbell 3 , Christy C. Ong 4 , Klaus P. Hoeflich 4 , Long-Sheng Chang 5, 6, 7 , D. Bradley Welling 7 , Zeng-jie Yang 1 , Jonathan Chernoff 1 1 Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA 2 Department of Microbiology and Immunology, Sol Sherry Thrombosis Research Center, Temple University, Philadelphia, Pennsylvania 19104, USA 3 Afraxis Inc., La Jolla, California 92037, USA 4 Department of Translational Oncology, Genentech, South San Francisco, California 94080, USA 5 Center for Childhood Cancer, The Research Institute at Nationwide Children’s Hospital, The Ohio State University College of Medicine, Columbus, Ohio 43205, USA 6 Department of Pediatrics, The Ohio State University College of Medicine, Columbus, Ohio 43205, USA 7 Department of Otolaryngology, The Ohio State University College of Medicine, Columbus, Ohio 43205, USA * These authors contributed equally to this work Correspondence to: Jonathan Chernoff, e-mail: Jonathan.Chernoff@fccc.edu Keywords: protein kinases, p21-activated kinase, neurofibromatosis, meningioma, signal transduction, small molecule inhibitors Received: October 02, 2014 Accepted: November 24, 2014 Published: January 24, 2015 ABSTRACT Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder characterized by the development of multiple tumors in the central nervous system, most notably schwannomas and meningiomas. Mutational inactivation of NF2 is found in 40–60% of sporadic meningiomas, but the molecular mechanisms underlying malignant changes of meningioma cells remain unclear. Because group I p21-activated kinases (Paks) bind to and are inhibited by the NF2 -encoded protein Merlin, we assessed the signaling and anti-tumor effects of three group-I specific Pak inhibitors - Frax597, 716 and 1036 - in NF2 −/− meningiomas in vitro and in an orthotopic mouse model. We found that these Pak inhibitors suppressed the proliferation and motility of both benign (Ben-Men1) and malignant (KT21-MG1) meningiomas cells. In addition, we found a strong reduction in phosphorylation of Mek and S6, and decreased cyclin D1 expression in both cell lines after treatment with Pak inhibitors. Using intracranial xenografts of luciferase-expressing KT21-MG1 cells, we found that treated mice showed significant tumor suppression for all three Pak inhibitors. Similar effects were observed in Ben-Men1 cells. Tumors dissected from treated animals exhibited an increase in apoptosis without notable change in proliferation. Collectively, these results suggest that Pak inhibitors might be useful agents in treating NF2 -deficient meningiomas.

Glucose is the principal cellular energy source in humans and maintenance of glucose homeostasis is critical for survival. Glucose uptake into peripheral skeletal muscle and adipose tissues requires the trafficking of vesicles containing glucose transporter-4 (GLUT4) from the intracellular storage compartments to the cell surface. Trafficking of GLUT4 storage vesicles is initiated via the canonical insulin signaling cascade in skeletal muscle and fat cells, as well as via exercise-induced contraction in muscle cells. Recent studies have elucidated steps in the signaling cascades that involve remodeling of the cytoskeleton, a process that underpins the mechanical movement of GLUT4 vesicles. This review is focused upon an alternate phosphoinositide-3 kinase-dependent pathway involving Ras-related C3 botulinum toxin substrate 1 signaling through the p21-activated kinase p21-activated kinase 1 and showcases related signaling events that co-regulate both the depolymerization and re-polymerization of filamentous actin. These new insights provide an enriched understanding into the process of glucose transport and yield potential new targets for interventions aimed to improve insulin sensitivity and remediate insulin resistance, pre-diabetes, and the progression to type 2 diabetes.

Defects in p21-activated kinase (PAK) lead to dendritic spine abnormalities and are sufficient to cause cognition impairment. The decrease in PAK in the brain of Alzheimer's disease (AD) patients is suspected to underlie synaptic and dendritic disturbances associated with its clinical expression, particularly with symptoms related to frontal cortex dysfunction. To investigate the role of PAK combined with Aβ and tau pathologies (3xTg-AD mice) in the frontal cortex, we generated a transgenic model of AD with a deficit in PAK activity (3xTg-AD-dnPAK mice). PAK inactivation had no effect on Aβ40 and Aβ42 levels, but increased the phosphorylation ratio of tau in detergent-insoluble protein fractions in the frontal cortex of 18-month-old heterozygous 3xTg-AD mice. Morphometric analyses of layer II/III pyramidal neurons in the frontal cortex showed that 3xTg-AD-dnPAK neurons exhibited significant dendritic attrition, lower spine density and longer spines compared to NonTg and 3xTg-AD mice. Finally, behavioral assessments revealed that 3xTg-AD-dnPAK mice exhibited pronounced anxious traits and disturbances in social behaviors, reminiscent of fronto-dependent symptoms observed in AD. Our results substantiate a critical role for PAK in the genesis of neuronal abnormalities in the frontal cortex underlying the emergence of psychiatric-like symptoms in AD.

Lethal Toxin from Clostridium sordellii (TcsL), which is casually involved in the toxic shock syndrome and in gas gangrene, enters its target cells by receptor-mediated endocytosis. Inside the cell, TcsL mono-O-glucosylates and thereby inactivates Rac/Cdc42 and Ras subtype GTPases, resulting in actin reorganization and an activation of p38 MAP kinase. While a role of p38 MAP kinase in TcsL-induced cell death is well established, data on a role of p38 MAP kinase in TcsL-induced actin reorganization are not available. In this study, TcsL-induced Rac/Cdc42 glucosylation and actin reorganization are differentially analyzed in p38alpha−/− MSCV empty vector MEFs and the corresponding cell line with reconstituted p38alpha expression (p38alpha−/− MSCV p38alpha MEFs). Genetic deletion of p38alpha results in reduced susceptibility of cells to TcsL-induced Rac/Cdc42 glucosylation and actin reorganization. Furthermore, SB203580, a pyridinyl imidazole inhibitor of p38alpha/beta MAP kinase, also protects cells from TcsL-induced effects in both p38−/− MSCV empty vector MEFs and in p38alpha−/− MSCV p38alpha MEFs, suggesting that inhibition of p38beta contributes to the protective effect of SB203580. In contrast, the effects of the related C. difficile Toxin B are responsive neither to SB203580 treatment nor to p38alpha deletion. In conclusion, the protective effects of SB203580 and of p38alpha deletion are likely not based on inhibition of the toxins’ glucosyltransferase activity rather than on inhibited endocytic uptake of specifically TcsL into target cells.

Activated G protein-coupled receptors (GPCRs) and receptor tyrosine kinases relay extracellular signals through spatial and temporal controlled kinase and GTPase entities. These enzymes are coordinated by multifunctional scaffolding proteins for precise intracellular signal processing. The cAMP-dependent protein kinase A (PKA) is the prime example for compartmentalized signal transmission downstream of distinct GPCRs. A-kinase anchoring proteins tether PKA to specific intracellular sites to ensure precision and directionality of PKA phosphorylation events. Here, we show that the Rho-GTPase Rac contains A-kinase anchoring protein properties and forms a dynamic cellular protein complex with PKA. The formation of this transient core complex depends on binary interactions with PKA subunits, cAMP levels and cellular GTP-loading accounting for bidirectional consequences on PKA and Rac downstream signaling. We show that GTP-Rac stabilizes the inactive PKA holoenzyme. However, β-adrenergic receptor-mediated activation of GTP-Rac-bound PKA routes signals to the Raf-Mek-Erk cascade, which is critically implicated in cell proliferation. We describe a further mechanism of how cAMP enhances nuclear Erk1/2 signaling: It emanates from transphosphorylation of p21-activated kinases in their evolutionary conserved kinase-activation loop through GTP-Rac compartmentalized PKA activities. Sole transphosphorylation of p21-activated kinases is not sufficient to activate Erk1/2. It requires complex formation of both kinases with GTP-Rac1 to unleash cAMP-PKA-boosted activation of Raf-Mek-Erk. Consequently GTP-Rac functions as a dual kinase-tuning scaffold that favors the PKA holoenzyme and contributes to potentiate Erk1/2 signaling. Our findings offer additional mechanistic insights how β-adrenergic receptor-controlled PKA activities enhance GTP-Rac-mediated activation of nuclear Erk1/2 signaling.

Lethal Toxin from Clostridium sordellii (TcsL), which is casually involved in the toxic shock syndrome and in gas gangrene, enters its target cells by receptor-mediated endocytosis. Inside the cell, TcsL mono-O-glucosylates and thereby inactivates Rac/Cdc42 and Ras subtype GTPases, resulting in actin reorganization and an activation of p38 MAP kinase. While a role of p38 MAP kinase in TcsL-induced cell death is well established, data on a role of p38 MAP kinase in TcsL-induced actin reorganization are not available. In this study, TcsL-induced Rac/Cdc42 glucosylation and actin reorganization are differentially analyzed in p38alpha−/− MSCV empty vector MEFs and the corresponding cell line with reconstituted p38alpha expression (p38alpha−/− MSCV p38alpha MEFs). Genetic deletion of p38alpha results in reduced susceptibility of cells to TcsL-induced Rac/Cdc42 glucosylation and actin reorganization. Furthermore, SB203580, a pyridinyl imidazole inhibitor of p38alpha/beta MAP kinase, also protects cells from TcsL-induced effects in both p38−/− MSCV empty vector MEFs and in p38alpha−/− MSCV p38alpha MEFs, suggesting that inhibition of p38beta contributes to the protective effect of SB203580. In contrast, the effects of the related C. difficile Toxin B are responsive neither to SB203580 treatment nor to p38alpha deletion. In conclusion, the protective effects of SB203580 and of p38alpha deletion are likely not based on inhibition of the toxins’ glucosyltransferase activity rather than on inhibited endocytic uptake of specifically TcsL into target cells.

The p21-activated kinases (PAKs) of group I are the main effectors for the small Rho GTPases, critically involved in neurodevelopment, plasticity and maturation of the nervous system. Moreover, the neuronal complexity controlled by PAK1/PAK3 signaling determines the postnatal brain size and synaptic properties. Stress induces alterations at the level of structural and functional synaptic plasticity accompanied by reductions in size and activity of the hippocampus and the prefrontal cortex (PFC). These abnormalities are likely to contribute to the pathology of depression and, in part, reflect impaired cytoskeleton remodeling pointing to the role of Rho GTPase signaling. Thus, the present study assessed the expression of the group I PAKs and their activators in the brain of depressed subjects. Using quantitative polymerase chain reaction (qPCR), mRNA levels and coexpression of the group I PAKs: PAK1, PAK2, and PAK3 as well as of their activators: RAC1, CDC42 and ARHGEF7 were examined in postmortem samples from the PFC (n = 25) and the hippocampus (n = 23) of subjects with depression and compared to control subjects (PFC n = 24; hippocampus n = 21). Results demonstrated that mRNA levels of PAK1 and PAK3, are significantly reduced in the brain of depressed subjects, with PAK1 being reduced in the PFC and PAK3 in the hippocampus. No differences were observed for the ubiquitously expressed PAK2. Following analysis of gene coexpression demonstrated disruption of coordinated gene expression in the brain of subjects with depression. Abnormalities in mRNA expression of PAK1 and PAK3 as well as their altered coexpression patterns were detected in the brain of subjects with depression. Fil: Fuchsova, Beata. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Alvarez Juliá, Anabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Rizavi, Hooriyah S.. University of Illinois; Estados Unidos Fil: Frasch, Alberto Carlos C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Pandey, Ghanshyam N.. University of Illinois; Estados Unidos

PAK1 inhibitors are known to markedly improve social and cognitive function in several animal models of brain disorders, including autism, but the underlying mechanisms remain elusive. We show here that disruption of PAK1 in mice suppresses inhibitory neurotransmission through an increase in tonic, but not phasic, secretion of endocannabinoids (eCB). Consistently, we found elevated levels of anandamide (AEA), but not 2-arachidonoylglycerol (2-AG) following PAK1 disruption. This increased tonic AEA signaling is mediated by reduced cyclooxygenase-2 (COX-2), and COX-2 inhibitors recapitulate the effect of PAK1 deletion on GABAergic transmission in a CB1 receptor-dependent manner. These results establish a novel signaling process whereby PAK1 upregulates COX-2, reduces AEA and restricts tonic eCB-mediated processes. Because PAK1 and eCB are both critically involved in many other organ systems in addition to the brain, our findings may provide a unified mechanism by which PAK1 regulates these systems and their dysfunctions including cancers, inflammations and allergies. DOI: http://dx.doi.org/10.7554/eLife.14653.001, eLife digest Brain cells communicate by sending chemical signals that activate or excite neighbouring cells. However, too much signalling can be harmful. As such the brain has systems in place to inhibit brain signals, and healthy brain activity relies striking a proper balance between excitation and inhibition. In some brain mental health conditions, like autism or schizophrenia, the balance is skewed which has an impact on the brain’s activity. A chemical produced by brain cells called endocannabinoid helps maintain the appropriate balance in brain excitation and inhibition. Endocannabinoid is similar to a chemical found in cannabis, but little is known about how it works and which proteins interact with endocannabinoid. A family of proteins called p21-activated kinases (PAKs) has been implicated in autism and other disorders like Huntingtin disease and Alzheimer disease, but it is not fully understood how these proteins interact with endocannabinoid. Now, Xia, Zhou et al. show that one member of this protein family called PAK1 plays a key role in controlling endocannabinoid activity. The experiments showed that mice genetically engineered to lack the PAK1 protein have higher levels of endocannabinoids and, as a consequence, the chemical signals that inhibit brain cells are affected more. The experiments also revealed that PAK1 does not interact directly with endocannabinoids. Instead PAK1 boosts levels of another protein called COX-2 and reduces levels of a molecule called anandamide, which together restrict endocannabinoid’s inhibitory effects. Scientists are currently interested in developing drugs that target the endocannabinoids and their regulators in the brain as a way to treat anxiety, pain and sleep problems. Drugs that block PAK1 are already being studied. Future studies are needed to determine if such PAK1-targeting drugs could be useful for restoring excitatory and inhibitory balance in brain diseases or for treating other diseases involving the PAK proteins. DOI: http://dx.doi.org/10.7554/eLife.14653.002

Rab7 regulates the biogenesis of late endosomes, lysosomes, and autophagosomes. It has been proposed that a functional and physical interaction exists between Rab7 and Rac1 GTPases in CDH1 endocytosis and ruffled border formation. In FRT cells over-expressing Rab7, increased expression and activity of Rac1 was observed, whereas a reduction of Rab7 expression by RNAi resulted in reduced Rac1 activity, as measured by PAK1 phosphorylation. We found that CDH1 endocytosis was extremely reduced only in Rab7 over-expressing cells but was unchanged in Rab7 silenced cells. In Rab7 under or over-expressing cells, Rab7 and LC3B-II co-localized and co-localization in large circular structures occurred only in Rab7 over-expressing cells. These large circular structures occurred in about 10% of the cell population; some of them (61%) showed co-localization of Rab7 with cortactin and f-actin and were identified as circular dorsal ruffles (CDRs), the others as mature autophagosomes. We propose that the over-expression of Rab7 is sufficient to induce CDRs. Furthermore, in FRT cells, we found that the expression of the insoluble/active form of Rab7, rather than Rab5, or Rab8, was inducible by cAMP and that cAMP-stimulated FRT cells showed increased PAK1 phosphorylation and were no longer able to endocytose CDH1. Finally, we demonstrated that Rab7 over-expressing cells are able to endocytose exogenous thyroglobulin via pinocytosis/CDRs more efficiently than control cells. We propose that the major thyroglobulin endocytosis described in thyroid autonomous adenomas due to Rab7 increased expression, occurs via CDRs. J. Cell. Physiol. 231: 1695-1708, 2016. © 2015 Wiley Periodicals, Inc.

During mitosis, microtubules (MTs) are massively rearranged into three sets of highly dynamic MTs that are nucleated from the centrosomes to form the mitotic spindle. Tight regulation of spindle positioning in the dividing cell and chromosome alignment at the center of the metaphase spindle are required to ensure perfect chromosome segregation and to position the cytokinetic furrow that will specify the two daughter cells. Spindle positioning requires regulation of MT dynamics, involving depolymerase activities together with cortical and kinetochore-mediated pushing and pulling forces acting on astral MTs and kinetochore fibres. These forces rely on MT motor activities. Cortical pulling forces exerted on astral MTs depend upon dynein/dynactin complexes and are essential in both symmetric and asymmetric cell division. A well-established spindle positioning pathway regulating the cortical targeting of dynein/dynactin involves the conserved LGN (Leu-Gly-Asn repeat-enriched-protein) and NuMA (microtubule binding nuclear mitotic apparatus protein) complex.1 Spindle orientation is also regulated by integrin-mediated cell adhesion2 and actin retraction fibres that respond to mechanical stress and are influenced by the microenvironment of the dividing cell.3 Altering the capture of astral MTs or modulating pulling forces affects spindle position, which can impair cell division, differentiation and embryogenesis. In this general scheme, the activity of mitotic kinases such as Auroras and Plk1 (Polo-like kinase 1) is crucial.4 Recently, the p21-activated kinases (PAKs) emerged as novel important players in mitotic progression. In our recent article, we demonstrated that PAK4 regulates spindle positioning in symmetric cell division.5 In this commentary, and in light of recent published studies, we discuss how PAK4 could participate in the regulation of mechanisms involved in spindle positioning and orientation.

The p21-activated kinase (PAK) family is homologous to the yeast sterile 20 (Ste20) and regulates a wide variety of cellular responses, including cell morphology, proliferation, and survival. In this study we examined the activation of PAK1 by Gbetagamma subunits. Co-transfection of COS7 cells with Gbeta1gamma2 or Gbeta1gamma5 was sufficient to induce agonist-independent activation of PAK1. Expression of dominant/negative Rac, Cdc42, or Ras did not inhibit this Gbetagamma-dependent activation. Wortmannin, which inhibits phosphoinositide 3-kinase (PI3-kinase) activity, and expression of a dominant/negative form of Akt were sufficient to abrogate the activation of PAK1 that was induced by Gbetagamma. These results reveal that stimulation of PAK1 by Gbetagamma can occur via a PI3-kinase and Akt pathway that does not require Rac1 or Cdc42.

Actin in migrating cells is regulated by Rho GTPases. However, Rho proteins might also affect microtubules (MTs). Here, we used time-lapse microscopy of PtK1 cells to examine MT regulation downstream of Rac1. In these cells, “pioneer” MTs growing into leading-edge protrusions exhibited a decreased catastrophe frequency and an increased time in growth as compared with MTs further from the leading edge. Constitutively active Rac1(Q61L) promoted pioneer behavior in most MTs, whereas dominant-negative Rac1(T17N) eliminated pioneer MTs, indicating that Rac1 is a regulator of MT dynamics in vivo. Rac1(Q61L) also enhanced MT turnover through stimulation of MT retrograde flow and breakage. Inhibition of p21-activated kinases (Paks), downstream effectors of Rac1, inhibited Rac1(Q61L)-induced MT growth and retrograde flow. In addition, Rac1(Q61L) promoted lamellipodial actin polymerization and Pak-dependent retrograde flow. Together, these results indicate coordinated regulation of the two cytoskeletal systems in the leading edge of migrating cells.

p21-activated kinase 1 (PAK1) can affect cell migration (Price et al., 1998; del Pozo et al., 2000) and modulate myosin light chain kinase and LIM kinase, which are components of the cellular motility machinery (Edwards, D.C., L.C. Sanders, G.M. Bokoch, and G.N. Gill. 1999. Nature Cell Biol. 1:253-259; Sanders, L.C., F. Matsumura, G.M. Bokoch, and P. de Lanerolle. 1999. SCIENCE: 283:2083-2085). We here present a novel cell motility pathway by demonstrating that PAK4 directly interacts with an integrin intracellular domain and regulates carcinoma cell motility in an integrin-specific manner. Yeast two-hybrid screening identified PAK4 binding to the cytoplasmic domain of the integrin beta 5 subunit, an association that was also found in mammalian cells between endogenous PAK4 and integrin alpha v beta 5. Furthermore, we mapped the PAK4 binding to the membrane-proximal region of integrin beta 5, and identified an integrin-binding domain at aa 505-530 in the COOH terminus of PAK4. Importantly, engagement of integrin alpha v beta 5 by cell attachment to vitronectin led to a redistribution of PAK4 from the cytosol to dynamic lamellipodial structures where PAK4 colocalized with integrin alpha v beta 5. Functionally, PAK4 induced integrin alpha v beta 5-mediated, but not beta1-mediated, human breast carcinoma cell migration, while no changes in integrin cell surface expression levels were observed. In conclusion, our results demonstrate that PAK4 interacts with integrin alpha v beta 5 and selectively promotes integrin alpha v beta 5-mediated cell migration.

Aims The MEK5/Erk5 pathway mediates beneficial effects of laminar flow, a major physiological factor preventing vascular dysfunction. Forced Erk5 activation induces a protective phenotype in endothelial cell (EC) that is associated with a dramatically decreased migration capacity of those cells. Transcriptional profiling identified the Kruppel-like transcription factors KLF2 and KLF4 as central mediators of Erk5-dependent gene expression. However, their downstream role regarding migration is unclear and relevant secondary effectors remain elusive. Here, we further investigated the mechanism underlying Erk5-dependent migration arrest in ECs.Methods and results Our experiments reveal KLF2-dependent loss of the pro-migratory Rac/Cdc42 mediator, p21-activated kinase 1 (PAK1), as an important mechanism of Erk5-induced migration inhibition. We show that endothelial Erk5 activation by expression of a constitutively active MEK5 mutant, by statin treatment, or by application of laminar shear stress strongly decreased PAK1 mRNA and protein expression. Knockdown of KLF2 but not of KLF4 prevented Erk5-mediated PAK1 mRNA inhibition, revealing KLF2 as a novel PAK1 repressor in ECs. Importantly, both PAK1 re-expression and KLF2 knockdown restored the migration capacity of Erk5-activated ECs underscoring their functional relevance downstream of Erk5.Conclusion Our data provide first evidence for existence of a previously unknown Erk5/KLF2/PAK1 axis, which may limit undesired cell migration in unperturbed endothelium and lower its sensitivity for migratory cues that promote vascular diseases including atherosclerosis.

The molecular mechanisms that govern thymocyte development and maturation are incompletely understood. The P21-activated kinase 2 (Pak2) is an effector for the Rho family GTPases Rac and Cdc42 that regulate actin cytoskeletal remodeling, but its role in the immune system remains poorly understood. In this study, we show that T-cell specific deletion of Pak2 gene in mice resulted in severe T cell lymphopenia accompanied by marked defects in development, maturation, and egress of thymocytes. Pak2 was required for pre-TCR β-selection and positive selection. Surprisingly, Pak2 deficiency in CD4 single positive thymocytes prevented functional maturation and reduced expression of S1P1 and KLF2. Mechanistically, Pak2 is required for actin cytoskeletal remodeling triggered by TCR. Failure to induce proper actin cytoskeletal remodeling impaired PLCγ1 and Erk1/2 signaling in the absence of Pak2, uncovering the critical function of Pak2 as an essential regulator that governs the actin cytoskeleton-dependent signaling to ensure normal thymocyte development and maturation. DOI: http://dx.doi.org/10.7554/eLife.02270.001, eLife digest T cells are a key element of the immune system. There are many different types of T cells, and they all have their origins in hematopoietic stem cells that are found in the bone marrow. These stem cells leave the bone marrow and circulate in the body until they reach an organ called the thymus, where they become early thymic progenitor cells. These progenitor cells then undergo a process called differentiation to become specific types of T cells, which mature in the thymus before moving to the blood. Although various molecules and mechanisms are known to be involved in the development of T cells, many details of this process are not understood. One group of molecules that has been implicated in the differentiation of T cells is the p21-activated kinases. Kinases are proteins that activate or deactivate other proteins by adding phosphate groups to specific amino acids. Pak2 adds phosphorylate groups to various proteins that are involved in the reorganization of an important structure inside the cell called the cytoskeleton. A kinase called Pak2 has an important role in the reorganization of the cytoskeleton, and since this reorganization is involved in almost all aspects of T cell biology, it seems plausible that Pak2 is also involved in the development of T cells. However, it has not been possible to test this idea because deleting the gene for Pak2 in mice results in their death. Now, Phee et al. have overcome this problem by performing experiments in which the gene for Pak2 was only deleted in T cells. These mice had significantly fewer mature T cells than healthy mice. In particular, the absence of Pak2 in thymocytes (the cells that become T cells) prevented them from maturing into T cells, and also prevented them from producing a receptor protein that is needed for mature T cells to leave the thymus. This work implies that disruption of the Pak2-mediated signaling pathway that regulates the cytoskeleton may weaken the immune system in humans. DOI: http://dx.doi.org/10.7554/eLife.02270.002

The p21 (Cdc42/Rac) activated kinase Pak1 regulates cell morphology and polarity in most, if not all, eukaryotic cells. We and others have established that Pak's effects on these parameters are mediated by changes in the organization of cortical actin. Because cell motility requires polarized rearrangements of the actin/myosin cytoskeleton, we examined the role of Pak1 in regulating cell movement. We established clonal tetracycline-regulated NIH-3T3 cell lines that inducibly express either wild-type Pak1, a kinase-dead, or constitutively-active forms of this enzyme, and examined the morphology, F-actin organization, and motility of these cells. Expression of any of these forms of Pak1 induced dramatic changes in actin organization which were not inhibited by coexpression of a dominant-negative form of Rac1. Cells inducibly expressing wild-type or constitutively-active Pak1 had large, polarized lamellipodia at the leading edge, were more motile than their normal counterparts when plated on a fibronectin-coated surface, and displayed enhanced directional movement in response to an immobilized collagen gradient. In contrast, cells expressing a kinase-dead form of Pak1 projected multiple lamellipodia emerging from different parts of the cell simultaneously. These cells, though highly motile, displayed reduced persistence of movement when plated on a fibronectin-coated surface and had defects in directed motility toward immobilized collagen. Expression of constitutively activated Pak1 was accompanied by increased myosin light chain (MLC) phosphorylation, whereas expression of kinase-dead Pak1 had no effect on MLC. These results suggest that Pak1 affects the phosphorylation state of MLC, thus linking this kinase to a molecule that directly affects cell movement.

Endothelial functions are highly regulated by imposed shear stress in vivo. The characteristics of shear stress determine mechanotransduction events that regulate phenotypic outcomes including redox and inflammatory states. Recent data indicate that microRNAs (miRs) in vascular endothelial cells play an essential role in shear stress-regulated endothelial responses. More specifically, atheroprotective pulsatile flow (PS) induces miRs that inhibit mediators of oxidative stress and inflammation while promoting those involved in maintaining vascular homeostasis. Conversely, oscillatory flow (OS) elicits the opposing networks. This is exemplified by the PS-responsive transcription factor Krüppel-like factor 2 (KLF2), which regulates miR expression but is also regulated by OS-sensitive miRs to ultimately regulate the oxidative and inflammatory state of the endothelium. In this review, we outline important findings demonstrating the multifaceted roles of shear stress-regulated miRs in endothelial redox and inflammatory balance. Furthermore, we discuss the use of algorithms in deciphering signaling networks differentially regulated by PS and OS.

Background Hypoxia is a major driving force in vascularization and vascular remodeling. Pharmacological inhibition of prolyl hydroxylases (PHDs) leads to an oxygen-independent and long-lasting activation of hypoxia-inducible factors (HIFs). Whereas effects of HIF-stabilization on transcriptional responses have been thoroughly investigated in endothelial cells, the molecular details of cytoskeletal changes elicited by PHD-inhibition remain largely unknown. To investigate this important aspect of PHD-inhibition, we used a spheroid-on-matrix cell culture model. Results Microvascular endothelial cells (glEND.2) were organized into spheroids. Migration of cells from the spheroids was quantified and analyzed by immunocytochemistry. The PHD inhibitor dimethyloxalyl glycine (DMOG) induced F-actin stress fiber formation in migrating cells, but only weakly affected microvascular endothelial cells firmly attached in a monolayer. Compared to control spheroids, the residual spheroids were larger upon PHD inhibition and contained more cells with tight VE-cadherin positive cell-cell contacts. Morphological alterations were dependent on stabilization of HIF-1α and not HIF-2α as shown in cells with stable knockdown of HIF-α isoforms. DMOG-treated endothelial cells exhibited a reduction of immunoreactive Rac-1 at the migrating front, concomitant with a diminished Rac-1 activity, whereas total Rac-1 protein remained unchanged. Two chemically distinct Rac-1 inhibitors mimicked the effects of DMOG in terms of F-actin fiber formation and orientation, as well as stabilization of residual spheroids. Furthermore, phosphorylation of p21-activated kinase PAK downstream of Rac-1 was reduced by DMOG in a HIF-1α-dependent manner. Stabilization of cell-cell contacts associated with decreased Rac-1 activity was also confirmed in human umbilical vein endothelial cells. Conclusions Our data demonstrates that PHD inhibition induces HIF-1α-dependent cytoskeletal remodeling in endothelial cells, which is mediated essentially by a reduction in Rac-1 signaling.

Eukaryotic, prokaryotic and viral pathogens are known to interfere with signaling pathways of their host to promote their own survival and proliferation. Here, we present selected examples of modulation of PAK activity in human cells by both intracellular and extracellular pathogens, focusing on one eukaryotic pathogen, the human malaria parasite Plasmodium falciparum, two Gram-negative bacteria (Helicobacter pylori and Pseudomonas aeruginosa), and two viruses belonging to distinct groups, the lentivirus HIV and the orthomyxovirus Influenza virus A.

Small GTPases function as molecular switches in cell signaling, alternating between an inactive, GDP-bound state, and active GTP-bound state. βPix is one of guanine nucleotide exchange factors (GEFs) that catalyze the exchange of bound GDP for ambient GTP. The central goal of this review article is to summarize recent findings on βPix and the role it plays in kidney pathology and physiology. Recent studies shed new light on several key questions concerning the signaling mechanisms mediated by βPix. This manuscript provides a review of the various mechanisms whereby βPix has been shown to function within the kidney through a wide range of actions. Both canonical GEF activity and non-canonical signaling pathways mediated by βPix are discussed. Distribution patterns of βPix in the kidney will be also covered. Much has yet to be discerned, but it is clear that βPix plays a significant role in the kidney.

Glomerulosclerosis (GS) is the irreversible scarring of glomerular tissue which underlies the development of chronic kidney disease (CKD). Increased intraglomerular capillary pressure (Pgc) is a major contributor to the development of GS and can occur in both hypertensive and diabetic patients. With elevated Pgc, in vitro and in vivo evidence suggest that mesangial cells (MC) experience cyclic stretch and secrete pro-fibrotic factors such as connective tissue growth factor (CTGF) which contributes to GS. The signaling pathways that are activated in response to elevated Pgc and lead to extracellular matrix (ECM) production in MCs are the main focus of this thesis. Previous data demonstrated activation of the Rho GTPase, Rac1, with cyclic stretch in MCs. Furthermore, the most characterized effector of Rac1, p21-activated kinase (PAK), has been observed to have a role in endothelial cells (ECs) exposed to mechanical stress. We thus proposed that the Rac1-PAK signaling pathway is involved in mechanical stress signaling in MCs. Our data demonstrate that Rac1-PAK signaling was activated in response to cyclic stretch and required for stretch-induced CTGF production in MCs. RhoA activation was also regulated by Rac1-PAK signaling, and RhoA/ROCK were observed to mediate CTGF upregulation with stretch. Further investigation on the role of Rac1-PAK signaling and how it regulates CTGF in MCs exposed to stretch, will provide insight into potential therapeutic targets to delay the progression of hypertension-mediated CKD. Master of Science in Medical Sciences (MSMS)

Dysfunctional regulation of airway smooth muscle tone is a feature of obstructive airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle contraction is directly associated with changes in the phosphorylation of myosin light chain (MLC), which is increased by Rho and decreased by Rac. Although cyclic adenosine monophosphate (cAMP)-elevating agents are believed to relieve bronchoconstriction mainly via activation of protein kinase A (PKA), here we addressed the role of the novel cAMP-mediated exchange protein Epac in the regulation of airway smooth muscle tone. Isometric tension measurements showed that specific activation of Epac led to relaxation of guinea pig tracheal preparations pre-contracted with methacholine, independently of PKA. In airway smooth muscle cells, Epac activation reduced methacholine-induced MLC phosphorylation. Moreover, when Epac was stimulated, we observed a decreased methacholine-induced RhoA activation, measured by both stress fibre formation and pull-down assay whereas the same Epac activation prevented methacholine-induced Rac1 inhibition measured by pull-down assay. Epac-driven inhibition of both methacholine-induced muscle contraction by Toxin B-1470, and MLC phosphorylation by the Rac1-inhibitor NSC23766, were significantly attenuated, confirming the importance of Rac1 in Epac-mediated relaxation. Importantly, human airway smooth muscle tissue also expresses Epac, and Epac activation both relaxed pre-contracted human tracheal preparations and decreased MLC phosphorylation. Collectively, we show that activation of Epac relaxes airway smooth muscle by decreasing MLC phosphorylation by skewing the balance of RhoA/Rac1 activation towards Rac1. Therefore, activation of Epac may have therapeutical potential in the treatment of obstructive airway diseases.

This article is available open access through the publisher’s website at the link below. Copyright @ 2009 The Authors. The conserved Rho-type GTPase Cdc42p is a key regulator of signal transduction and polarity in eukaryotic cells. In the yeast Saccharomyces cerevisiae, Cdc42p promotes polarized growth through the p21-activated kinases Ste20p and Cla4p. Previously, we demonstrated that Ste20p forms a complex with Erg4p, Cbr1p and Ncp1p, which all catalyze important steps in sterol biosynthesis. CLA4 interacts genetically with ERG4 and NCP1. Furthermore, Erg4p, Ncp1p and Cbr1p play important roles in cell polarization during vegetative growth, mating and filamentation. As Ste20p and Cla4p are involved in these processes it seems likely that sterol biosynthetic enzymes and p21-activated kinases act in related pathways. Here, we demonstrate that the deletion of either STE20 or CLA4 results in increased levels of sterols. In addition, higher concentrations of steryl esters, the storage form of sterols, were observed in cla4Δ cells. CLA4 expression from a multicopy plasmid reduces enzyme activity of Are2p, the major steryl ester synthase, under aerobic conditions. Altogether, our data suggest that Ste20p and Cla4p may function as negative modulators of sterol biosynthesis. Moreover, Cla4p has a negative effect on steryl ester formation. As sterol homeostasis is crucial for cell polarization, Ste20p and Cla4p may regulate cell polarity in part through the modulation of sterol homeostasis. Deutsche Forschungsgemeinschaft and the Austrian FWF.

Synapse loss occurs early in Alzheimer's disease (AD) and is considered thebest pathological correlate of cognitive decline. Ephrins and Eph receptorsare involved in regulation of excitatory neurotransmission and play a rolein cytoskeleton remodeling. We asked whether alterations in Eph receptorscould underlie cognitive impairment in an AD mouse model overexpressinghuman amyloid-β protein precursor (hAβPP) with familialmutations (hAβPPswe-ind mice). We found that EphA4 and EphB2receptors were reduced in the hippocampus before the development of impairedobject recognition and spatial memory. Similar results were obtained inanother line of transgenic AβPP mice, Tg2576. A reduction in Ephreceptor levels was also found in postmortem hippocampal tissue frompatients with incipient AD. At the time of onset of memory decline inhAβPPswe-ind mice, no change in surface expression of AMPA orNMDA receptor subunits was apparent, but we found changes in Eph-receptordownstream signaling, in particular a decrease in membrane-associatedphosho-cofilin levels that may cause cytoskeletal changes and disruptedsynaptic activity. Consistent with this finding, Eph receptor activation incell culture increased phosho-cofilin levels. The results suggest thatalterations in Eph receptors may play a role in synaptic dysfunction in thehippocampus leading to cognitive impairment in a model of AD., Supported by SAF2005-05086, SAF2008-02342 (Ministry of Science), CIBERNED (Ministry of Health), Government of Navarra and UTE project FIMA. We also thank Government of Navarra and La Rioja for fellowships (AMS, LE).

Tumorhead (TH) regulates neural plate cell proliferation during Xenopus early development, and gain or loss of function prevents neural differentiation. TH shuttles between the nuclear and cytoplasmic/cortical cell compartments in embryonic cells. In this study, we show that subcellular distribution of TH is important for its functions. Targeting TH to the cell cortex/membrane potentiates a TH gain of function phenotype and results in neural plate expansion and inhibition of neuronal differentiation. We have found that TH subcellular localization is regulated, and that its shuttling between the nucleus and the cell cortex/cytoplasm is controlled by the catalytic activity of p21-activated kinase, X-PAK1. The phenotypes of embryos that lack, or have excess, X-PAK1 activity mimic the phenotypes induced by loss or gain of TH functions, respectively. We provide evidence that X-PAK1 is an upstream regulator of TH and discuss potential functions of TH at the cell cortex/cytoplasmic membrane and in the nucleus.

Integrin-linked kinase (ILK) is a phosphorylated protein that regulates physiological processes that overlap with those regulated by p21-activated kinase 1 (PAK1). Here we report the possible role of ILK phosphorylation by PAK1 in ILK-mediated signaling and intracellular translocation. We found that PAK1 phosphorylates ILK at threonine-173 and serine-246 in vitro and in vivo. Depletion of PAK1 decreased the levels of endogenous ILK phosphorylation in vivo. Mutation of PAK1 phosphorylation sites on ILK to alanine reduced cell motility and cell proliferation. Biochemical fractionation, confocal microscopy, and chromatin-interaction analyses of human cells revealed that ILK localizes predominantly in the cytoplasm but also resides in the nucleus. Transfection of MCF-7 cells with point mutants ILK-T173A, ILK-S246A, or ILK-T173A; S246A (ILK-DM) altered ILK localization. Selective depletion of PAK1 dramatically increased the nuclear and focal point accumulation of ILK, further demonstrating a role for PAK1 in ILK translocation. We also identified functional nuclear localization sequence and nuclear export sequence motifs in ILK, delineated an apparently integral role for ILK in maintaining normal nuclear integrity, and established that ILK interacts with the regulatory region of the CNKSR3 gene chromatin to negatively modulate its expression. Together, these results suggest that ILK is a PAK1 substrate, undergoes phosphorylation-dependent shuttling between the cell nucleus and cytoplasm, and interacts with gene-regulatory chromatin.

The p21-activated kinases signal through a number of cellular pathways fundamental to growth, differentiation and apoptosis. A wealth of information has accumulated at an impressive pace in the recent past, both with regard to previously identified targets for p21-activated kinases that regulate the actin cytoskeleton and cellular stress pathways and with regard to newly identified targets and their role in cancer. Emerging data also provide new clues towards a previously unappreciated link between these various cellular processes. The present review attempts to provide a quick tutorial to the reader about the evolving significance of p21-activated kinases and small GTPases in breast cancer, using information from mouse models, tissue culture studies, and human materials.

p21-activated kinases (Paks) are effectors of the small GTPases Cdc42 and Rac, and are thought to mediate some of the cytoskeletal and transcriptional activities of these proteins. To localize activated Pak1 in cells, we developed an antibody directed against a phosphopeptide that is contained within the activation loop of Pak1. This antibody specifically recognizes the activated form of Pak1. Immunofluorescence analysis of NIH-3T3 cells coexpressing activated Cdc42 or Rac1 plus wild-type Pak1 shows that activated Pak1 accumulates at sites of focal adhesion, throughout filopodia and within the body and edges of lamellipodia. Platelet-derived growth factor stimulation of NIH-3T3 cells shows a pattern of Pak1 activation similar to that observed with Rac1. During closure of a fibroblast monolayer wound, Pak1 is rapidly activated and localizes to the leading edge of motile cells, then gradually tapers off as the wound closes. The activation of Pak1 by wounding is blocked by inhibitors of phosphatidylinositol 3-kinase, and Src family kinases, but not by an inhibitor of the epidermal growth factor receptor. These findings indicate that activated Pak1, and by extension, probably activated Cdc42 or Rac, accumulates at sites of cortical actin remodeling in motile fibroblasts.

During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and cell surface growth are polarized, mediating bud emergence, bud growth, and cytokinesis. We have determined whether p21-activated kinase (PAK)-family kinases regulate cell and actin polarization at one or several points during the yeast cell cycle. Inactivation of the PAK homologues Ste20 and Cla4 at various points in the cell cycle resulted in loss of cell and actin cytoskeletal polarity, but not in depolymerization of F-actin. Loss of PAK function in G1 depolarized the cortical actin cytoskeleton and blocked bud emergence, but allowed isotropic growth and led to defects in septin assembly, indicating that PAKs are effectors of the Rho–guanosine triphosphatase Cdc42. PAK inactivation in S/G2 resulted in depolarized growth of the mother and bud and a loss of actin polarity. Loss of PAK function in mitosis caused a defect in cytokinesis and a failure to polarize the cortical actin cytoskeleton to the mother-bud neck. Cla4–green fluorescent protein localized to sites where the cortical actin cytoskeleton and cell surface growth are polarized, independently of an intact actin cytoskeleton. Thus, PAK family kinases are primary regulators of cell and actin cytoskeletal polarity throughout most or all of the yeast cell cycle. PAK-family kinases in higher organisms may have similar functions.

Neisseria gonorrhoeae (Ngo), the etiologic agent of gonorrhea, induce a number of proinflammatory cytokines by contact to epithelial cells. Cytokine genes and a variety of other immune response genes are activated as a result of the regulatory function of immediate early response transcription factors including activator protein 1 (AP-1). Since it is established that phosphorylation of c-Jun, the central component of AP-1, by the stress-activated c-Jun NH2-terminal kinase (JNK) increases the transcriptional activity of AP-1, we studied whether Ngo could induce stress response pathways involving JNK. We found that virulent Ngo strains induce phosphorylation and activation of JNK but not of p38 kinase. Analysis of a nonpathogenic Ngo strain revealed only weak JNK activation. In respect to the molecular components upstream of the JNK signaling cascade, we show that a dominant negative mutant of MAP kinase kinase 4 (MKK4) represses transcription of an AP-1–dependent reporter gene. Regarding upstream stress response factors involved in Ngo-induced MKK4/JNK/AP-1 activation, we identified p21-activated kinase (PAK) but not MAPK/ERK kinase kinase (MEKK1). Inhibition of small GTPases including Rac1 and Cdc42 by Toxin B prevented JNK and AP-1 activation. Our results indicate that Ngo induce the activation of proinflammatory cytokines via a cascade of cellular stress response kinases involving PAK, which directs the signal from the Rho family of small GTPases to JNK/AP-1 activation.

The Plenty of SH3 domains protein (POSH) is an E3 ligase and a scaffold in the JNK mediated apoptosis, linking Rac1 to downstream components.We here describe POSH2 which was identified from a p21-activated kinase 2 (PAK2) interactor screen. POSH2 is highly homologous with other members of the POSH family; it contains four Src homology 3 (SH3) domains and a RING finger domain which confers E3 ligase activity to the protein. In addition POSH2 contains an N-terminal extension which is conserved among its mammalian counterparts. POSH2 interacts with GTP-loaded Rac1. We have mapped this interaction to a previously unrecognized partial Cdc42/Rac1-interactive binding domain.Structured summaryMINT-7987761: POSH1 (uniprotkb:Q9HAM2) physically interacts (MI:0915) with Ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)MINT-7987932: PAK2 (uniprotkb:Q13177) binds (MI:0407) to CDC42 (uniprotkb:Q07912) by solid phase assay (MI:0892)MINT-7987908: POSH1 (uniprotkb:Q9HAM2) binds (MI:0407) to Rac1 (uniprotkb:P63000) by solid phase assay (MI:0892)MINT-7987880: POSH2 (uniprotkb:Q8TEJ3) binds (MI:0407) to Rac1 (uniprotkb:P63000) by solid phase assay (MI:0892)MINT-7987734: POSH2 (uniprotkb:Q8TEJ3) physically interacts (MI:0915) with Ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)MINT-7987779, MINT-7987804, MINT-7987824, MINT-7987838, MINT-7987853: Rac1 (uniprotkb:P63000) physically interacts (MI:0915) with POSH2 (uniprotkb:Q8TEJ3) by anti tag coimmunoprecipitation (MI:0007)MINT-7987920: PAK2 (uniprotkb:Q13177) binds (MI:0407) to Rac1 (uniprotkb:P63000) by solid phase assay (MI:0892)

Members of the Cool protein family contain SH3, Dbl, and pleckstrin homology domains and are binding partners for the p21-activated kinase (PAK). Using the yeast two-hybrid screen, we identified Cbl-b as a Cool family binding partner. We co-immunoprecipitated endogenous Cool and Cbl-b from a variety of breast cancer cell lines. The Cool–Cbl-b interaction requires the SH3 domain of Cool and competes with the binding of PAK to Cool proteins. Expression of Cbl-b effectively blocks the ability of Cool-2 to stimulate PAK, thus providing an additional mechanism, aside from catalyzing receptor ubiquitination, by which Cbl-b acts as a negative regulator for signaling activities requiring PAK activation.