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P21-activated kinases (PAK) regulate processes associated with cytoskeleton dynamics. PAK expression in leukemia cells was measured on protein and mRNA levels. In functional assays, we analyzed the effect of PAK inhibitors IPA-3 and FRAX597 on cell adhesivity and viability. PAK2 was dominant in cell lines, whereas primary cells also expressed comparable amount of PAK1 transcription isoforms: PAK1-full and PAK1Δ15. PAK1Δ15 and PAK2 levels correlated with surface density of integrins β1 and αVβ3. PAK1-full, but not PAK2, was present in membrane protrusions. IPA-3, which prevents PAK activation, induced cell contraction in semi-adherent HEL cells only. FRAX597, which inhibits PAK kinase activity, increased cell-surface contact area in all leukemia cells. Both inhibitors reduced the stability of cell attachment and induced cell death.
Influenza A virus infection stimulates a wide range of virus-supportive or antiviral mechanisms in host cells. p21-Activated kinase 1 (PAK1) is a serine/threonine kinase that regulates a number of fundamental cellular processes and has been implicated in the modulation of virus replication. Here, we investigated the role of PAK1 activation during influenza A virus infection and found that virus propagation corresponded to stimulated PAK1 phosphorylation. Moreover, transfection of the active form of PAK1 (PAK1-T423E) in A549 cells induced higher viral titers (∼10-fold differences) compared to that in the control vector or inactive PAK1 (PAK1-K299R)-transfected cells. PAK1-specific siRNA knockdown also resulted in 10-100-fold reductions in virus yields compared to that in the control siRNA-treatment (p<0.05). We further showed that treatment with PAK18, a PAK1 peptide inhibitor, resulted in marked suppression of both ERK 1/2 phosphorylation and infectious virus production, which was comparable to that by U0126, a specific MEK/ERK inhibitor. These results provide evidence for the importance of PAK1 activation during influenza virus infection and its association with ERK in regulating virus replication. The present study also implicates PAK1 as a potential therapeutic target for managing influenza virus infections.
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.
Correct positioning of organelles is essential to eukaryotic cells. Molecular motors transport organelles to their proper destinations, yet little is known about the pathways that define these destinations. In Saccharomyces cerevisiae, the myosin V motor Myo2 binds the vacuole-specific adapter Vac17 to attach to the vacuole/lysosome and initiate transport. After arrival in the bud, Myo2 releases the vacuole, and Vac17 is degraded. However, the mechanisms that spatially regulate this release were not established. In this study, we report that the bud cortex is a landmark that signals a successful delivery of the vacuole to the bud. We demonstrate that upon arrival at the bud cortex, Vac17 is phosphorylated by Cla4. Cla4-dependent phosphorylation is required for the ubiquitylation and subsequent degradation of Vac17 and the release of the vacuole from Myo2. Our study reveals a critical step in the spatial regulation of myosin V-dependent organelle transport and may reveal common mechanisms for how molecular motors accurately deposit cargoes at the correct locations.
Pancreatic cancer is one of the most lethal of human malignancies. Nearly 100% cases of pancreatic cancer carry mutations in KRas. P-21-activated kinases (PAKs) are activated by and act downstream of KRas. Glaucarubinone, a natural product first isolated from the seeds of the tree Simarouba glauca, was originally developed as an antimalarial drug, and has more recently been recognised as an anticancer agent. The aims of this study were to determine whether glaucarubinone, alone or in combination with the front-line chemotherapeutic agent gemcitabine, would inhibit the growth of pancreatic cancer cells in vitro or in vivo and the mechanism involved. Growth of the human pancreatic cancer cell lines PANC-1 and MiaPaCa-2 was measured by (3)H-thymidine incorporation in vitro, and by volume as xenografts in SCID mice. The expression and activities of the two serine/threonine kinases PAK1 and PAK4, which are key regulators of cancer progression, were measured by Western blotting. Here we report that glaucarubinone decreased proliferation and migration of pancreatic cancer cells in vitro, and reduced their growth as xenografts in vivo. Treatment with glaucarubinone and gemcitabine reduced proliferation in vitro and tumor growth in vivo more than treatment with either glaucarubinone or gemcitabine alone. Treatment with glaucarubinone reduced PAK1 and PAK4 activities, which were further decreased by the combination of glaucarubinone and gemcitabine. These results indicate that glaucarubinone reduced pancreatic cancer cell growth at least in part via inhibition of pathways involving PAK1 and PAK4. The synergistic inhibition by glaucarubinone and gemcitabine observed both in vitro and in vivo suggests that glaucarubinone may be a useful adjunct to current regimes of chemotherapy.
Theoretically, small molecule CDK4/6 inhibitors (CDK4/6is) represent a logical therapeutic option in non-small cell lung cancers since most of these malignancies have wildtype RB, the key target of CDKs and master regulator of the cell cycle. Unfortunately, CDK4/6is are found to have limited clinical activity as single agents in non-small cell lung cancer. To address this problem and to identify effective CDK4/6i combinations, we screened a library of targeted agents for efficacy in four non-small cell lung cancer lines treated with CDK4/6 inhibitors Palbociclib or Abemaciclib. The pan-PAK (p21-activated kinase) inhibitor PF03758309 emerged as a promising candidate with viability ratios indicating synergy in all 4 cell lines and for both CDK4/6is. It is noteworthy that the PAKs are downstream effectors of small GTPases Rac1 and Cdc42 and are overexpressed in a wide variety of cancers. Individually the compounds primarily induced cell cycle arrest; however, the synergistic combination induced apoptosis, accounting for the synergy. Surprisingly, while the pan-PAK inhibitor PF03758309 synergizes with CDK4/6is, no synergy occurs with group I PAK inhibitors FRAX486 or FRAX597. Cell lines treated only with Ribociclib, FRAX486 or FRAX597 underwent G1/G0 arrest, whereas combination treatment with these compounds predominantly resulted in autophagy. Combining high concentrations of FRAX486, which weakly inhibits PAK4, and Ribociclib, mimics the autophagy and apoptotic effect of PF03758309 combined with Ribociclib. FRAX597, a PAKi that does not inhibit PAK4 did not reduce autophagy in combination with Ribociclib. Our results suggest that a unique combination of PAKs plays a crucial role in the synergy of PAK inhibitors with CDK4/6i. Targeting this unique PAK combination, could greatly improve the efficacy of CDK4/6i and broaden the spectrum of cancer treatment.
NF2 is an autosomal dominant disease characterized by development of bilateral vestibular schwannomas and other benign tumors in central nervous system. Loss of the NF2 gene product, Merlin, leads to aberrant Schwann cell proliferation, motility, and survival, but the mechanisms by which this tumor suppressor functions remain unclear. One well-defined target of Merlin is the group I family of p21-activated kinases, which are allosterically inhibited by Merlin and which, when activated, stimulate cell cycle progression, motility, and increased survival. Here, we examine the effect of Pak inhibition on cells with diminished Merlin function.
The matricellular protein periostin (PN) promotes postnatal valve remodeling and maturation. Incomplete remodeling of the valve can trigger degenerative processes that lead to a myxomatous phenotype that includes loss of PN. However, signaling pathways involved that link valvular-interstitial-fibroblast cells (VICs) to proliferation, migration and actin remodeling functions are unclear. The p21-activated kinases (Paks) have been shown to regulate cytoskeleton rearrangements and cell proliferation/adhesion/migration functions in a variety of cellular contexts, including normal cells and cancer cells. This study shows that Pak1, but not Pak2 and Pak4, is a critical mediator of VIC survival and actin organization, and that the molecular signaling regulating actin-remodeling is initiated upon PN/beta-integrin-induced phosphorylation of the focal-adhesion-kinase (Fak) (Y397). Molecular and pharmacological inhibition of key components of PN/Fak/Akt1 signaling abolished the PN-induced actin polymerization and the activation of mTOR, p70S6K and Pak1. Similarly, blocking mTOR inhibited p70S6K, Pak1 phosphorylation and consequently actin-polymerization. Accordingly, inhibiting p70S6K blocked Pak1 phosphorylation and actin polymerization, and subsequently inhibited adhesion and growth of VICs. Periostin-induced Akt1 activation of Pak1 is independent of Cdc42 and Rac1 GTPases, and Akt1 is both downstream and upstream of Pak1. Further, the PN-Pak1-induced Akt1 protects cells from apoptosis through suppression of transcriptional activation of Forkhead-Transcription-Factor (FKHR). In contrast, kinase deficient Pak1 increases apoptosis by increasing FKHR-mediated transcriptional activation. These studies define new functional significance of PN-Fak-Akt1-Pak1 signaling that at least partly regulates Akt1-induced actin polymerization and FKHR-mediated transcriptional activation, which may eventually regulate the mature-valve-leaflet remodeling function, and also FKHR-mediated transcriptional activation for pro-survival of VICs.
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.
p21-activated kinases have been classified into two groups based on their domain architecture. Group II PAKs (PAK4-6) regulate a wide variety of cellular functions, and PAK deregulation has been linked to tumor development. Structural comparison of five high-resolution structures comprising all active, monophosphorylated group II catalytic domains revealed a surprising degree of domain plasticity, including a number of catalytically productive and nonproductive conformers. Rearrangements of helix alphaC, a key regulatory element of kinase function, resulted in an additional helical turn at the alphaC N terminus and a distortion of its C terminus, a movement hitherto unseen in protein kinases. The observed structural changes led to the formation of interactions between conserved residues that structurally link the glycine-rich loop, alphaC, and the activation segment and firmly anchor alphaC in an active conformation. Inhibitor screening identified six potent PAK inhibitors from which a tri-substituted purine inhibitor was cocrystallized with PAK4 and PAK5.
Inflammasomes are signalling platforms that are assembled in response to infection or sterile inflammation by cytosolic pattern recognition receptors. The consequent inflammasome-triggered caspase-1 activation is critical for the host defence against pathogens. During infection, NLRP3, which is a pattern recognition receptor that is also known as cryopyrin, triggers the assembly of the inflammasome-activating caspase-1 through the recruitment of ASC and Nek7. The activation of the NLRP3 inflammasome is tightly controlled both transcriptionally and post-translationally. Despite the importance of the NLRP3 inflammasome regulation in autoinflammatory and infectious diseases, little is known about the mechanism controlling the activation of NLRP3 and the upstream signalling that regulates the NLRP3 inflammasome assembly. We have previously shown that the Rho-GTPase-activating toxin from Escherichia coli cytotoxic necrotizing factor-1 (CNF1) activates caspase-1, but the upstream mechanism is unclear. Here, we provide evidence of the role of the NLRP3 inflammasome in sensing the activity of bacterial toxins and virulence factors that activate host Rho GTPases. We demonstrate that this activation relies on the monitoring of the toxin's activity on the Rho GTPase Rac2. We also show that the NLRP3 inflammasome is activated by a signalling cascade that involves the p21-activated kinases 1 and 2 (Pak1/2) and the Pak1-mediated phosphorylation of Thr 659 of NLRP3, which is necessary for the NLRP3-Nek7 interaction, inflammasome activation and IL-1β cytokine maturation. Furthermore, inhibition of the Pak-NLRP3 axis decreases the bacterial clearance of CNF1-expressing UTI89 E. coli during bacteraemia in mice. Taken together, our results establish that Pak1 and Pak2 are critical regulators of the NLRP3 inflammasome and reveal the role of the Pak-NLRP3 signalling axis in vivo during bacteraemia in mice.
Breast cancer, the most common cause of cancer-related deaths worldwide among women, is a molecularly and clinically heterogeneous disease. Extensive genetic and epigenetic profiling of breast tumors has recently revealed novel putative driver genes, including p21-activated kinase (PAK)1. PAK1 is a serine/threonine kinase downstream of small GTP-binding proteins, Rac1 and Cdc42, and is an integral component of growth factor signaling networks and cellular functions fundamental to tumorigenesis.
Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and tropical spastic paraparesis. HTLV-1 encodes transactivator protein Tax that interacts with various cellular factors to modulate transcription and other biological functions. Additional cellular mediators of Tax-mediated transcriptional activation of HTLV-1 long terminal repeats (LTR) remain to be identified and characterized.
p21-activated kinases (PAKs) are key regulators of actin dynamics, cell proliferation and cell survival. Deregulation of PAK activity contributes to the pathogenesis of various human diseases, including cancer and neurological disorders. Using an ELISA-based screening protocol, we identified naphtho(hydro)quinone-based small molecules that allosterically inhibit PAK activity. These molecules interfere with the interactions between the p21-binding domain (PBD) of PAK1 and Rho GTPases by binding to the PBD. Importantly, they inhibit the activity of full-length PAKs and are selective for PAK1 and PAK3 in vitro and in living cells. These compounds may potentially be useful for determining the details of the PAK signaling pathway and may also be used as lead molecules in the development of more selective and potent PAK inhibitors.
p21-activated kinase 7 (PAK7), also named as PAK5, is a member of Rac/Cdc42-associated Ser/Thr protein kinases. It is overexpressed in some types of cancer such as colorectal and pancreatic cancers. However, the expression status and biological function of PAK7 in osteosarcoma are still ambiguous. To evaluate the expression levels of PAK7 in osteosarcoma tissues and cell lines, immunohistochemistry was used. To investigate the role of PAK7 in cell proliferation, apoptosis and tumorigenicity in vitro and vivo, a recombinant lentivirus expressing PAK7 short hairpin RNA (Lv-shPAK7) was developed and transfected into Saos-2 cells. The silencing effect of PAK7 was confirmed by quantitative real-time PCR (qRT-PCR) and Western blot technique. PAK7 was overexpressed in osteosarcoma tissue and cell line. By knocking-down of PAK7, the proliferation and colony formation of Saos-2 cells were inhibited and apoptosis enhanced significantly. The in vivo tumorigenic ability in xenograft model of Saos-2 cells was also notably inhibited when PAK7 was knocked down. Our results imply that PAK7 promotes cell proliferation and tumorigenesis and may be an attractive candidate for the therapeutic target of osteosarcoma.
p21-activated kinases (PAKs) play an important role in diverse cellular processes. Full activation of PAKs requires autophosphorylation of a critical threonine/serine located in the activation loop of the kinase domain. Here we report crystal structures of the phosphorylated and unphosphorylated PAK1 kinase domain. The phosphorylated PAK1 kinase domain has a conformation typical of all active protein kinases. Interestingly, the structure of the unphosphorylated PAK1 kinase domain reveals an unusual dimeric arrangement expected in an authentic enzyme-substrate complex, in which the activation loop of the putative "substrate" is projected into the active site of the "enzyme." The enzyme is bound to AMP-PNP and has an active conformation, whereas the substrate is empty and adopts an inactive conformation. Thus, the structure of the asymmetric homodimer mimics a trans-autophosphorylation complex, and suggests that unphosphorylated PAK1 could dynamically adopt both the active and inactive conformations in solution.
The p21-activated kinases (Paks) interact with Rac/Cdc42 GTPases to regulate the actin cytoskeleton as well as various signaling pathways. Although activation of Paks in many human cancers is known to mediate cancer progression, the role of Pak proteins in hypoxia is poorly understood. In this study, we found that both Pak1 and Pak4 are highly expressed in HeLa cervical cancer cells, but only Pak4 knockdown attenuates expression of hypoxia-inducible factor-1α (HIF-1α) in hypoxia. We further discovered that Pak4 regulates HIF-1α translation via the Akt-mTOR-4E-BP1 pathway under hypoxic conditions. These results support a novel connection between HIF-1α and Pak4 in hypoxic cancer cells, and provide insights into mechanisms whereby tumors respond to and thrive under oxygen-deficient conditions.
Raf-1 is an important effector of Ras mediated signaling and is a critical regulator of the ERK/MAPK pathway. Raf-1 activation is controlled in part by phosphorylation on multiple residues, including an obligate phosphorylation site at serine 338. Previously PAK1 and casein kinase II have been implicated as serine 338 kinases. To identify novel kinases that phosphorylate this site, we tested the ability of group II PAKs (PAKs 4-6) to control serine 338 phosphorylation. We observed that all group II PAKs were efficient serine 338 kinases, although only PAK1 and PAK5 significantly stimulated Raf-1 kinase activity. We also showed that PAK5 forms a tight complex with Raf-1 in the cell, but not A-Raf or B-Raf. Importantly, we also demonstrated that the association of Raf-1 with PAK5 targets a subpopulation of Raf-1 to mitochondria. These data indicate that PAK5 is a potent regulator of Raf-1 activity and may control Raf-1 dependent signaling at mitochondria.
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.
Asexual spores (conidia) are the infectious propagules of many pathogenic fungi, and the capacity to sense the host environment and trigger conidial germination is a key pathogenicity determinant. Germination of conidia requires the de novo establishment of a polarised growth axis and consequent germ tube extension. The molecular mechanisms that control polarisation during germination are poorly understood. In the dimorphic human pathogenic fungus Penicillium marneffei, conidia germinate to produce one of two cell types that have very different fates in response to an environmental cue. At 25 degrees C, conidia germinate to produce the saprophytic cell type, septate, multinucleate hyphae that have the capacity to undergo asexual development. At 37 degrees C, conidia germinate to produce the pathogenic cell type, arthroconidiating hyphae that liberate uninucleate yeast cells. This study shows that the p21-activated kinase pakA is an essential component of the polarity establishment machinery during conidial germination and polarised growth of yeast cells at 37 degrees C but is not required for germination or polarised growth at 25 degrees C. Analysis shows that the heterotrimeric G protein alpha subunit GasC and the CDC42 orthologue CflA lie upstream of PakA for germination at both temperatures, while the Ras orthologue RasA only functions at 25 degrees C. These findings suggest that although some proteins that regulate the establishment of polarised growth in budding yeast are conserved in filamentous fungi, the circuitry and downstream effectors are differentially regulated to give rise to distinct cell types.
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