Activation Of P21-activated Kinase Research Articles

Learning-induced enduring changes in inhibitory synaptic transmission in lateral amygdala are mediated by p21-activated kinase.

In this study we explored whether learning leads to enduring changes in inhibitory synaptic transmission in lateral amygdala (LA). We revealed that olfactory discrimination (OD) learning in rats led to a long-lasting increase in postsynaptic GABAA channel-mediated miniature inhibitory postsynaptic currents (mIPSCs) in LA. Olfactory fear conditioning, but not auditory fear conditioning, also led to enduring enhancement in GABAA-mediated mIPSCs. Auditory fear conditioning, but not olfactory fear conditioning or OD learning, induced an enduring reduction in the frequency but not the current of mIPSC events. We found that p21-activated kinase (PAK) activity is needed to maintain OD and olfactory fear conditioning learning-induced enduring enhancement of mIPSCs. Further analysis revealed that OD led to an increase in GABAA channel conductance whereas olfactory fear conditioning increased the number of GABAA channels. These alterations in GABAA channels conductance and level are controlled by PAK activity. Our study shows that the learning-induced increase in postsynaptic inhibitory transmission in LA is specific to the sensory modality. However, the mechanism that mediates the increase in inhibitory transmission, namely the increase in the conductance or in the level of GABAA channel, is learning specific.NEW & NOTEWORTHY Here we studied whether learning leads to long-lasting alterations in inhibitory synaptic transmission in lateral amygdala (LA). We revealed that learning led to enduring changes in inhibitory synaptic transmission in LA that are affected by the sensory modality (auditory or olfaction) used during learning. However, the mechanism that mediated the changes in inhibitory transmission (alterations in GABAA channel level or conductance) depended on the type of learning. These long-lasting alterations are maintained by p21-activated kinase.

The Role of PAK1 in the Maturation of Invadopodia During Transient Mechanical Stimulation.

Cancer cells are affected by a wide range of mechanical forces within their extracellular environment. It has been widely shown that these forces can lead to increased metastatic activity of these cells. One such force is a transient tugging-like force that results from contractile forces generated by cells within the tumor microenvironment. When this force is simulated in vitro with a mechano-invasion assay, human fibrosarcoma cells exhibit enhanced cell invasion in a 3D collagen-fibronectin matrix by downregulating the expression of integrin β3. Furthermore, this force stimulates the maturation of invadopodia in an integrin β3-dependent manner that includes an increase in the active form of cofilin and MMP-2 secretion. In the present study we discovered that the decrease in integrin β3 signaling in response to mechanical stimulation is coupled to the activity of p21-activated kinase 1 (PAK1). It was found that PAK1 has decreased activity, as detected by a decrease in Ser144 phosphorylation, with mechanical stimulation. However, this loss in phosphorylation can be reversed if integrin β3 is overexpressed. Furthermore, PAK1 mutants show a correlated response in MMP-2 enzyme expression and activity, in addition to the lengthening of invadopodia, in response to stimulation. These results identify a novel mechano-sensitive response in human fibrosarcoma that utilizes PAK1 as a signaling player positioned downstream of integrin β3.

P21-Activated Kinase 1 Overactivates in Eutopic Endometrium of Adenomyosis.

Adenomyosis is a common gynecological disease, characterized by the existence of endometrium in the myometrium. The pathogenesis of adenomyosis is not fully understood. P21-activated kinase 1 (PAK1) is an effector of small Rho GTPases including CDC42 and RAC1 and plays various roles in cellular biology, especially cytoskeletal remodeling. This study aimed to evaluate whether the expression and activation of PAK1 in adenomyosis were different from normal. Immunohistochemistry was performed to evaluate the expression of PAK1 and its active form phosphorylated-PAK1 (pPAK1) semi-quantitatively in women with and without adenomyosis. Immunofluorescence was performed to locate the distribution of pPAK1. This study found that PAK1 in eutopic endometrium of adenomyosis was overactivated compared to normal. Phosphorylated-PAK1 assembled along the apical surface of glandular cell membrane. In ectopic lesions, PAK1 expression decreased and its activation returned to the baseline. The expression of pPAK1 correlated with the frequency of reproduction. These findings suggest that PAK1 overactivation in the endometrium may be an important event during the development of adenomyosis, meanwhile, decreased phosphorylation may assist to form lesions.

Pathogenic Escherichia coli Hijacks GTPase-Activated p21-Activated Kinase for Actin Pedestal Formation.

Enteropathogenic Escherichia coli and enterohemorrhagic E. coli (EPEC and EHEC, respectively) are extracellular pathogens that reorganize the host cell cytoskeleton to form "actin pedestals" beneath the tightly adherent bacteria, a critical step in pathogenesis. EPEC and EHEC inject effector proteins that manipulate host cell signaling cascades to trigger pedestal assembly. One such effector, EspG, has been reported to bind and activate p21-activated kinase (PAK), a key cytoskeletal regulator, but the function of this interaction and whether it impacts pedestal assembly are unknown. Here, we demonstrate that deletion of espG significantly impairs pedestal formation and attachment by both EPEC and EHEC. This role of EspG is shown to be dependent on its interaction with PAK. Unexpectedly, EspG was able to subvert PAK only in the presence of Rho family small GTPases, which function to both concentrate PAK at the membrane and stimulate PAK activation. Our findings reveal a novel mechanism by which EspG hijacks PAK and sustains its active state to drive bacterial attachment to host cells.IMPORTANCE Enteropathogenic E. coli and enterohemorrhagic E. coli (EPEC and EHEC, respectively) remain a significant global health problem. Both EPEC and EHEC initiate infection by attaching to cells in the host intestine, triggering the formation of actin-rich "pedestal" structures directly beneath the adherent pathogen. These bacteria inject their own receptor into host cells, which upon binding to a protein on the pathogen surface triggers pedestal formation. Multiple other proteins are also delivered into the cells of the host intestine, but how they contribute to disease is often less clear. Here, we show how one of these injected proteins, EspG, hijacks a host signaling pathway for pedestal production. This provides new insights into this essential early stage in EPEC and EHEC disease.

MAPK- and AKT-activated thyroid cancers are sensitive to group I PAK inhibition.

The number of individuals who succumb to thyroid cancer has been increasing and those who are refractory to standard care have limited therapeutic options, highlighting the importance of developing new treatments for patients with aggressive forms of the disease. Mutational activation of MAPK signaling, through BRAF and RAS mutations and/or gene rearrangements, and activation of PI3K signaling, through mutational activation of PIK3CA or loss of PTEN, are well described in aggressive thyroid cancer. We previously reported overactivation and overexpression of p21-activated kinases (PAKs) in aggressive human thyroid cancer invasive fronts and determined that PAK1 functionally regulated thyroid cancer cell migration. We reported mechanistic crosstalk between the MAPK and PAK pathways that are BRAF-dependent but MEK independent, suggesting that PAK and MEK inhibition might be synergistic. In the present study, we tested this hypothesis. Pharmacologic inhibition of group I PAKs using two PAK kinase inhibitors, G-5555 or FRAX1036, reduced thyroid cancer cell viability, cell cycle progression and migration and invasion, with greater potency for G-5555. Combination of G-5555 with vemurafenib was synergistic in BRAFV600E-mutated thyroid cancer cell lines. Finally, G-5555 restrained thyroid size of BRAFV600E-driven murine papillary thyroid cancer by >50% (P < 0.0001) and reduced carcinoma formation (P = 0.0167), despite maintenance of MAPK activity. Taken together, these findings suggest both that group I PAKs may be a new therapeutic target for thyroid cancer and that PAK activation is functionally important for BRAFV600E-mediated thyroid cancer development.

Mo1398 – Activation of P21-Activated Kinase 4 (PAK4) is Needed to Stimulate Na+,K+Atpase Which Mediates Fluid/Electrolyte Secretion in Pancreatic Acini

Mo1398 – Activation of P21-Activated Kinase 4 (PAK4) is Needed to Stimulate Na+,K+Atpase Which Mediates Fluid/Electrolyte Secretion in Pancreatic Acini

Hypoxia aggravates Aβ1-42-induced toxicity in human neuroblastoma cells through PAK1/LIMK1/cofilin pathway

Objective To investigate the pathways by which hypoxia aggravates the neurotoxic effects of amyloid-beta protein(Aβ)on neurons. Methods Human neuroblastoma cells(SH-SY5Y cells)were cultured in vitro, and were divided into four groups: the control group, Aβ intervention group, hypoxia group, Aβ intervention plus hypoxia group.Quantitative real time polymerase chain reaction(qRT-PCR)was adopted to detect the mRNA expression levels of p21-activated kinase 1(PAK1), LIM kinase 1 protein(LIMK1)and cofilin.Western blot was used to measure the protein levels of PAK1, LIMK1, phosphate-LIMK1(P-LIMK1), cofilin and phosphate-cofilin(P-cofilin). Results After Aβ treatment, the activity of SH-SY5Y cells was decreased.Compared with the control group, the protein levels of PAK1, LIMK1, P-LIMK1, P-cofilin, and the mRNA expression levels of PAK1 and LIMK1 were decreased(all P<0.05), but the protein and mRNA expression of cofilin had no significant changes after 24 h of treatment with 10 μmol/L Aβ.Compared with the Aβ intervention group, the protein levels of PAK1, LIMK1, P-LIMK1 and P-cofilin were decreased(all P<0.05), and the mRNA expression levels of PAK1 and LIMK1 were decreased(both P<0.05), but the protein and mRNA expression of cofilin had no significant changes after 24 h of treatment of SH-SY5Y cells with 10 μmol/L Aβ plus 2% oxygen. Conclusions Aβ may reduce P-LIMK1 expression by inhibiting the activity of PAK1, thereby reducing the P-cofilin, increasing the formation of dephosphorylated cofilin, leading to neural cells damage, and hypoxia aggravates the neurotoxicity of Aβ through this pathway. Key words: Alzheimer disease; Amyloid beta-protein; Anoxia

Targeting PAK1 with the Small Molecule Drug AK963/40708899 Suppresses Gastric Cancer Cell Proliferation and Invasion by Downregulation of PAK1 Activity and PAK1-Related Signaling Pathways.

PAK1 (p21-activated kinase 1) is a serine/threonine protein kinase which has been initially identified as downstream effector of the Rho GTPase family. In previous research, PAK1 has been involved in the regulation of diverse cellular processes, such as cell motility, cell proliferation, gene transcription, cytoskeletal rearrangement, and cell invasion. Hyper-activation of PAK1 was constantly observed in a variety of human cancer which make it a potential target of novel anti-tumor drugs. To date, a great number of attentions focus on identifying the PAK1 inhibitors in medical and pharmaceutical fields. In this article, we found that a novel and potent PAK1 inhibitor, AK963/40708899, suppressed the proliferation of human gastric cancer cells significantly by downregulation of PAK1-NF-κB-cyclinB1 pathway. In addition, AK963/40708899 inhibited the formation of filopodia and promoted cell adhesion which in turn inhibited invasive potential of gastric cells by negatively regulating PAK1-LIMKl-cofilin and PAK1-ERK-FAK pathways. Considering our result, AK963/40708899 would be a possible candidate for PAK1 targeted anti-tumor drug. Anat Rec, 302:1571-1579, 2019. © 2019 American Association for Anatomy.

Mixed lineage kinase 3 promotes breast tumorigenesis via phosphorylation and activation of p21-activated kinase 1.

Mixed lineage kinase 3 (MLK3), a MAP3K member has been envisioned as a viable drug target in cancer, yet its detailed function and signaling is not fully elucidated. We identified that MLK3 tightly associates with an oncogene, PAK1. Mammalian PAK1 being a Ste20 (MAP4K) member, we tested whether it is an upstream regulator of MLK3. In contrast to our hypothesis, MLK3 activated PAK1 kinase activity directly, as well as in the cells. Although, MLK3 can phosphorylate PAK1 on Ser133 and Ser204 sites, PAK1S133A mutant is constitutively active, whereas, PAK1S204A is not activated by MLK3. Stable overexpression of PAK1S204A in breast cancer cells, impedes migration, invasion, and NFĸB activity. In vivo breast cancer cell tumorigenesis is significantly reduced in tumors expressing PAK1S204A mutant. These results suggest that mammalian PAK1 does not act as a MAP4K and MLK3-induced direct activation of PAK1 plays a key role in breast cancer tumorigenesis.

Rho kinase collaborates with p21-activated kinase to regulate actin polymerization and contraction in airway smooth muscle.

The mechanisms by which Rho kinase (ROCK) regulates airway smooth muscle contraction were determined in tracheal smooth muscle tissues. ROCK may mediate smooth muscle contraction by inhibiting myosin regulatory light chain (RLC) phosphatase. ROCK can also regulate F-actin dynamics during cell migration, and actin polymerization is critical for airway smooth muscle contraction. Our results show that ROCK does not regulate airway smooth muscle contraction by inhibiting myosin RLC phosphatase or by stimulating myosin RLC phosphorylation. We find that ROCK regulates airway smooth muscle contraction by activating the serine-threonine kinase Pak, which mediates the activation of Cdc42 and neuronal Wiskott-Aldrich syndrome protein (N-WASp). N-WASP transmits signals from Cdc42 to the Arp2/3 complex for the nucleation of actin filaments. These results demonstrate a novel molecular function for ROCK in the regulation of Pak and Cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle. Rho kinase (ROCK), a RhoA GTPase effector, can regulate the contraction of airway and other smooth muscle tissues. In some tissues, ROCK can inhibit myosin regulatory light chain (RLC) phosphatase, which increases the phosphorylation of myosin RLC and promotes smooth muscle contraction. ROCK can also regulate cell motility and migration by affecting F-actin dynamics. Actin polymerization is stimulated by contractile agonists in airway smooth muscle tissues and is required for contractile tension development in addition to myosin RLC phosphorylation. We investigated the mechanisms by which ROCK regulates the contractility of tracheal smooth muscle tissues by expressing a kinase-inactive mutant of ROCK, ROCK-K121G, in the tissues or by treating them with the ROCK inhibitor H-1152P. Our results show no role for ROCK in the regulation of non-muscle or smooth muscle myosin RLC phosphorylation during contractile stimulation in this tissue. We found that ROCK regulates airway smooth muscle contraction by mediating activation of p21-activated kinase (Pak), a serine-threonine kinase, to promote actin polymerization. Pak catalyses paxillin phosphorylation on Ser273 and coupling of the GIT1-βPIX-Pak signalling module to paxillin, which activates the guanine nucleotide exchange factor (GEF) activity of βPIX towards Cdc42. Cdc42 is required for the activation of neuronal Wiskott-Aldrich syndrome protein (N-WASp), which transmits signals from Cdc42 to the Arp2/3 complex for the nucleation of actin filaments. Our results demonstrate a novel molecular function for ROCK in the regulation of Pak and Cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle.

The actin-related p41ARC subunit contributes to p21-activated kinase-1 (PAK1)–mediated glucose uptake into skeletal muscle cells

Defects in translocation of the glucose transporter GLUT4 are associated with peripheral insulin resistance, preclinical diabetes, and progression to type 2 diabetes. GLUT4 recruitment to the plasma membrane of skeletal muscle cells requires F-actin remodeling. Insulin signaling in muscle requires p21-activated kinase-1 (PAK1), whose downstream signaling triggers actin remodeling, which promotes GLUT4 vesicle translocation and glucose uptake into skeletal muscle cells. Actin remodeling is a cyclic process, and although PAK1 is known to initiate changes to the cortical actin-binding protein cofilin to stimulate the depolymerizing arm of the cycle, how PAK1 might trigger the polymerizing arm of the cycle remains unresolved. Toward this, we investigated whether PAK1 contributes to the mechanisms involving the actin-binding and -polymerizing proteins neural Wiskott-Aldrich syndrome protein (N-WASP), cortactin, and ARP2/3 subunits. We found that the actin-polymerizing ARP2/3 subunit p41ARC is a PAK1 substrate in skeletal muscle cells. Moreover, co-immunoprecipitation experiments revealed that insulin stimulates p41ARC phosphorylation and increases its association with N-WASP coordinately with the associations of N-WASP with cortactin and actin. Importantly, all of these associations were ablated by the PAK inhibitor IPA3, suggesting that PAK1 activation lies upstream of these actin-polymerizing complexes. Using the N-WASP inhibitor wiskostatin, we further demonstrated that N-WASP is required for localized F-actin polymerization, GLUT4 vesicle translocation, and glucose uptake. These results expand the model of insulin-stimulated glucose uptake in skeletal muscle cells by implicating p41ARC as a new component of the insulin-signaling cascade and connecting PAK1 signaling to N-WASP-cortactin-mediated actin polymerization and GLUT4 vesicle translocation.

Affecting long-term fear memory formation through optical control of Rac1 GTPase and PAK activity in lateral amygdala

Fear conditioning, a behavioral model for studying fear-related disorders, is believed to be formed by alterations of synaptic efficacy mediated by changes in synaptic transmission and neuronal morphology in lateral amygdala (LA). Rac GTPase and its downstream effector p21-activated kinase (PAK) are involved in such key neuronal functions. Here we show that optical activation of Rac1 GTPase using photoactivatable form of Rac1 (PA-Rac1) in amygdala led to phosphorylation of PAK and inhibition of long-term but not short-term auditory fear conditioning memory formation. Activation of PA-Rac1 in LA one day after fear conditioning had no effect on long-term fear memory tested 24 hrs after PA-Rac1 activation. Inhibition of PAK in LA by microinjection of the PAK inhibitor IPA-3 30 minutes before fear conditioning enhanced long-term but not short-term fear memory formation. Our results demonstrate that photoactivation of Rac1 GTPase in lateral amygdala impairs fear memory formation. Moreover, Rac1 effector PAK activity during fear conditioning constrains the formation of fear memory in LA. Thus, Rac GTPase and PAK proteins may serve as targets for treatment of fear and anxiety disorders.

Transgenic autoinhibition of p21-activated kinase exacerbates synaptic impairments and fronto-dependent behavioral deficits in an animal model of Alzheimer's disease.

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.

Resveratrol regulates neuronal glucose uptake and insulin sensitivity via P21-activated kinase 2 (PAK2)

We have recently reported P21-activated kinase 2 (PAK2), a serine/threonine kinase as a negative regulator of neuronal glucose uptake and insulin sensitivity. Resveratrol (RSV), a natural polyphenol with anti-oxidative, anti-inflammatory and anti-diabetic properties, regulates PAK2 activity in HepG2 and ESC-B5 cell apoptosis. However, regulation of PAK2 by RSV in neuronal insulin signaling pathway, if any, is still unknown. In the present study, RSV treatment significantly increased PAK2 activity under insulin-sensitive and insulin-resistant condition, along with a marked decrease in glucose uptake in differentiated N2A cells. Pretreatment with AMPK inhibitor, followed by RSV treatment resulted in reduction in PAK2 activity whereas glucose uptake showed an increase. However, pretreatment with Akt inhibitor and then RSV exposure significantly increased PAK2 activity, with a corresponding decrease in glucose uptake. RSV treatment increased AMPK activity and decreased Akt activity. In conclusion, RSV negatively regulates neuronal glucose uptake and insulin sensitivity via PAK2.

Inhibition of group 1 p21-activated kinases suppresses pancreatic stellate cell activation and increases survival of mice with pancreatic cancer.

Pancreatic cancer remains one of the most lethal of all solid tumors. Pancreatic stellate cells (PSCs) are primarily responsible for the fibrosis that constitutes the stroma and p21-activated kinase 1 (PAK1) may have a role in signalling pathways involving PSCs. This study aimed to examine the role of PAK1 in PSCs and in the interaction of PSCs with pancreatic cancer cells. Human PSCs were isolated using the modified outgrowth method. The effect of inhibiting PAK1 with group 1 PAK inhibitor, FRAX597, on cell proliferation and apoptosis in vitro was measured by thymidine incorporation and annexin V assays, respectively. The effect of depleting host PAK1 on the survival of mice with pancreatic Pan02 cell tumors was evaluated using PAK1 knockout (KO) mice. PAK1 was expressed in isolated PSCs. FRAX597 reduced the activation of PSCs, inhibited PSC proliferation, and increased PSC apoptosis at least in partial by inhibiting PAK1 activity. The decreased expression and activity of PAK1 in PAK1 KO mice tumors was associated with an increased mouse survival. These results implicate PAK1 as a regulator of PSC activation, proliferation and apoptosis. Targeting stromal PAK1 could increase therapeutic response and survival of patients with pancreatic cancer.

Reduced PAK1 activity sensitizes FA/BRCA-proficient breast cancer cells to PARP inhibition.

Cells that are deficient in homologous recombination, such as those that have mutations in any of the Fanconi Anemia (FA)/BRCA genes, are hypersensitive to inhibition of poly(ADP-ribose) polymerase (PARP). However, FA/BRCA-deficient tumors represent a small fraction of breast cancers, which might restrict the therapeutic utility of PARP inhibitor monotherapy. The gene encoding the serine-threonine protein kinase p21-activated kinase 1 (PAK1) is amplified and/or overexpressed in several human cancer types including 25-30% of breast tumors. This enzyme controls many cellular processes by phosphorylating both cytoplasmic and nuclear substrates. Here, we show that depletion or pharmacological inhibition of PAK1 down-regulated the expression of genes involved in the FA/BRCA pathway and compromised the ability of cells to repair DNA by Homologous Recombination (HR), promoting apoptosis and reducing colony formation. Combined inhibition of PAK1 and PARP in PAK1 overexpressing breast cancer cells had a synergistic effect, enhancing apoptosis, suppressing colony formation, and delaying tumor growth in a xenograft setting. Because reduced PAK1 activity impaired FA/BRCA function, inhibition of this kinase in PAK1 amplified and/or overexpressing breast cancer cells represents a plausible strategy for expanding the utility of PARP inhibitors to FA/BRCA-proficient cancers.

Tuning PAK Activity to Rescue Abnormal Myelin Permeability in HNPP.

Schwann cells in the peripheral nervous systems extend their membranes to wrap axons concentrically and form the insulating sheath, called myelin. The spaces between layers of myelin are sealed by myelin junctions. This tight insulation enables rapid conduction of electric impulses (action potentials) through axons. Demyelination (stripping off the insulating sheath) has been widely regarded as one of the most important mechanisms altering the action potential propagation in many neurological diseases. However, the effective nerve conduction is also thought to require a proper myelin seal through myelin junctions such as tight junctions and adherens junctions. In the present study, we have demonstrated the disruption of myelin junctions in a mouse model (Pmp22+/-) of hereditary neuropathy with liability to pressure palsies (HNPP) with heterozygous deletion of Pmp22 gene. We observed a robust increase of F-actin in Pmp22+/- nerve regions where myelin junctions were disrupted, leading to increased myelin permeability. These abnormalities were present long before segmental demyelination at the late phase of Pmp22+/- mice. Moreover, the increase of F-actin levels correlated with an enhanced activity of p21-activated kinase (PAK1), a molecule known to regulate actin polymerization. Pharmacological inhibition of PAK normalized levels of F-actin, and completely prevented the progression of the myelin junction disruption and nerve conduction failure in Pmp22+/- mice. Our findings explain how abnormal myelin permeability is caused in HNPP, leading to impaired action potential propagation in the absence of demyelination. We call it “functional demyelination”, a novel mechanism upstream to the actual stripping of myelin that is relevant to many demyelinating diseases. This observation also provides a potential therapeutic approach for HNPP.

Vaccinia virus dissemination requires p21-activated kinase 1.

The orthopoxvirus vaccinia virus (VACV) interacts with both actin and microtubule cytoskeletons in order to generate and spread progeny virions. Here, we present evidence demonstrating the involvement of PAK1 (p21-activated kinase 1) in the dissemination of VACV. Although PAK1 activation has previously been associated with optimal VACV entry via macropinocytosis, its absence does not affect the production of intracellular mature virions (IMVs) and extracellular enveloped virions (EEVs). Our data demonstrate that low-multiplicity infection of PAK1(-/-) MEFs leads to a reduction in plaque size followed by decreased production of both IMVs and EEVs, strongly suggesting that virus spread was impaired in the absence of PAK1. Confocal and scanning electron microscopy showed a substantial reduction in the amount of VACV-induced actin tails in PAK1(-/-) MEFs, but no significant alteration in the total amount of cell-associated enveloped virions (CEVs). Furthermore, the decreased VACV dissemination in PAK1(-/-) cells was correlated with the absence of phosphorylated ARPC1 (Thr21), a downstream target of PAK1 and a key regulatory subunit of the ARP2/3 complex, which is necessary for the formation of actin tails and viral spread. We conclude that PAK1, besides its role in virus entry, also plays a relevant role in VACV dissemination.

Up-regulation of stem cell markers by P21-activated kinase 1 contributes to 5-fluorouracil resistance of colorectal cancer

ABSTRACTCancer stem cells (CSC) are tumorigenic and resistant to chemotherapy. In colorectal cancer (CRC), CSCs have been identified by the expression of specific markers, including CD44, Bmi1 and Nanog. Although p21-activated kinase 1 (PAK1), acting downstream of Ras, stimulates Wnt/β-catenin signaling and is known to play an important role in CRC development and progression, the role of PAK1 in the expression of CSC markers has not previously been investigated. The effect of PAK1 over-expression, knockdown or inhibition on the expression or alteration (in the case of CD44) of CSC markers in human CRC cell lines was measured by immunofluorescence and Western blotting. The effect of PAK1 modulation on tumorigenesis, and on resistance to treatment with 5-fluorouracil (5-FU), was measured by sphere formation in vitro and by growth of xenografted tumors in vivo. The results show that PAK1 activity correlated with the expression of CSC markers and the CD44 isoform profile, and with tumor growth both in vitro and in vivo. Furthermore PAK overexpression partially overcame the inhibition of CRC growth by 5-FU, and PAK inhibition was synergistic with 5-FU treatment. Our findings lay the foundation for a combination therapy in which PAK1 inhibitors targeting CSCs may be combined with conventional 5-FU-based chemotherapy for the treatment of CRC.

A novel PAK1/Notch-1 axis contributes to intestinal inflammation and cancer

Background: PAK1 belongs to a family of serine-threonine kinase and contributes to various oncogenic pathways like MAPK and Wnt-β catenin. Our lab recently linked activation of p21-activated kinase (PAK1) to IBD as well as colitis-associated cancer (Khare V, Inflamm Bowel Dis 2015). Here we tested the effects of PAK1 deletion on intestinal inflammation and carcinogenesis in IL-10 KO mice. Methods: IL-10 KO mice were crossed with Pak1 KO mice to generate double-knockout mice (DKO). Inflammation was triggered with piroxicam (200ppm mixed to chow for 14 d). After 12 wks mice were euthanized, intestines were prepared and length was measured. Paraffin embedded Swiss rolls were stained with HE MAPK pathway activation, as shown by increased pERK1/2 staining and an increase in γH2AX positive nuclei, indicating more DNA double-strand breaks. A reduced differentiation of goblet cells and Paneth cells was marked by PAS- and Lysozyme staining, together with an expansion of the stem cell compartment (shown by increased activated Notch1 and LGR5 staining) in the colonic crypts of DKOs. Interestingly NFκB was found to be reduced in DKO mice. Conclusion: Our data suggest a novel interaction of PAK-1 with the Notch pathway in regulating crypt cell stemness, proliferation and differentiation. Lack of mature goblet cells and mucus can exacerbate colitis in IL-10 KO mice. Hyperproliferation may promote dysplastic lesions independent of NFκB.