Fibrinogen Activates PAK1/Cofilin Signaling Pathway to Protect Endothelial Barrier Integrity.

INTRODUCTION: We recently demonstrated that fibrinogen stabilizes syndecan-1 on the endothelial cell (EC) surface and contributes to EC barrier protection, though the intracellular signaling pathway remains unclear. P21 (Rac1) activated kinase 1 (PAK1) is a protein kinase involved in intracellular signaling leading to actin cytoskeleton rearrangement and plays an important role in maintaining endothelial barrier integrity. We therefore hypothesized that fibrinogen binding to syndecan-1 activated the PAK1 pathway. METHODS: Primary human lung microvascular endothelial cells were incubated in 10% lactated Ringers (LR) solution or 10% fibrinogen saline solution (5 mg/mL). Protein phosphorylation was determined by Western blot analysis and endothelial permeability measured by fluorescein isothiocyanate (FITC)-dextran. Cells were silenced by siRNA transfection. Protein concentration was measured in the lung lavages of mice. RESULTS: Fibrinogen treatment resulted in increased syndecan-1, PAK1 activation (phosphorylation), cofilin activation (dephosphorylation), as well as decreased stress fibers and permeability when compared with LR treatment. Cofilin is an actin-binding protein that depolymerizes F-actin to decrease stress fiber formation. Notably, fibrinogen did not influence myosin light chain activation (phosphorylation), a mediator of EC tension. Silencing of PAK1 prevented fibrinogen-induced dephosphorylation of cofilin and barrier integrity. Moreover, to confirm the in vitro findings, mice underwent hemorrhagic shock and were resuscitated with either LR or fibrinogen. Hemorrhage shock decreased lung p-PAK1 levels and caused significant lung vascular leakage. However, fibrinogen administration increased p-PAK1 expression to near sham levels and remarkably prevented the lung leakage. CONCLUSION: We have identified a novel pathway by which fibrinogen activates PAK1 signaling to stimulate/dephosphorylate cofilin, leading to disassembly of stress fibers and reduction of endothelial permeability.

Cannabinoids Inhibited Pancreatic Cancer via P-21 Activated Kinase 1 Mediated Pathway.

The anti-cancer effects of cannabinoids including CBD (Cannabidiol) and THC ((-)-trans-∆9-tetrahydrocannabinol) have been reported in the case of pancreatic cancer (PC). The connection of these cannabinoids to KRas oncogenes that mutate in more than 90% of PC, and their effects on PD-L1, a key target of immune checkpoint blockade, have not been thoroughly investigated. Using cell lines and mouse models of PC, the effects of CBD and THC on cancer growth, the interaction between PC cells and a stromal cell, namely pancreatic stellate cells (PSCs), and the mechanism(s) involved were determined by cell-based assays and mouse study in vivo. CBD and THC inhibited the proliferation of PC, PSC, and PSC-stimulated PC cells. They also suppressed pancreatic tumour growth in mice. Furthermore, CBD and/or THC reduced the expression of PD-L1 by either PC or PSC cells. Knockout of p-21 activated kinase 1 (PAK1, activated by KRas) in PC and PSC cells and, in mice, dramatically decreased or blocked these inhibitory effects of CBD and/or THC. These results indicated that CBD and THC exerted their inhibitions on PC and PSC via a p-21 activated kinase 1 (PAK1)-dependent pathway, suggesting that CBD and THC suppress Kras activated pathway by targeting PAK1. The inhibition by CBD and THC of PD-L1 expression will enhance the immune checkpoint blockade of PC.

PAK Membrane Translocation and Phosphorylation Regulate Platelet Aggregation Downstream of Gi and G12/13 Pathways.

Platelet activation plays a pivotal role in physiological hemostasis and pathological thrombosis causing heart attack and stroke. Previous studies conclude that simultaneous activation of Gi and G12/13 signaling pathways is sufficient to cause platelet aggregation. However, using Gq knockout mice and Gq-specific inhibitors, we here demonstrated that platelet aggregation downstream of coactivation of Gi and G12/13 depends on agonist concentrations; coactivation of Gi and G12/13 pathways only induces platelet aggregation under higher agonist concentrations. We confirmed Gi and G12/13 pathway activation by showing cAMP (cyclic adenosine monophosphate) decrease and RhoA activation in platelets stimulated at both low and high agonist concentrations. Interestingly, we found that though Akt and PAK (p21-activated kinase) translocate to the platelet membrane upon both low and high agonist stimulation, membrane-translocated Akt and PAK only phosphorylate at high agonist concentrations, correlating well with platelet aggregation downstream of concomitant Gi and G12/13 pathway activation. PAK inhibitor abolishes Akt phosphorylation, inhibits platelet aggregation in vitro and arterial thrombus formation in vivo. We propose that the PAK-PI3K/Akt pathway mediates platelet aggregation downstream of Gi and G12/13, and PAK may represent a potential antiplatelet and antithrombotic target.

Knockdown of RhoGDI2 represses human gastric cancer cell proliferation, invasion and drug resistance via the Rac1/Pak1/LIMK1 pathway.

Gastric cancer (GC) is the fifth most common primary malignancy in humans. Rho GDP dissociation inhibitor 2 (RhoGDI2) is overexpressed in multiple cancer types, but the role of RhoGDI2 in GC has not been elucidated. This study aims to determine the level of RhoGDI2 in GC and to confirm the effect of its inhibition or overexpression on GC cell migration, invasion and chemosensitivity. RhoGDI2 level is significantly enhanced in human GC tissue samples in comparison with normal gastric epithelium and corresponding para-cancerous samples. The expression of RhoGDI2 is correlated with clinicopathological parameters and prognosis. Transfection in combination with miRNA targeting of RhoGDI2 in GC cell lines remarkably downregulates GC cell migration and invasion and reduces the mRNA levels of Rac1, Pak1 and LIMK1. The inhibition of RhoGDI2 downregulates GC cell migration and invasion by attenuating the EMT cascade via the Rac1/Pak1/LIMK1 pathway. Knockdown of RhoGDI2 is a potential therapeutic strategy for GC.

p21-Activated Kinases in Thyroid Cancer.

The family of p21-activated kinases (PAKs) are oncogenic proteins that regulate critical cellular functions. PAKs play central signaling roles in the integrin/CDC42/Rho, ERK/MAPK, PI3K/AKT, NF-κB, and Wnt/ß-catenin pathways, functioning both as kinases and scaffolds to regulate cell motility, mitosis and proliferation, cytoskeletal rearrangement, and other cellular activities. PAKs have been implicated in both the development and progression of a wide range of cancers, including breast cancer, pancreatic melanoma, thyroid cancer, and others. Here we will discuss the current knowledge on the structure and biological functions of both group I and group II PAKs, as well as the roles that PAKs play in oncogenesis and progression, with a focus on thyroid cancer and emerging data regarding BRAF/PAK signaling.

PAK1 silencing is synthetic lethal with CDK4/6 inhibition in gastric cancer cells via regulating PDK1 expression.

Gastric cancer (GC) is one of the most common malignancies worldwide. The prognosis of GC is unsatisfied owning to widespread metastasis. P21-activated kinase 1 (PAK1), a member of serine/threonine kinases, is associated with the progression of multiple types of human cancers. Here, we demonstrated that CDK4/6 inhibitor reduced GC cell viability and decreased PAK1 expression. Consistently, PAK1 ablation increased GC cell sensitivity exposed to CDK4/6 inhibitor and promoted DNA damage. We also revealed PAK1 depletion notably affected PDK1-AKT pathway, and PDK1 overexpression totally abrogated the effect of PAK1 deletion on DNA damage in GC cells. Additionally, PDK1 overexpression also rescued the increased GC cell sensitivity towards CDK4/6 inhibitor and the cell cycle arrest caused by PAK1 depletion. Our findings, therefore, suggested that PAK1 silencing increased sensitivity to CDK4/6 inhibition in gastric cancer cells via PDK1-AKT pathway. We, therefore, thought PAK1 as a promising therapeutic target for the treatment of CDK4/6 inhibitor-resistant gastric cancer.

ROCK1 but not LIMK1 or PAK2 is a key regulator of apoptotic membrane blebbing and cell disassembly.

Many cell types are known to undergo a series of morphological changes during the progression of apoptosis, leading to their disassembly into smaller membrane-bound vesicles known as apoptotic bodies (ApoBDs). In particular, the formation of circular bulges called membrane blebs on the surface of apoptotic cells is a key morphological step required for a number of cell types to generate ApoBDs. Although apoptotic membrane blebbing is thought to be regulated by kinases including ROCK1, PAK2 and LIMK1, it is unclear whether these kinases exhibit overlapping roles in the disassembly of apoptotic cells. Utilising both pharmacological and CRISPR/Cas9 gene editing based approaches, we identified ROCK1 but not PAK2 or LIMK1 as a key non-redundant positive regulator of apoptotic membrane blebbing as well as ApoBD formation. Functionally, we have established an experimental system to either inhibit or enhance ApoBD formation and demonstrated the importance of apoptotic cell disassembly in the efficient uptake of apoptotic materials by various phagocytes. Unexpectedly, we also noted that ROCK1 could play a role in regulating the onset of secondary necrosis. Together, these data shed light on both the mechanism and function of cell disassembly during apoptosis.

Normal mammary gland development after MMTV-Cre mediated conditional PAK4 gene depletion.

p21-activated kinases (PAKs) are serine/threonine kinases functioning as downstream effectors of the small GTPases Rac1 and Cdc42. Members of the PAK family are overexpressed in human breast cancer, but their role in mammary gland development is not fully explored. Here we examined the functional role of PAK4 in mammary gland development by creating a mouse model of MMTV-Cre driven conditional PAK4 gene depletion in the mammary gland. The PAK4 conditional knock-out mice were born healthy, with no observed developmental deficits. Mammary gland whole-mounts revealed no defects in ductal formation or elongation of the mammary tree through the fat pad. PAK4 gene depletion also did not alter proliferation and invasion of the mammary epithelium in young virgin mice. Moreover, adult mice gave birth to healthy pups with normal body weight upon weaning. This implies that MMTV-Cre induced gene depletion of PAK4 in mice does not impair normal mammary gland development and thereby provides an in vivo model that can be explored for examination of the potential function of PAK4 in breast cancer.

MiR-1261/circ-PTPRZ1/PAK1 pathway regulates glioma cell growth and invasion.

Glioma is the most common primary brain tumor in adults, with high malignancy and poor prognosis. According to the research in these years, the relationship between circular RNAs (circRNAs) and glioma development is abnormally close. Studies on circRNAs in glioma cells revealed that miR-1261 had almost completely matched binding site on the circ-PTPRZ1 sequence, and dual-luciferase reporter gene assay confirmed that circ-PTPRZ1 was a target gene of miR-1261. MiR-1261 inhibited circ-PTPRZ1 expression in glioma cells, while circ-PTPRZ1 did not affect miR-1261 expression. At the same time, circ-PTPRZ1 could promote p-PAK1 expression, while miR-1261 suppressed the activation of PAK1 by regulating the expression of circ-PTPRZ1. Biological behaviors of glioma cells were detected, circ-PTPRZ1 enhanced cell proliferation and invasion, and inhibited cell apoptosis; miR-1261 had the opposite effects, and could terminate the above effects of circ-PTPRZ1. When co-transfected with PAK1 siRNAs and circ-PTPRZ1 over-expression vector, the changes of above biological behaviors were not obvious. Therefore, in glioma cells, the expression of circ-PTPRZ1/PAK1 is regulated by miR-1261, which affects the proliferation, apoptosis, and invasion. This finding provides another powerful evidence for the role of circRNAs in glioma.

Pak2 kinase promotes cellular senescence and organismal aging.

Cellular senescence defines an irreversible cell growth arrest state linked to loss of tissue function and aging in mammals. This transition from proliferation to senescence is typically characterized by increased expression of the cell-cycle inhibitor p16INK4a and formation of senescence-associated heterochromatin foci (SAHF). SAHF formation depends on HIRA-mediated nucleosome assembly of histone H3.3, which is regulated by the serine/threonine protein kinase Pak2. However, it is unknown if Pak2 contributes to cellular senescence. Here, we show that depletion of Pak2 delayed oncogene-induced senescence in IMR90 human fibroblasts and oxidative stress-induced senescence of mouse embryonic fibroblasts (MEFs), whereas overexpression of Pak2 accelerated senescence of IMR90 cells. Importantly, depletion of Pak2 in BubR1 progeroid mice attenuated the onset of aging-associated phenotypes and extended life span. Pak2 is required for expression of genes involved in cellular senescence and regulated the deposition of newly synthesized H3.3 onto chromatin in senescent cells. Together, our results demonstrate that Pak2 is an important regulator of cellular senescence and organismal aging, in part through the regulation of gene expression and H3.3 nucleosome assembly.

Polarization and sprouting of endothelial cells by angiopoietin-1 require PAK2 and paxillin-dependent Cdc42 activation.

Binding of angiopoietin-1 (Ang-1) to its receptor Tie2 on endothelial cells (ECs) promotes vessel barrier integrity and angiogenesis. Here, we identify PAK2 and paxillin as critical targets of Ang-1 responsible for EC migration, polarization, and sprouting. We found that Ang-1 increases PAK2-dependent paxillin phosphorylation and remodeling of focal adhesions and that PAK2 and paxillin are required for EC polarization, migration, and angiogenic sprouting in response to Ang-1. Our findings show that Ang-1 triggers Cdc42 activation at the leading edges of migrating ECs, which is dependent on PAK2 and paxillin expression. We also established that the polarity protein Par3 interacts with Cdc42 in response to Ang-1 in a PAK2- and paxillin-dependent manner. Par3 is recruited at the leading edges of migrating cells and in focal adhesion, where it forms a signaling complex with PAK2 and paxillin in response to Ang-1. These results show that Ang-1 triggers EC polarization and angiogenic sprouting through PAK2-dependent paxillin activation and remodeling of focal adhesions, which are necessary for local activation of Cdc42 and the associated polarity complex. We have shown that PAK2 controls a signaling pathway important for angiogenic sprouting that links focal adhesions to polarity signaling in ECs.

Pak1 gene functioned differentially in different BCR-ABL subtypes in leukemiagenesis and treatment response through STAT5 pathway.

The BCR-ABL fusion gene (BCR-ABL) has different subtypes such as p210 and p190 with p190 appear to lead to a worse prognosis. To explore the mechanism of difference in pathogenesis and prognosis in different BCR-ABL subtype-related leukemia, expression profile microarray analysis was conducted between p190 and p210 patients and verified by RT-PCR. The p21-activated kinase (PAK1) gene was chosen and regulation of the PAK1-STAT5 biological axis and its influence on proliferation and apoptosis in leukemia cells were also analyzed. The results showed that PAK1 might be an important molecular mechanism of the pathogenic difference between different BCR-ABL subtypes. In P210 (+) chronic myelogenous leukemia (CML), down-regulated PAK1 gene expressions may lead to the suppression of cell proliferation and promotion of apoptosis through phosphorylation of STAT5, with a reverse effect in P190 (+) acute lymphoblastic leukemia(ALL), especially acute B lymphoblastic leukemia (B-ALL). Additionally, in P210 (+) CML, down-regulated PAK1 expression may enhance the effect of TKI, whereas the reverse is true in P190 (+) B-ALL, demonstrating that PAK1 might also be an important therapeutic target between different BCR-ABL subtypes.

The p21-activated kinase 1 (Pak1) signalling pathway in cardiac disease: from mechanistic study to therapeutic exploration.

p21-activated kinase 1 (Pak1) is a member of the highly conserved family of serine/threonine protein kinases regulated by Ras-related small G-proteins, Cdc42/Rac1. It has been previously demonstrated to be involved in cardiac protection. Based on recent studies, this review provides an overview of the role of Pak1 in cardiac diseases including disrupted Ca2+ homoeostasis-related cardiac arrhythmias, adrenergic stress- and pressure overload-induced hypertrophy, and ischaemia/reperfusion injury. These findings demonstrate the important role of Pak1 mediated through the phosphorylation and transcriptional modification of hypertrophy and/or arrhythmia-related genes. This review also discusses the anti-arrhythmic and anti-hypertrophic, protective function of Pak1 and the beneficial effects of fingolimod (an FDA-approved sphingolipid drug), a Pak1 activator, and its ability to prevent arrhythmias and cardiac hypertrophy. These findings also highlight the therapeutic potential of Pak1 signalling in the treatment and prevention of cardiac diseases. LINKED ARTICLES: This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

A key role for Rac and Pak signaling in neutrophil extracellular traps (NETs) formation defines a new potential therapeutic target.

NET formation in mice (NETosis) is supported by reactive oxygen species (ROS) production by NADPH oxidase and histone hypercitrullination by peptidylarginine deiminase 4 (PAD4). Rac1 and Rac2, expressed in polymorphonuclear neutrophils (PMNs), regulate the cytoskeleton, cell shape, adhesion, and migration and are also essential components of the NADPH oxidase complex. We aimed to explore the role of the Rac signaling pathway including the upstream guanosine exchange factor (GEF) activator, Vav, and a downstream effector, the p21-activated kinase, Pak, on NETosis in PMNs using a previously described flow-cytometry-based assay. Rac2-/- PMNs showed reduced levels of citrullinated histone H3 (H3Cit)-positive cells and defective NETosis. Rac1Δ/Δ ; Rac2-/- PMNs demonstrated a further reduction in PMA-induced H3Cit levels and a more profound impairment of NETosis than deletion of Rac2 alone, suggesting an overlapping role of these two highly related proteins. Genetic knockouts of Vav1, or Vav2, did not impair H3Cit response to phorbol myristate ester (PMA) or NETosis. Combined, Vav1 and Vav3 deletions decreased H3Cit response and caused a modest but significant impairment of NETosis. Pharmacologic inhibition of Pak by two inhibitors with distinct mechanisms of action, led to reduced H3Cit levels after PMA stimulation, as well as significant inhibition of NETosis. We validated the importance of Pak using Pak2Δ/Δ PMNs, which demonstrated significantly impaired histone H3 citrullination and NETosis. These data confirm and more comprehensively define the key role of the Rac signaling pathway in PMN NETosis. The Rac signaling cascade may represent a valuable target for inhibition of NETosis and related pathological processes.

Kalirin reduction rescues psychosis-associated behavioral deficits in APPswe/PSEN1dE9 transgenic mice.

Psychosis in Alzheimer's disease (AD+P) represents a distinct clinical and neurobiological AD phenotype and is associated with more rapid cognitive decline, higher rates of abnormal behaviors, and increased caregiver burden compared with AD without psychosis. On a molecular level, AD+P is associated with greater reductions in the protein kalirin, a guanine exchange factor which has also been linked to the psychotic disease, schizophrenia. In this study, we sought to determine the molecular and behavioral consequences of kalirin reduction in APPswe/PSEN1dE9 mice. We evaluated mice with and without kalirin reduction during tasks measuring psychosis-associated behaviors and spatial memory. We found that kalirin reduction in APPswe/PSEN1dE9 mice significantly attenuated psychosis-associated behavior at 12 months of age without changing spatial memory performance. The 12-month-old APPswe/PSEN1dE9 mice with reduced kalirin levels also had increased levels of the active, phosphorylated forms of p21 protein (Cdc42/Rac)-activated kinases (PAKs), which function in signaling pathways for maintenance of dendritic spine density, morphology, and function.

PAK4 Phosphorylates p53 at Serine 215 to Promote Liver Cancer Metastasis.

PAK4 kinase contributes to signaling pathways controlling cancer cell transformation, invasion, and survival, but its clinicopathological impact has begun to emerge only recently. Here we report that PAK4 overexpression in hepatocellular carcinoma (HCC) conveys aggressive metastatic properties. A novel nuclear splice isoform of PAK4 lacking exon 2 sequences was isolated as part of our studies. By stably overexpressing or silencing PAK4 in HCC cells, we showed that it was critical for their migration. Mechanistic investigations in this setting revealed that PAK4 directly phosphorylated p53 at S215, which not only attenuated transcriptional transactivation activity but also inhibited p53-mediated suppression of HCC cell invasion. Taken together, our results showed how PAK4 overexpression in HCC promotes metastatic invasion by regulating p53 phosphorylation. Cancer Res; 76(19); 5732-42. ©2016 AACR.

p21-Activated kinase (Pak) regulates airway smooth muscle contraction by regulating paxillin complexes that mediate actin polymerization.

KEY POINTS: In airway smooth muscle, tension development caused by a contractile stimulus requires phosphorylation of the 20 kDa myosin light chain (MLC), which activates crossbridge cycling and the polymerization of a pool of submembraneous actin. The p21-activated kinases (Paks) can regulate the contractility of smooth muscle and non-muscle cells, and there is evidence that this occurs through the regulation of MLC phosphorylation. We show that Pak has no effect on MLC phosphorylation during the contraction of airway smooth muscle, and that it regulates contraction by mediating actin polymerization. We find that Pak phosphorylates the adhesion junction protein, paxillin, on Ser273, which promotes the formation of a signalling complex that activates the small GTPase, cdc42, and the actin polymerization catalyst, neuronal Wiskott-Aldrich syndrome protein (N-WASP). These studies demonstrate a novel role for Pak in regulating the contractility of smooth muscle by regulating actin polymerization. ABSTRACT: The p21-activated kinases (Pak) can regulate contractility in smooth muscle and other cell and tissue types, but the mechanisms by which Paks regulate cell contractility are unclear. In airway smooth muscle, stimulus-induced contraction requires phosphorylation of the 20 kDa light chain of myosin, which activates crossbridge cycling, as well as the polymerization of a small pool of actin. The role of Pak in airway smooth muscle contraction was evaluated by inhibiting acetylcholine (ACh)-induced Pak activation through the expression of a kinase inactive mutant, Pak1 K299R, or by treating tissues with the Pak inhibitor, IPA3. Pak inhibition suppressed actin polymerization and contraction in response to ACh, but it did not affect myosin light chain phosphorylation. Pak activation induced paxillin phosphorylation on Ser273; the paxillin mutant, paxillin S273A, inhibited paxillin Ser273 phosphorylation and inhibited actin polymerization and contraction. Immunoprecipitation analysis of tissue extracts and proximity ligation assays in dissociated cells showed that Pak activation and paxillin Ser273 phosphorylation triggered the formation of an adhesion junction signalling complex with paxillin that included G-protein-coupled receptor kinase-interacting protein (GIT1) and the cdc42 guanine exchange factor, ßPIX (Pak interactive exchange factor). Assembly of the Pak-GIT1-ßPIX-paxillin complex was necessary for cdc42 and neuronal Wiskott-Aldrich syndrome protein (N-WASP) activation, actin polymerization and contraction in response to ACh. RhoA activation was also required for the recruitment of Pak to adhesion junctions, Pak activation, paxillin Ser273 phosphorylation and paxillin complex assembly. These studies demonstrate a novel role for Pak in the regulation of N-WASP activation, actin dynamics and cell contractility.

p21-activated kinase 2 regulates HSPC cytoskeleton, migration, and homing via CDC42 activation and interaction with ß-Pix.

Cytoskeletal remodeling of hematopoietic stem and progenitor cells (HSPCs) is essential for homing to the bone marrow (BM). The Ras-related C3 botulinum toxin substrate (Rac)/cell division control protein 42 homolog (CDC42) effector p21-activated kinase (Pak2) has been implicated in HSPC homing and engraftment. However, the molecular pathways mediating Pak2 functions in HSPCs are unknown. Here, we demonstrate that both Pak2 kinase activity and its interaction with the PAK-interacting exchange factor-ß (ß-Pix) are required to reconstitute defective ITALIC! Pak2 (ITALIC! Δ/Δ)HSPC homing to the BM. Pak2 serine/threonine kinase activity is required for stromal-derived factor-1 (SDF1α) chemokine-induced HSPC directional migration, whereas Pak2 interaction with ß-Pix is required to regulate the velocity of HSPC migration and precise F-actin assembly. Lack of SDF1α-induced filopodia and associated abnormal cell protrusions seen in ITALIC! Pak2 (ITALIC! Δ/Δ)HSPCs were rescued by wild-type (WT) Pak2 but not by a Pak2-kinase dead mutant (KD). Expression of a ß-Pix interaction-defective mutant of Pak2 rescued filopodia formation but led to abnormal F-actin bundles. Although CDC42 has previously been considered an upstream regulator of Pak2, we found a paradoxical decrease in baseline activation of CDC42 in ITALIC! Pak2 (ITALIC! Δ/Δ)HSPCs, which was rescued by expression of Pak2-WT but not by Pak2-KD; defective homing of ITALIC! Pak2-deleted HSPCs was rescued by constitutive active CDC42. These data demonstrate that both Pak2 kinase activity and its interaction with ß-Pix are essential for HSPC filopodia formation, cytoskeletal integrity, and homing via activation of CDC42. Taken together, we provide mechanistic insights into the role of Pak2 in HSPC migration and homing.

Neuronal filopodium formation induced by the membrane glycoprotein M6a (Gpm6a) is facilitated by coronin-1a, Rac1, and p21-activated kinase 1 (Pak1).

Stress-responsive neuronal membrane glycoprotein M6a (Gpm6a) functions in neurite extension, filopodium and spine formation and synaptogenesis. The mechanisms of Gpm6a action in these processes are incompletely understood. Previously, we identified the actin regulator coronin-1a (Coro1a) as a putative Gpm6a interacting partner. Here, we used co-immunoprecipitation assays with the anti-Coro1a antibody to show that Coro1a associates with Gpm6a in rat hippocampal neurons. By immunofluorescence microscopy, we demonstrated that in hippocampal neurons Coro1a localizes in F-actin-enriched regions and some of Coro1a spots co-localize with Gpm6a labeling. Notably, the over-expression of a dominant-negative form of Coro1a as well as its down-regulation by siRNA interfered with Gpm6a-induced filopodium formation. Coro1a is known to regulate the plasma membrane translocation and activation of small GTPase Rac1. We show that Coro1a co-immunoprecipitates with Rac1 together with Gpm6a. Pharmacological inhibition of Rac1 resulted in a significant decrease in filopodium formation by Gpm6a. The same was observed upon the co-expression of Gpm6a with the inactive GDP-bound form of Rac1. In this case, the elevated membrane recruitment of GDP-bound Rac1 was detected as well. Moreover, the kinase activity of the p21-activated kinase 1 (Pak1), a main downstream effector of Rac1 that acts downstream of Coro1a, was required for Gpm6a-induced filopodium formation. Taken together, our results provide evidence that a signaling pathway including Coro1a, Rac1, and Pak1 facilitates Gpm6a-induced filopodium formation. Formation of filopodia by membrane glycoprotein M6a (Gpm6a) requires actin regulator coronin-1a (Coro1a), known to regulate plasma membrane localization and activation of Rac1 and its downstream effector Pak1. Coro1a associates with Gpm6a. Blockage of Coro1a, Rac1, or Pak1 interferes with Gpm6a-induced filopodium formation. Moreover, Gpm6a facilitates Rac1 membrane recruitment. Altogether, a mechanistic insight into the process of Gpm6a-induced neuronal filopodium formation is provided.

p-21 activated kinase 4 (PAK4) maintains stem cell-like phenotypes in pancreatic cancer cells through activation of STAT3 signaling.

Pancreatic cancer (PC) remains a highly lethal malignancy due to its unusual chemoresistance and high aggressiveness. A subpopulation of pancreatic tumor cells, known as cancer stem cells (CSCs), is considered responsible not only for tumor-maintenance, but also for its widespread metastasis and therapeutic failure. Here we investigated the role of p-21 activated kinase 4 (PAK4) in driving PC stemness properties. Our data demonstrate that triple-positive (CD24(+)/CD44(+)/EpCAM(+)) subpopulation of pancreatic CSCs exhibits greater level of PAK4 as compared to triple-negative (CD24(-)/CD44(-)/EpCAM(-)) cells. Moreover, PAK4 silencing in PC cells leads to diminished fraction of CD24, CD44, and EpCAM positive cells. Furthermore, we show that PAK4-silenced PC cells exhibit decreased sphere-forming ability and increased chemosensitivity to gemcitabine toxicity. PAK4 expression is also associated with enhanced levels of stemness-associated transcription factors (Oct4/Nanog/Sox2 and KLF4). Furthermore, our data show decreased nuclear accumulation and transcriptional activity of STAT3 in PAK4-silenced PC cells and restitution of its activity leads to restoration of stem cell phenotypes. Together, our findings deliver first experimental evidence for the involvement of PAK4 in PC stemness and support its clinical utility as a novel therapeutic target in PC.