The up-regulation of PAK2 indicates unfavorable prognosis in patients with serous epithelial ovarian cancer and contributes to paclitaxel resistance in ovarian cancer cells.

BACKGROUND: The main challenge in treating ovarian cancer is chemotherapy resistance. Previous studies have shown that PAK2 is highly expressed in various cancers. This research investigates whether increased PAK2 expression contributes to chemo-resistance and poor prognosis in ovarian cancer. METHODS: Initially, bioinformatics analysis was used to assess the importance of PAK2 mRNA up-regulation in ovarian cancer. This was then validated using tissue microarray to confirm PAK2 protein expression and localization in clinical samples. Univariate and multivariate logistic regression analyses were carried out to identify potential risk factors for chemo-resistance in serous epithelial ovarian cancer (EOC), while multivariate Cox regression and Kaplan-Meier analysis were conducted to ascertain prognostic factors for overall survival (OS) and disease-free survival (DFS) in patients with serous EOC. In vitro experiments were conducted to verify if inhibiting PAK2 expression could increase A2780/Taxol cells' sensitivity to paclitaxel, as shown by evaluating cell proliferation, apoptosis, transwell, and clone formation. Additionally, the interaction between PAK2, lnc-SNHG1, and miR-216b-5p was verified using RIP and luciferase reporter assays. Rescue experiments were undertaken to examine the influence of the lnc-SNHG1/miR-216b-5p/PAK2 axis on the development of paclitaxel resistance in A2780/Taxol cells. RESULTS: The bioinformatics analysis indicated a notable increase in PAK2 expression in ovarian malignant tumors compared to adjacent tissues, particularly in patients with stage III-IV disease compared to those with stage I-II disease (P = 0.0056). Elevated levels of PAK2 were linked to reduced OS in ovarian cancer patients, although no significant association was observed with DFS. Immunohistochemistry findings further supported these results, showing positive PAK2 protein expression in chemo-resistant serous EOC tissues, predominantly localized in the cytoplasm, which correlated with poorer OS and DFS outcomes. In vitro experiments demonstrated that the downregulation of PAK2 in A2780/Taxol cells led to a reduction in colony formation, an increase in apoptosis, and a diminished capacity for cell invasion. Subsequent analysis confirmed that lnc-SNHG1 functions as a competitive endogenous RNA (ceRNA) by interacting with miR-216b-5p and regulating PAK2 expression. Rescue experiments demonstrated that lnc-SNHG1 induces resistance to paclitaxel in A2780/Taxol cells by modulating the miR-216b-5p/PAK2 axis. CONCLUSIONS: PAK2 shows promise as a predictor of chemotherapy resistance and poor outcomes in ovarian cancer, indicating its potential use as a treatment target to overcome this resistance.

Study of the genetic association between selected 3q29 region genes and schizophrenia and autism spectrum disorder in the Japanese population.

Psychiatric disorders are highly inheritable, and most psychiatric disorders exhibit genetic overlap. Recent studies associated the 3q29 recurrent deletion with schizophrenia (SCZ) and autism spectrum disorder (ASD). In this study, we investigated the association of genes in the 3q29 region with SCZ and ASD. TM4SF19 and PAK2 were chosen as candidate genes for this study based on evidence from previous research. We sequenced TM4SF19 and PAK2 in 437 SCZ cases, 187 ASD cases and 524 controls in the Japanese population. Through targeted sequencing, we identified 6 missense variants among the cases (ASD & SCZ), 3 missense variants among controls, and 1 variant common to both cases and controls; however, no loss-of-function variants were identified. Fisher's exact test showed a significant association of variants in TM4SF19 among cases (p=0.0160). These results suggest TM4SF19 variants affect the etiology of SCZ and ASD in the Japanese population. Further research examining 3q29 region genes and their association with SCZ and ASD is thus needed.

New kinase-deficient PAK2 variants associated with Knobloch syndrome type 2.

The p21-activated kinase (PAK) family of proteins regulates various processes requiring dynamic cytoskeleton organization such as cell adhesion, migration, proliferation, and apoptosis. Among the six members of the protein family, PAK2 is specifically involved in apoptosis, angiogenesis, or the development of endothelial cells. We report a novel de novo heterozygous missense PAK2 variant, p.(Thr406Met), found in a newborn with clinical manifestations of Knobloch syndrome. In vitro experiments indicated that this and another reported variant, p.(Asp425Asn), result in substantially impaired protein kinase activity. Similar findings were described previously for the PAK2 p.(Glu435Lys) variant found in two siblings with proposed Knobloch syndrome type 2 (KNO2). These new variants support the association of PAK2 kinase deficiency with a second, autosomal dominant form of Knobloch syndrome: KNO2.

PAK2 promotes proliferation, migration, and invasion of lung squamous cell carcinoma through LIMK1/cofilin signaling pathway.

Although the p21-activated kinase 2 (PAK2) is an essential serine/threonine protein kinase, its role in lung squamous cell carcinoma (LUSC) progression has yet to be fully understood. We analyzed PAK2 mRNA levels and DNA copy numbers as well as protein levels by quantitative real-time PCR and immunohistochemical staining, respectively, in human LUSC tissues and adjacent normal tissues. Then, we used colony formation assays, cell counting kit-8 assays, matrigel invasion assays, wound healing assays and xenograft models in nude mice to investigate the functions of PAK2 in LUSC progression. We demonstrated that the mRNA levels, DNA copy numbers, and protein levels of PAK2 were up-regulated in human LUSC tissues than in adjacent normal tissues. In addition, a higher PAK2 expression was correlated with a poorer prognosis in LUSC patients. In the in vitro study, we found that PAK2 promoted cell growth, migration, invasion, EMT process, and cell morphology regulation in LUSC cells. Furthermore, PAK2 enhanced tumor cell proliferation, migration, and invasion by regulating actin dynamics through the LIMK1/cofilin signaling. Our findings implicated that the PAK2/LIMK1/cofilin signaling pathway is likely a potential clinical marker and therapeutic target for LUSC.

Genetic mutation and immune infiltration in embryonal tumor with multilayered rosettes.

PURPOSE: Genetic mutations stand as pivotal factors leading to the occurrence of embryonal tumor with multilayered rosettes (ETMR). This study aims to identify improved treatment approaches by unraveling the genetic drivers and immune infiltration in ETMR. METHODS: Two siblings with ETMR, treated at the General Hospital of Ningxia Medical University, were enrolled. Diagnosis involved MRI, Hematoxylin and Eosin (HE), and immunohistochemical (IHC) staining. Differentially expressed genes (DEGs) in ETMR were identified using GSE122077 and GSE14296 datasets. GO and KEGG analyses were used to determine ETMR-related pathways. Whole exome sequencing (WES) was utilized to annotate genetic variations in ETMR. Core genes, identified by protein-protein interaction (PPI), formed a diagnostic model evaluated by Logistic Regression. Single-sample Gene Set Enrichment Analysis (ssGSEA) assessed immune infiltration in ETMR, examining correlations between immune cells and core genes. RESULTS: Two siblings were diagnosed with ETMR. In ETMR, 135 DEGs were identified, of which 25 genes were annotated with 28 mutation sites. Moreover, ETMR-related pathways included cell cycle, synaptic functions, and neurodegeneration. Three ETMR-related core genes (ALB, PSMD1, and PAK2) were screened by protein-protein interaction (PPI). The diagnostic model constructed using these genes demonstrated an AUC value of 0.901 (95% CI: 0.811-0.991) in the training set, indicating accurate predictions in ETMR. Enhanced ssGSEA scores for 16 immune cells in ETMR tissues suggested a strong immune response. CONCLUSION: This study identifies diagnostic models associated with three core variant genes (ALB, PSMD1, PAK2) and enhanced immune cell activity in ETMR. It reveals crucial genetic features and significant immune responses in ETMR.

ACSL4 promotes malignant progression of Hepatocellular carcinoma by targeting PAK2 transcription.

Long-chain fatty acyl-Coa ligase 4 (ACSL4) is an important enzyme that converts fatty acids to fatty acyl-Coa esters, there is increasing evidence for its role in carcinogenesis. However, the precise role of ACLS4 in hepatocellular carcinoma (HCC) is not clearly understood. In the present study, we provide evidence that ACSL4 expression was specifically elevated in HCC and is associated with poor clinical outcomes. ACSL4 significantly promotes the growth and metastasis of HCC both in vitro and in vivo. RNA sequencing and functional experiments showed that the effect of ACSL4 on HCC development was heavily dependent on PAK2. ACSL4 expression is well correlated with PAK2 in HCC, and ACSL4 even transcriptionally increased PAK2 gene expression mediated by Sp1. In addition, emodin, a naturally occurring anthraquinone derivative, inhibited HCC cell growth and tumor progression by targeting ACSL4. In summary, ACSL4 plays a novel oncogene in HCC development by regulating PAK2 transcription. Targeting ACSL4 could be useful in drug development and therapy for HCC.

Engineered hsa-miR-455-3p-Abundant Extracellular Vesicles Derived from 3D-Cultured Adipose Mesenchymal Stem Cells for Tissue-Engineering Hyaline Cartilage Regeneration.

Efforts are made to enhance the inherent potential of extracellular vesicles (EVs) by utilizing 3D culture platforms and engineered strategies for functional cargo-loading. Three distinct types of adipose mesenchymal stem cells-derived EVs (ADSCs-EVs) are successfully isolated utilizing 3D culture platforms consisting of porous gelatin methacryloyl (PG), PG combined with sericin methacryloyl (PG/SerMA), or PG combined with chondroitin sulfate methacryloyl (PG/ChSMA). These correspond to PG-EVs, PG/SerMA-EVs, and PG/ChSMA-EVs, respectively. Unique microRNA (miRNA) profiles are observed in each type of ADSCs-EVs. Notably, PG-EVs encapsulate higher levels of hsa-miR-455-3p and deliver more hsa-miR-455-3p to chondrocytes, which results in the activation of the hsa-miR-455-3p/PAK2/Smad2/3 axis and the subsequent hyaline cartilage regeneration. Furthermore, the functionality of PG-EVs is optimized through engineered strategies, including agomir/lentivirus transfection, electroporation, and Exo-Fect transfection. These strategies, referred to as Agomir-EVs, Lentivirus-EVs, Electroporation-EVs, and Exo-Fect-EVs, respectively, are ranked based on their efficacy in encapsulating hsa-miR-455-3p, delivering hsa-miR-455-3p to chondrocytes, and promoting cartilage formation via the hsa-miR-455-3p/PAK2/Smad2/3 axis. Notably, Exo-Fect-EVs exhibit the highest efficiency. Collectively, the 3D culture conditions and engineered strategies have an impact on the miRNA profiles and cartilage regeneration capabilities of ADSCs-EVs. The findings provide valuable insights into the mechanisms underlying the promotion of cartilage regeneration by ADSCs-EVs.

Icariin Attenuates Human Renal Tubular Epithelial Cell Senescence by Targeting PAK2 via miR-23b-3p.

BACKGROUND: Renal tubular epithelial cells (RTECs) senescence is crucial in kidney diseases. Icariin is shown to have protective effects against renal fibrosis, acute kidney injury, and proteinuria. We aimed to explore the role of icariin in protecting RTECs from senescence and the underlying mechanism involved. METHODS: An in vitro model of RTEC senescence was established by incubating HK-2 cells with urine exosomes from patients with diabetic kidney disease. Stimulated cells were treated with icariin at various doses to evaluate the compound's therapeutic effects. After RNA transfection, cell cycle arrest and senescence, flow cytometry, and SA-ß-Gal staining were analyzed. At the same time, quantitative real-time PCR examined microRNA expression. Biochemical assays. RESULTS: Urine exosomes induced senescence and cell cycle arrest in the G1 stage in HK-2 cells, which were inhibited by icariin. Urine exosome stimulation up-regulated miR-23b-3p expression, which in turn suppressed PAK2 expression. Significantly, the induced and inhibited miR- 23b-3p expressions weakened and augmented the resistance of cells against urine exosome stimulation, respectively, while PAK2 overexpression provided additional protection. Icariin suppressed miR-23b-3p expression, and miR-23b-3p induction blocked the effects of icariin and promoted RTEC senescence. CONCLUSION: miR-23b-3p and PAK2 form a signaling axis that regulates RTEC senescence upon urine exosome stimulation. Icariin can increase the resistance of RTECs against senescence via miR-23b-3p/PAK2. Our findings shed light on the mechanism of the clinical effects of icariin on renal diseases, which can be exploited to develop effective drugs targeting RTEC senescence in the future.

Gene fusions in poroma, porocarcinoma and related adnexal skin tumours: An update.

Poroma is a benign sweat gland tumour showing morphological features recapitulating the superficial portion of the eccrine sweat coil. A subset of poromas may transform into porocarcinoma, its malignant counterpart. Poroma and porocarcinoma are characterised by recurrent gene fusions involving YAP1, a transcriptional co-activator, which is controlled by the Hippo signalling pathway. The fusion genes frequently involve MAML2 and NUTM1, which are also rearranged in other cutaneous and extracutaneous neoplasms. We aimed to review the clinical, morphological and molecular features of this category of adnexal neoplasms with a special focus upon emerging differential diagnoses, and discuss how their systematic molecular characterisation may contribute to a standardisation of diagnosis, more accurate classification and, ultimately, refinement of their prognosis and therapeutic modalities.

A novel pipeline for prioritizing cancer type-specific therapeutic vulnerabilities using DepMap identifies PAK2 as a target in head and neck squamous cell carcinomas.

There is limited guidance on exploiting the genome-wide loss-of-function CRISPR screens in cancer Dependency Map (DepMap) to identify new targets for individual cancer types. This study integrated multiple tools to filter these data in order to seek new therapeutic targets specific to head and neck squamous cell carcinoma (HNSCC). The resulting pipeline prioritized 143 targetable dependencies that represented both well-studied targets and emerging target classes like mitochondrial carriers and RNA-binding proteins. In total, 14 targets had clinical inhibitors used for other cancers or nonmalignant diseases that hold near-term potential to repurpose for HNSCC therapy. Comparing inhibitor response data that were publicly available for 13 prioritized targets between the cell lines with high vs. low dependency on each target uncovered novel therapeutic potential for the PAK2 serine/threonine kinase. PAK2 gene dependency was found to be associated with wild-type p53, low PAK2 mRNA, and diploid status of the 3q amplicon containing PAK2. These findings establish a generalizable pipeline to prioritize clinically relevant targets for individual cancer types using DepMap. Its application to HNSCC highlights novel relevance for PAK2 inhibition and identifies biomarkers of PAK2 inhibitor response.

PAK2 is necessary for myelination in the peripheral nervous system.

Myelination enables electrical impulses to propagate on axons at the highest speed, encoding essential life functions. The Rho family GTPases, RAC1 and CDC42, have been shown to critically regulate Schwann cell myelination. P21-activated kinase 2 (PAK2) is an effector of RAC1/CDC42, but its specific role in myelination remains undetermined. We produced a Schwann cell-specific knockout mouse of Pak2 (scPak2-/-) to evaluate PAK2's role in myelination. Deletion of Pak2 specifically in mouse Schwann cells resulted in severe hypomyelination, slowed nerve conduction velocity, and behavior dysfunctions in the scPak2-/- peripheral nerve. Many Schwann cells in scPak2-/-sciatic nerves were arrested at the stage of axonal sorting. These abnormalities were rescued by reintroducing Pak2, but not the kinase-dead mutation of Pak2, via lentivirus delivery to scPak2-/- Schwann cells in vivo. Moreover, ablation of Pak2 in Schwann cells blocked the promyelinating effect driven by neuregulin-1, prion protein, and inactivated RAC1/CDC42. Conversely, the ablation of Pak2 in neurons exhibited no phenotype. Such PAK2 activity can also be either enhanced or inhibited by different myelin lipids. We have identified a novel promyelinating factor, PAK2, that acts as a critical convergence point for multiple promyelinating signaling pathways. The promyelination by PAK2 is Schwann cell-autonomous. Myelin lipids, identified as inhibitors or activators of PAK2, may be utilized to develop therapies for repairing abnormal myelin in peripheral neuropathies.

Circular RNA circ_0000119 promotes cervical cancer cell growth and migration via miR-433-3p/PAK2 axis.

The purpose of this study was to investigate the role of circ_0000119 on CC progression and its molecular mechanism. The expression levels of circ_0000119, miR-433-3p, and p21-activated kinase 2 (PAK2) in CC tissues and cell lines were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was assessed using 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, 5-Ethynyl-2'-deoxyuridine (EdU) assay and colony formation assay. Cell cycle and apoptosis were assessed by flow cytometry. Cell migration and invasive ability were examined by Transwell assays. Downstream binding targets of circ_0000119 were predicted by online bioinformatics tools and confirmed by dual luciferase reporter gene assay, RNA immunoprecipitation (RIP) assay, and RNA pull-down assay. The role of circ_0000119/miR-433-3p/PAK2 axis in regulating the CC process was explored by rescue experiments. A xenograft model was constructed to further determine the effect of circ_0000119 on CC tumor growth in vivo. Immunohistochemistry (IHC) assay was conducted for Ki67 expression. Circ_0000119 was aberrantly upregulated in CC tissues and cell lines. Knockdown of circ_0000119 inhibited CC cell proliferation, cell cycle progress, migration, invasion, and promoted apoptosis of CC cells. MiR-433-3p was a binding target of circ_0000119, and PAK2 was a downstream gene of miR-433-3p. MiR-433-3p inhibition reversed the inhibitory effect of silencing circ_0000119 on CC progression. In addition, PAK2 overexpression reversed the effect of miR-433-3p on CC progression. PAK2 expression was regulated by circ_0000119 and miR-433-3p. Moreover, circ_0000119 knockdown reduced tumor growth of CC in vivo. Circ_0000119 was upregulated in CC, and circ_0000119 knockdown suppressed CC malignant development through the miR-433-3p/PAK2 axis.

PAK2/beta-catenin/c-Myc/PKM2 signal transduction suppresses ovarian granulosa cell apoptosis in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) exhibits the highest morbidity among endocrine diseases in women ranging from age 18 to 44. However, its pathogenesis remains unclear. The imbalance between systemic and ovarian oxidative stress (OS) is a key characteristic of PCOS, and accumulating evidence indicates that the antioxidative protein nuclear factor erythroid-2-related factor 2 (Nrf2) is implicated in cell apoptosis and inflammation caused by OS. The activated kinase 2 (PAK2)/-catenin/c-Myc/pyruvate kinase M2 (PKM2) axis is a newly identified signaling pathway that may regulate Nrf2 expression and thereby influence OS. In this study, we sought to identify PAK2 expression and function in PCOS cells. PAK2 and downstream PKM2 expression in KGN cells and tissues were detected by microarray and qPCR. Cell viability was determined using CCK-8 and colony formation assays (CFAs). Apoptosis was examined by flow cytometry. qPCR and ELISA were used to examine cell inflammation. Oxidant and OS-related enzymes were examined by ELISA. We found that PAK2 and PKM2 expression levels were reduced in KGN cells and PCOS ovarian cortex tissues. PAK2 overexpression activated ß-catenin/c-Myc/PKM2 while PAK2 silencing deactivated it. PAK2 overexpression was reduced, whereas PAK2 silencing promoted, KGN cell proliferation and colony formation. Cell apoptosis and inflammation were also induced by PAK2 overexpression but were alleviated by its silencing. Furthermore, increased peroxidation product levels decreased antioxidative protein activities, and deactivated antioxidative Nrf2/HO-1 pathway were detected in PAK2-overexpressing KGN cells, whereas these effects were counteracted in PAK2 silenced cells. Our data suggest that PAK2 and its associated ß-catenin/c-Myc/PKM2 inhibited cell viability and induced apoptosis and inflammation by triggering OS by deactivating the Nrf2/HO-1 pathway, suggesting the potential of PAK2 as a therapeutic PCOS treatment target.

Role of activated p21-activated kinase 2 in methylmercury-induced embryotoxic effects on mouse blastocysts.

Methylmercury (MeHg), a biotransformation product derived from mercury or inorganic mercury compounds in waterways, is a potent toxin that exerts hazardous effects on human health via environmental contamination. Previous studies have reported MeHg-induced impairment of nerve development in embryogenesis and placental development. However, the potential deleterious effects and regulatory mechanisms of action of MeHg on pre- and post-implantation embryo development are yet to be established. Experiments from the current study clearly demonstrate that MeHg exerts toxic effects on early embryonic development processes, including the zygote to blastocyst stage. Induction of apoptosis and decrease in embryo cell number were clearly detected in MeHg-treated blastocysts. Additionally, intracellular reactive oxygen species (ROS) generation and activation of caspase-3 and p21-activated protein kinase 2 (PAK2) were observed in MeHg-treated blastocysts. Importantly, prevention of ROS generation by pre-treatment with Trolox, a potent antioxidant, significantly attenuated MeHg-triggered caspase-3 and PAK2 activation as well as apoptosis. Notably, the downregulation of PAK2 via transfection of specifically targeted siRNA (siPAK2) led to marked attenuation of PAK2 activity and apoptosis and the deleterious effects of MeHg on embryonic development in blastocysts. Our findings strongly suggest that ROS serve as an important upstream regulator to trigger the activation of caspase-3, which further cleaves and activates PAK2 in MeHg-treated blastocysts. Activated PAK2 promotes apoptotic processes that, in turn, cause sequent impairment of embryonic and fetal development.

Recurrent PAK2 rearrangements in poroma with folliculo-sebaceous differentiation.

AIMS: Poroma is a benign adnexal neoplasm with differentiation towards the upper portion of the sweat gland apparatus. In 2019, Sekine et al. demonstrated recurrent YAP1::MAML2 and YAP1::NUTM1 fusion in poroma and porocarcinoma. Follicular, sebaceous and/or apocrine differentiation has been reported in rare cases of poroma and whether these tumours constitute a variant of poroma or represent a distinctive tumour is a matter to debate. Herein we describe the clinical, immunophenotypic, and molecular features of 13 cases of poroma with folliculo-sebaceous differentiation. METHODS AND RESULTS: Most of the tumours were located on the head and neck region (n = 7), and on the thigh (n = 3). All presented were adults with a slight male predilection. The median tumour size was 10 mm (range: 4-25). Microscopically, lesions displayed features of poroma with nodules of monotonous basophilic cells associated with a second population of larger eosinophilic cells. In all cases, ducts and scattered sebocytes were identified. Infundibular cysts were present in 10 cases. In two cases high mitotic activity was noted, and in three cases cytologic atypia and areas of necrosis were identified. Whole transcriptome RNA sequencing demonstrated in-frame fusion transcripts involving RNF13::PAK2 (n = 4), EPHB3::PAK2 (n = 2), DLG1::PAK2 (n = 2), LRIG1::PAK2 (n = 1), ATP1B3::PAK2 (n = 1), TM9SF4::PAK2 (n = 1), and CTNNA1::PAK2 (n = 1). Moreover, fluorescence in situ hybridisation (FISH) analysis revealed PAK2 rearrangement in an additional case. No YAP1::MAML2 or YAP1::NUTM1 fusion was detected. CONCLUSION: Recurrent fusions involving the PAK2 gene in all analysed poroma with folliculo-sebaceous differentiation in this study confirms that this neoplasm represents a separate tumour entity distinct from YAP1::MAML2 or YAP1::NUTM1 rearranged poromas.

AP-2 Adaptor Complex-Dependent Enhancement of HIV-1 Replication by Nef in the Absence of the Nef/AP-2 Targets SERINC5 and CD4.

Human immunodeficiency virus type 1 (HIV-1) Nef hijacks the clathrin adaptor complex 2 (AP-2) to downregulate the viral receptor CD4 and the antiviral multipass transmembrane proteins SERINC3 and SERINC5, which inhibit the infectivity of progeny virions when incorporated. In Jurkat Tag T lymphoid cells lacking SERINC3 and SERINC5, Nef is no longer required for full progeny virus infectivity and for efficient viral replication. However, in MOLT-3 T lymphoid cells, HIV-1 replication remains highly dependent on Nef even in the absence of SERINC3 and SERINC5. Using a knockout (KO) approach, we now show that the Nef-mediated enhancement of HIV-1 replication in MOLT-3 cells does not depend on the Nef-interacting kinases LCK and PAK2. Furthermore, Nef substantially enhanced HIV-1 replication even in triple-KO MOLT-3 cells that simultaneously lacked the three Nef/AP-2 targets, SERINC3, SERINC5, and CD4, and were reconstituted with a Nef-resistant CD4 to permit HIV-1 entry. Nevertheless, the ability of Nef mutants to promote HIV-1 replication in the triple-KO cells correlated strictly with the ability to bind AP-2. In addition, knockdown and reconstitution experiments confirmed the involvement of AP-2. These observations raise the possibility that MOLT-3 cells express a novel antiviral factor that is downregulated by Nef in an AP-2-dependent manner. IMPORTANCE The HIV-1 Nef protein hijacks a component of the cellular endocytic machinery called AP-2 to downregulate the viral receptor CD4 and the antiviral cellular membrane proteins SERINC3 and SERINC5. In the absence of Nef, SERINC3 and SERINC5 are taken up into viral particles, which reduces their infectivity. Surprisingly, in a T cell line called MOLT-3, Nef remains crucial for HIV-1 spreading in the absence of SERINC3 and SERINC5. We now show that this effect of Nef also does not depend on the cellular signaling molecules and Nef interaction partners LCK and PAK2. Nef was required for efficient HIV-1 spreading even in triple-knockout cells that completely lacked Nef/AP-2-sensitive CD4, in addition to the Nef/AP-2 targets SERINC3 and SERINC5. Nevertheless, our results indicate that the enhancement of HIV-1 spreading by Nef in the triple-knockout cells remained AP-2 dependent, which suggests the presence of an unknown antiviral factor that is sensitive to Nef/AP-2-mediated downregulation.

Loss-of-Function of p21-Activated Kinase 2 Links BMP Signaling to Neural Tube Patterning Defects.

Closure of the neural tube represents a highly complex and coordinated process, the failure of which constitutes common birth defects. The serine/threonine kinase p21-activated kinase 2 (PAK2) is a critical regulator of cytoskeleton dynamics; however, its role in the neurulation and pathogenesis of neural tube defects (NTDs) remains unclear. Here, the results show that Pak2-/- mouse embryos fail to develop dorsolateral hinge points (DLHPs) and exhibit craniorachischisis, a severe phenotype of NTDs. Pak2 knockout activates BMP signaling that involves in vertebrate bone formation. Single-cell transcriptomes reveal abnormal differentiation trajectories and transcriptional events in Pak2-/- mouse embryos during neural tube development. Two nonsynonymous and one recurrent splice-site mutations in the PAK2 gene are identified in five human NTD fetuses, which exhibit attenuated PAK2 expression and upregulated BMP signaling in the brain. Mechanistically, PAK2 regulates Smad9 phosphorylation to inhibit BMP signaling and ultimately induce DLHP formation. Depletion of pak2a in zebrafish induces defects in the neural tube, which are partially rescued by the overexpression of wild-type, but not mutant PAK2. The findings demonstrate the conserved role of PAK2 in neurulation in multiple vertebrate species, highlighting the molecular pathogenesis of PAK2 mutations in NTDs.

miR-128-3p inhibits high-glucose-induced peritoneal mesothelial cells fibrosis via PAK2/SyK/TGF-ß1 axis.

AIM: To elucidate the mechanism of miR-128-3p in peritoneal fibrosis (PF). METHODS: Peritoneal mesothelial cells (PMCs) were dealt with high glucose (HG) for 3 days. The expressions of miR-128-3p, p21-activated kinase 2 (PAK2), spleen tyrosine kinase (SyK), and transforming growth factor-ß1 (TGF-ß1) were detected with quantitative real-time reverse transcription polymerase chain reaction. The levels of IL-1ß, TNF-α, IL-6, and monocyte chemotactic protein-1 in supernatant were measured by ELISA. Proteins of TGF-ß1, SyK, PAK2, α-SMA, collagen I, vimentin, ERK/AP-1, and IκBα/NF-κB pathway related proteins were measured by Western blot. The correlation between miR-128-3p and PAK2 was found by bioinformatics analysis and luciferase reporter gene analysis. RESULTS: miR-128-3p was decreased while PAK2, SyK, and TGF-ß1 were increased in HG-induced PMCs. Moreover, miR-128-3p inhibited HG-induced fibrosis and inflammation in PMCs by targeting PAK2. PAK2 activated SyK, which induced TGF-ß1 expression through ERK/AP-1 and IκBα/NF-κB pathways to promote HG-induced fibrosis of PMCs. CONCLUSION: miR-128-3p inhibited HG-induced PMCs fibrosis via PAK2/SyK/TGF-ß1 axis.

Role of caspase-3-cleaved/activated PAK2 in brusatol-triggered apoptosis of human lung cancer A549 cells.

Brusatol, a major quassinoid extract of Bruceae fructus, is an important bioactive component with antineoplastic capacity. Several beneficial pharmacological and biological properties of brusatol have been uncovered to date, including anti-inflammatory, anticolitis, antimalarial, and anticancer activities. To confer anticancer benefits, brusatol is reported to effectively inhibit the Nrf2-mediated antioxidant response and trigger apoptotic signaling. In this study, we investigated the regulatory mechanisms underlying apoptotic processes in brusatol-treated A549 cells in detail. Our experiments showed that brusatol induces cell death through intracellular ROS-triggered mitochondria-dependent apoptotic events and does not involve necrosis. Mechanistically, p21-activated protein kinase 2 (PAK2) was cleaved by caspase-3 to generate an activated p34 fragment involved in brusatol-induced apoptosis of A549 cells. Notably, PAK2 knockdown led to downregulation of caspase-3-mediated PAK2 activity, in turn, effectively attenuating brusatol-induced apoptosis, highlighting a crucial role of caspase-3-activated PAK2 in this process. Moreover, knockdown of PAK2 resulted in significant inhibition of c-Jun N-terminal kinase (JNK) activity in brusatol-treated A549 cells, clearly suggesting that JNK serves as a downstream substrate of caspase-3-cleaved/activated PAK2 in the apoptotic cascade. SP600125, a specific JNK inhibitor, significantly suppressed brusatol-induced JNK activity but only partially prevented apoptosis, implying that JNK serves as only one of a number of substrates for PAK2 in the brusatol-triggered apoptotic cascade. Based on the collective results, we propose a signaling cascade model for brusatol-induced apoptosis in human A549 cells involving ROS, caspases, PAK2, and JNK.

Quantitative Phosphoproteomics Analysis Uncovers PAK2- and CDK1-Mediated Malignant Signaling Pathways in Clear Cell Renal Cell Carcinoma.

Clear cell Renal Cell Carcinoma (ccRCC) is among the 10 most common cancers in both men and women and causes more than 140,000 deaths worldwide every year. In order to elucidate the underlying molecular mechanisms orchestrated by phosphorylation modifications, we performed a comprehensive quantitative phosphoproteomics characterization of ccRCC tumor and normal adjacent tissues. Here, we identified 16,253 phosphopeptides, of which more than 9000 were singly quantified. Our in-depth analysis revealed 600 phosphopeptides to be significantly differentially regulated between tumor and normal tissues. Moreover, our data revealed that significantly up-regulated phosphoproteins are associated with protein synthesis and cytoskeletal re-organization which suggests proliferative and migratory behavior of renal tumors. This is supported by a mesenchymal profile of ccRCC phosphorylation events. Our rigorous characterization of the renal phosphoproteome also suggests that both epidermal growth factor receptor and vascular endothelial growth factor receptor are important mediators of phospho signaling in RCC pathogenesis. Furthermore, we determined the kinases p21-activated kinase 2, cyclin-dependent kinase 1 and c-Jun N-terminal kinase 1 to be master kinases that are responsible for phosphorylation of many substrates associated with cell proliferation, inflammation and migration. Moreover, high expression of p21-activated kinase 2 is associated with worse survival outcome of ccRCC patients. These master kinases are targetable by inhibitory drugs such as fostamatinib, minocycline, tamoxifen and bosutinib which can serve as novel therapeutic agents for ccRCC treatment.