P21-activated Kinase Research Articles

PAK inhibitor FRAX486 decreases the metastatic potential of triple-negative breast cancer cells by blocking autophagy

BackgroundTriple-negative breast cancer (TNBC) is a unique breast cancer subtype with a high risk of metastasis and recurrence and a poor prognosis. Epithelial-mesenchymal transition (EMT) endows epithelial cells with the ability to move to distant sites, which is essential for the metastasis of TNBC to organs, including the lung. Autophagy, an intracellular degradation process that involves formation of double-layered lipid autophagosomes that transport cytosolic cargoes into lysosomes via autophagosome–lysosome fusion, is involved in various diseases, including cancer and neurodegenerative, metabolic, cardiovascular, and infectious diseases. The relationship between autophagy and cancer has become relatively clear. However, research on pharmacological drugs that block cancer EMT by targeting autophagy is still in the initial stages. Therefore, the re-evaluation of old drugs for their potential in blocking both autophagy and EMT was conducted.MethodsMore than 2000 small molecule chemicals were screened for dual autophagy/EMT inhibitors, and FRAX486 was identified as the best candidate inhibitor of autophagy/EMT. The functions of FRAX486 in TNBC metastasis were detected by CCK-8, migration and wound healing assays. The effects of FRAX486 on autophagy and its target PAK2 were determined by immunoblotting, immunofluorescence, immunoprecipitation analysis and transmission electron microscopy. The findings were validated in mouse models.ResultsHere, we report that FRAX486, a potent P21-activated kinase 2 (PAK2) inhibitor, facilitates TNBC suppression both in vitro and in vivo by blocking autophagy. Mechanistically, FRAX486 inhibits autophagy in TNBC cells by targeting PAK2, leading to the ubiquitination and proteasomal degradation of STX17, which mediates autophagosome–lysosome fusion. The inhibition of autophagy by FRAX486 causes upregulation of the epithelial marker protein E-cadherin and thus suppresses the migration and metastasis of TNBC cells.ConclusionsThe effects of FRAX486 on TNBC metastasis suppression were verified to be dependent on PAK2 and autophagy inhibition. Together, our results provide a molecular basis for the application of FRAX486 as a potential treatment for inhibiting the metastasis of TNBC.

Role of MAPKs in TGF-β1-induced maturation and mineralization in human osteoblast-like cells

Objectives: Our study aimed to clarify the role of mitogen-activated protein kinases (MAPKs) in transforming growth factor (TGF)-β1-stimulated mineralization in the human osteoblast-like MG63 cells. MethodsThe viability of MG63 cells under TGF-β1 stimulation was assessed by MTS assay. Western blotting determined TGF-β1-mediated activation of extracellular signal-related protein kinase (ERK), p38, and c-Jun amino-terminal kinase (JNK). Mineralization-related gene expression was examined by quantitative real-time PCR, and mineral deposition levels were evaluated by alizarin red S staining. ResultsTGF-β1 had no effect on MG63 cell proliferation. Activation of p38 was observed at 3 h post TGF-β1 stimulation. Moreover, JNK phosphorylation was upregulated by TGF-β1 from 1 to 6 h post stimulation, but had no activation on ERK phosphorylation throughout the experimental period. Treatment with JNK inhibitor diminished the alizarin red S-stained area in a dose-dependent manner. Mineral deposition was unaffected by MEK inhibitor, whereas p38 inhibitor increased the red-stained area. Gene expression levels of ALP and BSP were significantly decreased under treatment with JNK inhibitor and p38 inhibitor. The MEK inhibitor had no effect on the TGF-β1–mediated upregulation of ALP and BSP. Although all three inhibitors suppressed expression of COL I, none were found to stimulate expression of OCN. ConclusionsHuman osteoblast-like MG63 cells maturation and mineralization are induced through JNK activation of MAPK signaling in response to TGF-β1.

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 behaviour 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 signalling 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.

NAK-associated protein 1/NAP1 activates TBK1 to ensure accurate mitosis and cytokinesis.

Subcellular location and activation of Tank Binding Kinase 1 (TBK1) govern precise progression through mitosis. Either loss of activated TBK1 or its sequestration from the centrosomes causes errors in mitosis and growth defects. Yet, what regulates its recruitment and activation on the centrosomes is unknown. We identified that NAK-associated protein 1 (NAP1) is essential for mitosis, binding to and activating TBK1, which both localize to centrosomes. Loss of NAP1 causes several mitotic and cytokinetic defects due to inactivation of TBK1. Our quantitative phosphoproteomics identified numerous TBK1 substrates that are not only confined to the centrosomes but are also associated with microtubules. Substrate motifs analysis indicates that TBK1 acts upstream of other essential cell cycle kinases like Aurora and PAK kinases. We also identified NAP1 as a TBK1 substrate phosphorylating NAP1 at S318 to promote its degradation by the ubiquitin proteasomal system. These data uncover an important distinct function for the NAP1-TBK1 complex during cell division.

Dark kinase annotation, mining, and visualization using the Protein Kinase Ontology.

The Protein Kinase Ontology (ProKinO) is an integrated knowledge graph that conceptualizes the complex relationships among protein kinase sequence, structure, function, and disease in a human and machine-readable format. In this study, we have significantly expanded ProKinO by incorporating additional data on expression patterns and drug interactions. Furthermore, we have developed a completely new browser from the ground up to render the knowledge graph visible and interactive on the web. We have enriched ProKinO with new classes and relationships that capture information on kinase ligand binding sites, expression patterns, and functional features. These additions extend ProKinO's capabilities as a discovery tool, enabling it to uncover novel insights about understudied members of the protein kinase family. We next demonstrate the application of ProKinO. Specifically, through graph mining and aggregate SPARQL queries, we identify the p21-activated protein kinase 5 (PAK5) as one of the most frequently mutated dark kinases in human cancers with abnormal expression in multiple cancers, including a previously unappreciated role in acute myeloid leukemia. We have identified recurrent oncogenic mutations in the PAK5 activation loop predicted to alter substrate binding and phosphorylation. Additionally, we have identified common ligand/drug binding residues in PAK family kinases, underscoring ProKinO's potential application in drug discovery. The updated ontology browser and the addition of a web component, ProtVista, which enables interactive mining of kinase sequence annotations in 3D structures and Alphafold models, provide a valuable resource for the signaling community. The updated ProKinO database is accessible at https://prokino.uga.edu.

Inhibition of BMP signaling delays tauopathy and cognitive deficits in P301S mouse model

AbstractBackgroundAging is the highest risk factor for sporadic Alzheimer’s disease (AD). Expression of bone morphogenic protein 4 (BMP4) increases 10‐fold with age, while expression of the BMP antagonist, noggin, decreases. We have observed that activating BMP signaling in induced human neurons increased levels of phosphorylated tau. The increase of p‐tau has been correlated with the activated non‐canonical BMP pathway leading to p38 activity. We hypothesized that modulating BMP signaling in an AD mouse model of tau would rescue disease phenotype.MethodP301S (PS19) mice were injected with lentivirus overexpressing noggin (LV‐noggin) or lentivirus mcherry (LV‐mcherry) control in the dentate gyrus. Cognitive behavioral testing was run at 7 months of age and brain tissue was collected for protein and immunofluorescent imaging.ResultWe observe that levels of BMP4, as well as BMP signaling, are increased in the brains of PS19 mice compared to littermate wildtype mice. Analysis of noggin injected PS19 brains show downregulation of BMP signaling in the DG shown by decreased pSMAD1/5/8, as well as non‐canonical signaling through p38 kinase activity. Inhibition of BMP signaling through overexpression of noggin improves cognitive ability shown through Barnes maze testing. Downregulation of BMP signaling in P301S mice led to a decrease in tau phosphorylation seen both in immunoblotting, as well as immunocytochemical analysis in the DG.ConclusionPS19 model of tauopathy show increased levels of BMP signaling. We have shown that increased levels of BMP signaling lead to increased tau phosphorylation and reduced degradation of p‐tau. We show that by inhibiting BMP signaling in vivo we can delay cognitive behavioral deficits seen in these mice by decreasing tau phosphorylation. This suggests that the age‐related increase in BMP signaling may participate in the initiation and progression of AD pathology and help to explain why age is the largest risk factor for AD.

Identification of Genetic Diversity of P21-Activated Kinases (PAK1) Gene in Senduro Goats and Boerawa Goats using PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism)

This study aimed to detect genetic diversity associated with litter size traits of the P21-activated kinases (PAK1) gene in Senduro and Boerawa goats using the PCR-RFLP method. The restriction enzyme used is MSP1 (C*CGG). A pair of primers used in this study, namely Forward ((5'-GCTCAGTGTTGTATTAGCAG-3') and Reverse (5'- CAAGATATAAAGCACAGCCG-3') product length 713 bp (access code ENSCHIG00000003071). The samples used in this study consisted of 80 samples of Senduro goats and 20 samples of Boerawa goats from UPT PT-HMT Singosari, Malang Regency. The results showed that the PAK1 exon 9 gene in Senduro goats and Boerawa goats UPT PT-HMT Singosari was uniform (monomorphic), it was because all samples had GG genotype and G allele frequency of 100%. In conclusion, the PAK1 exon 9 / MSP1 gene is monomorphic and does not associate with litter size in Senduro goats and Boerawa goats, so it cannot be used as a genetic marker.

DUSP1 Specific Protac Selectively Targets the JAK2 V617F Expressing Cells

Constitutive JAK-STAT signaling resulting from mutations in JAK2 V617F and certain growth-factor receptors has provided a strong rationale for targeting JAK2 kinase activity in myeloproliferative neoplasms (MPNs). Although several JAK2 inhibitors have been approved for MPN treatment, their clinical efficacy is limited to alleviating the constitutional symptoms associated with inflammatory cytokine signaling. These inhibitors are not cytotoxic and only exert a cytostatic response, failing to eradicate the malignant clone, induce molecular remission, or prevent progression to acute myeloid leukemia (AML). The inability of JAK2 TKIs to fully suppress the inflammatory cytokine overproduction, a key feature of MPN development and transition to AML, contributes to clonal persistence and TKI refractoriness. Recently, we reported that the cytokine signaling, in the context of JAK2 V617F signaling induces the expression of DUSP1, which enhances P53 degradation that leads to TKI refractoriness. Enhanced DUSP1 expression blocks P53 phosphorylation at Serine 15 (S15 phosphorylation prevents its interaction with MDM2) by inhibiting the P38 kinase activity. Consequently, reduced phosphorylation under JAK2 V617F signaling enhances P53 turnover. Deletion of DUSP1 enhances P38 kinase activity, leading to greater P53 stabilization and expression, which selectively induce cell death in JAK2 V617F expressing MPN cells while sparing normal cells. However, chemical inhibition of DUSP1 using a small molecule inhibitor (BCI) was ineffective in achieving synthetic lethality to JAK2 V617F cells, mainly due to pERK1/2 rebound caused by the off-target inhibition of DUSP6. Reintroduction of DUSP6 and BCI treatment restored JAK2 V617F clonal selectivity. This study provided the first mechanistic basis of TKI refractoriness in JAK2 targeted MPNs and suggested that selectively targeting DUSP1 could offer a stable or curative response in JAK2 V617F -driven MPNs. To address this, we developed a DUSP1-protac, a novel and highly potent DUSP1 selective PROTAC. Treatment of BaF3 cells expressing JAK2 V617F with DUSP1-protac resulted in dose- and time-dependent degradation of DUSP1, accompanied by increased P38 activity and P53 stabilization. The maximal level of DUSP1 degradation (Dmax) achieved was 95% with a DC50 of 70 nM/L within 24 hours. Pre-treatment with the proteasome inhibitor MG132, but not the lysosomal inhibitor chloroquine, reversed DUSP1 degradation, confirming that degradation occurs via the 26S-proteasome. As expected, DUSP1-protac induced cytotoxicity specifically in JAK2 V617F expressing cells, with a cellular IC50 of 50 nM. No toxicity was observed in BaF3 cells or those expressing MPL or CALR variants at concentrations up to 1000 nM, confirming its selectivity. In conclusion, our data suggest that DUSP1-protac is an exceptionally potent first-in-class DUSP1 selective PROTAC. It exerts a cytotoxic response specifically in JAK2 V617F expressing cells by enhancing P53 expression. Cellular uptake experiments and efficacy studies in primary cells are currently underway and will be presented in subsequent reports. The development of DUSP1-protac represents a promising approach for targeting JAK2 V617F -driven MPNs, offering potential benefits in achieving a stable or curative therapeutic response.

Network pharmacology‑based investigation of potential targets of triptonodiol acting on non-small-cell lung cancer

BackgroundTriptonodiol is a very promising antitumor drug candidate extracted from the Chinese herbal remedy Tripterygium wilfordii Hook. F., and related studies are underway.MethodsTo explore the mechanism of triptonodiol for lung cancer treatment, we used network pharmacology, molecular docking, and ultimately protein validation. Gene ontology (GO) analysis and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analysis were performed through the David database. Molecular docking was performed using PyMoL2.3.0 and AutoDock Vina software. After screening, the major targets of triptonodiol were identified for the treatment of lung cancer. Target networks were established, Protein–protein interaction (PPI) network topology was analyzed, then KEGG pathway enrichment analysis was performed. Useful proteins were screened by survival analysis, and Western blot analysis was performed.ResultsTriptonodiol may regulate cell proliferation, drug resistance, metastasis, anti-apoptosis, etc., by acting on glycogen synthase kinase 3 beta (GSK3B), protein kinase C (PKC), p21-activated kinase (PAK), and other processes. KEGG pathway enrichment analysis showed that these targets were associated with tumor, erythroblastic oncogene B (ErbB) signaling, protein phosphorylation, kinase activity, etc. Molecular docking showed that the target protein GSK has good binding activity to the main active component of triptonodiol. The protein abundance of GSK3B was significantly downregulated in non-small-cell lung cancer cells H1299 and A549 treated with triptonodiol for 24 h.ConclusionThe cellular-level studies combined with network pharmacology and molecular docking approaches provide new ideas for the development and therapeutic application of triptonodiol, and identify it as a potential GSK inhibitor.

The Splicing-Associated Network PAK1-Clk-SRRM1 Is a Critical Vulnerability to Overcome Chemoresistance in Acute Myeloid Leukemia

In Acute Myeloid Leukemia (AML), distinguishing between chemoresistance driver mechanisms and cell plasticity in response to chemotherapy is challenging due to their interconnectedness. To address this issue, we generated an MLL-AF9-driven syngeneic mouse model of AML, which we evolved to develop resistance to a front-line chemotherapy regimen by repetitive exposure to a maximally-tolerated combination of cytarabine and doxorubicin. An RNA sequencing (RNAseq)-based profiling of the chemoresistant (Chemo R) AML subpopulation and its naive (Chemo N) counterpart revealed a significant upregulation in the expression of 326 genes in Chemo R cells which were markedly enriched in gene sets related to splicing/mRNA processing. Notably, single-sample GSEA analysis of relapsed AML samples from the BEAT-AML cohort supported these findings, as they were enriched for the upregulation of genes in our chemoresistance gene signature. A combined in vivo functional screening endeavor using a pool of 2098 shRNAs targeting these upregulated genes and a motif discovery analysis performed on the set of dysregulated transcripts in Chemo R cells identified three candidate hits, Srrm1, Sf3b3, and Prpf8, which scored as chemotherapy-specific dependencies and were found to interact with central regulators of alternative splicing ( Figure 1A). Silencing these three candidate genes in our chemoresistant mouse model and the human AML cell lines, MOLM-14, MV4-11, and U937, evolved to become resistant to the combination of cytarabine and daunorubicin, revealed that the splicing regulator, SRRM1, is a critical vulnerability of chemoresistant leukemic cells. An orthogonal phospho-proteomic approach identified that PAK1 and CLK kinases are regulators of SRRM1 in chemoresistant AML cells, through the phosphorylation of multiple serine and threonine phosphosites in its arginine-rich domain. Human and murine chemoresistant cells were found to be markedly more sensitive to inhibitors of PAKs (FRAX597) and CLKs (TG003 and ML167) compared to their naive counterparts, an effect which was even further enhanced by treatment with cytarabine and daunorubicin. Notably, a whole-exome sequencing pipeline at our institution revealed a clonal variant of uncertain significance (c.1429G>T p.(Ala477Ser)) in PAK1 in an AML patient at relapse. Absence of this variant at diagnosis suggested its association with chemotherapy resistance. Biochemical and cellular assays confirmed that the A477S variant increased PAK1 activation as well as chemotherapy resistance in AML cell lines. This effect could be counteracted by suppressing SRRM1, indicating that PAK1 activation in relapsed AML confers increased vulnerability to SRRM1 suppression. Simultaneous suppression of Clk1 and Pak1 or Clk4 and Pak1 using validated shRNAs and combinations of FRAX597 and TG003, or FRAX597 and ML167 synergized to reduce the growth and the colony-forming capacity of chemoresistant AML cells and sensitized them to chemotherapy. These observations were supported by the RNAseq-based estimate of the inclusion ratios of alternative exons and introns which showed that the combination of PAK and CLK inhibitors phenocopied partially the effects of SRRM1 silencing on splicing perturbations. In preclinical studies, the combined treatment improved overall mouse survival and reduced disease burden in our chemoresistant AML mouse models compared to single-drug treatments ( Figure 1B). Moreover, AML patient cells that were primarily refractory or from a post chemotherapy relapse (n=21) exhibited a higher ex vivo sensitivity to the FRAX597 + TG003 combination in comparison with chemosensitive patients at diagnosis (n=28). Moreover, in NSG-S mice transplanted with matched samples from the same AML patient at diagnosis and relapse post-chemotherapy, combined PAK1 and CLK inhibition more effectively reduced disease progression in animals transplanted with the relapse sample than in those engrafted with the sample taken at diagnosis. In conclusion, our study advances the rationale that combined inhibition of PAK1 and CLK kinases effectively curtails malignant cell splicing adaptation to chemotherapy by affecting SRRM1 function. Combined PAK1 and CLK inhibitors either concomitant with or sequential to chemotherapy may offer greater clinical benefit and significantly improve AML patients' long-term outcomes.

PAK in Pancreatic Cancer-Associated Vasculature: Implications for Therapeutic Response.

Angiogenesis has been associated with numbers of solid tumours. Anti-angiogenesis drugs starve tumours of nutrients and oxygen but also make it difficult for a chemo reagent to distribute into a tumour, leading to aggressive tumour growth. Anti-angiogenesis drugs do not appear to improve the overall survival rate of pancreatic cancer. Vessel normalisation is merging as one of the new approaches for halting tumour progression by facilitating the tumour infiltration of immune cells and the delivery of chemo reagents. Targeting p21-activated kinases (PAKs) in cancer has been shown to inhibit cancer cell growth and improve the efficacy of chemotherapy. Inhibition of PAK enhances anti-tumour immunity and stimulates the efficacy of immune checkpoint blockades. Inhibition of PAK also improves Car-T immunotherapy by reprogramming the vascular microenvironment. This review summarizes current research on PAK's role in tumour vasculature and therapeutical response, with a focus on pancreatic cancer.

Endochondral Repair of Jawbone Defects Using Periosteal Cell Spheroids

Recapitulation of the natural healing process is receiving increasing recognition as a strategy to induce robust tissue regeneration. Endochondral ossification has been recognized as an essential reparative approach in natural jawbone defect healing. However, such an approach has been overlooked in the recent development of cell-based therapeutics for jawbone repair. Therefore, this study aimed to explore a bioinspired stem cell–based strategy for jawbone repair by mimicking the mesenchymal condensation of progenitor cells during the early endochondral ossification process. For this purpose, passage 3 of jawbone periosteum-derived cells (jb-PDCs) was cultured in our previously reported nonadherent microwells (200 µm in diameter, 148 µm in depth, and 100 µm space in between) and self-assembled into spheroids with a diameter of 96.4 ± 5.8 µm after 48 h. Compared to monolayer culture, the jb-PDC spheroids showed a significant reduction of stemness marker expression evidenced by flow cytometry. Furthermore, a significant upregulation of chondrogenic transcription factor SOX9 in both gene and protein levels was observed in the jb-PDC spheroids after 48 h of chondrogenic induction. RNA sequencing and Western blotting analysis further suggested that the enhanced SOX9-mediated chondrogenic differentiation in jb-PDC spheroids was attributed to the activation of the p38 MAPK pathway. Impressively, inhibition of p38 kinase activity significantly attenuated chondrogenic differentiation jb-PDC spheroids, evidenced by a significant decline of SOX9 in both gene and protein levels. Strikingly, the jb-PDC spheroids implanted in 6- to 8-wk-old male C57BL/6 mice with critical-size jawbone defects (1.8 mm in diameter) showed an evident contribution to cartilaginous callus formation after 1 wk, evidenced by histological analysis. Furthermore, micro–computed tomography analysis showed that the jb-PDC spheroids significantly accelerated bone healing after 2 wk in the absence of exogenous growth factors. In sum, the presented findings represent the successful development of cell-based therapeutics to reengineer the endochondral bone repair process and illustrate the potential application to improve bone repair and regeneration in the craniofacial skeleton.

Abstract 14169: Deletion of P21 Activated Kinase-1 Modulates Energy Homeostasis and Affects Cardiac Function

Introduction: We investigated the consequences of P21-activated kinase-1 (PAK1) deletion on cardiac function, body weight, body composition, and energy balance using a mouse model of global PAK1 knock out. PAK1 is a protein kinase that plays a role in multiple aspects of cardiac health. We hypothesize that PAK1 plays a role in cardiometabolic health including cardiac function and adipose tissue homeostasis. Methods: We used PAK1 -/- in the FVB/N background. We included 10-12-week-old male and female mice. Age and sex-matched FVB/N mice were used as wild-type (WT) controls. Most results included both male and female mice together, as no differences were found between sexes. Ca 2+ kinetics was measured in neonatal cardiomyocytes. Results: Deletion of PAK1 was found to have a significant impact on cardiac function. Specifically, slower Ca 2+ release and reuptake kinetics accompanied with decreased phospholamban phosphorylation was observed in PAK1 -/- mice. These changes affected cardiac systolic (EF, s’) and lusitropy parameters (E/e’, IVRT). Moreover, our findings revealed body weight differences between PAK1 -/- and WT at 12 weeks, accompanied with increased % fat mass and decreased % lean mass in PAK1 -/- , indicating a shift towards adiposity. Further analysis revealed that PAK1 -/- mice had increased weights of white adipose tissue (WAT), and brown adipose tissue (BAT). Moreover, we found increased food intake, increased number of feeding bouts and decreased energy expenditure accompanied with decreased thermogenic markers in subcutaneous WAT and BAT in PAK1 -/- , further contributing to the obesogenic phenotype (see table for results). Conclusions: Deletion of PAK1 affected systolic and diastolic functions, and resulted in increased energy intake, decreased energy expenditure, and increased adiposity. Therefore, targeting PAK1 signaling pathways may represent a promising therapeutic strategy for improving cardiometabolic diseases.

A Protein-Protein Interaction Analysis Suggests a Wide Range of New Functions for the p21-Activated Kinase (PAK) Ste20.

The p21-activated kinases (PAKs) are important signaling proteins. They contribute to a surprisingly wide range of cellular processes and play critical roles in a number of human diseases including cancer, neurological disorders and cardiac diseases. To get a better understanding of PAK functions, mechanisms and integration of various cellular activities, we screened for proteins that bind to the budding yeast PAK Ste20 as an example, using the split-ubiquitin technique. We identified 56 proteins, most of them not described previously as Ste20 interactors. The proteins fall into a small number of functional categories such as vesicle transport and translation. We analyzed the roles of Ste20 in glucose metabolism and gene expression further. Ste20 has a well-established role in the adaptation to changing environmental conditions through the stimulation of mitogen-activated protein kinase (MAPK) pathways which eventually leads to transcription factor activation. This includes filamentous growth, an adaptation to nutrient depletion. Here we show that Ste20 also induces filamentous growth through interaction with nuclear proteins such as Sac3, Ctk1 and Hmt1, key regulators of gene expression. Combining our observations and the data published by others, we suggest that Ste20 has several new and unexpected functions.

Dietary potassium stimulates Ppp1Ca-Ppp1r1a dephosphorylation of kidney NaCl cotransporter and reduces blood pressure.

Consumption of low dietary potassium, common with ultraprocessed foods, activates the thiazide-sensitive sodium chloride cotransporter (NCC) via the with no (K) lysine kinase/STE20/SPS1-related proline-alanine-rich protein kinase (WNK/SPAK) pathway to induce salt retention and elevate blood pressure (BP). However, it remains unclear how high-potassium "DASH-like" diets (dietary approaches to stop hypertension) inactivate the cotransporter and whether this decreases BP. A transcriptomics screen identified Ppp1Ca, encoding PP1A, as a potassium-upregulated gene, and its negative regulator Ppp1r1a, as a potassium-suppressed gene in the kidney. PP1A directly binds to and dephosphorylates NCC when extracellular potassium is elevated. Using mice genetically engineered to constitutively activate the NCC-regulatory kinase SPAK and thereby eliminate the effects of the WNK/SPAK kinase cascade, we confirmed that PP1A dephosphorylated NCC directly in a potassium-regulated manner. Prior adaptation to a high-potassium diet was required to maximally dephosphorylate NCC and lower BP in constitutively active SPAK mice, and this was associated with potassium-dependent suppression of Ppp1r1a and dephosphorylation of its cognate protein, inhibitory subunit 1 (I1). In conclusion, potassium-dependent activation of PP1A and inhibition of I1 drove NCC dephosphorylation, providing a mechanism to explain how high dietary K+ lowers BP. Shifting signaling of PP1A in favor of activation of WNK/SPAK may provide an improved therapeutic approach for treating salt-sensitive hypertension.

TrCla4 promotes actin polymerization at the hyphal tip and mycelial growth in Trichophyton rubrum.

Dermatophytes invade and colonize host superficial tissues via hyphal growth. Although cytoskeletal reorganization and its regulation are essential for hyphal growth, the molecular mechanisms in dermatophytes and their applicability as antifungal drug targets remain poorly understood. The p21-activated kinase (PAK) is a downstream effector of the small GTPases Rac and CDC42, also known as p21, and is involved in various molecular and cellular functions, including actin polymerization and cell morphogenesis. In this study, we investigated the contribution of the PAK protein TrCla4 to morphogenesis and mycelial growth in Trichophyton rubrum, the most frequently isolated fungus in dermatophytosis (athlete's foot). The actin polymerization inhibitor, cytochalasin A inhibited actin accumulation at the hyphal tip and mycelial growth of T. rubrum, suggesting the involvement of the actin cytoskeleton in mycelial growth. In the Trcla4 knockout strain (ΔTrcla4), we observed defects in mycelial growth, hyphal branching, and the accumulation of polymerized actin at the hyphal tip. Chemical inhibitors of TrRac-dependent TrCla4 kinase activity, FRAX486 and IPA-3, also inhibited spore germination and mycelial growth. Interestingly, ΔTrcla4 showed no additional inhibition of mycelial growth when treated with these inhibitors, indicating that their inhibitory effects are primarily mediated through TrCla4. In an invertebrate dermatophyte infection model, animals infected with ΔTrcla4 had higher survival rates than those infected with the wild-type, and IPA-3 and FRAX486 treatments both significantly improved animal survival rates. These results suggest that the dermatophyte PAK promotes mycelial growth by facilitating actin polymerization at the hyphal tip, making it a potential therapeutic target for dermatophytosis. IMPORTANCE Superficial fungal infections, such as athlete's foot, affect more than 10% of the world's population and have a significant impact on quality of life. Despite the fact that treatment-resistant fungi are a concern, there are just a few antifungal drug targets accessible, as opposed to the wide range of therapeutic targets found in bacterial infections. As a result, additional alternatives are sought. In this study, we generated a PAK TrCla4 deletion strain (∆Trcla4) of Trichophyton rubrum. The ∆Trcla4 strain exhibited deficiencies in mycelial growth, hyphal morphology, and polarized actin localization at the hyphal tip. IPA-3 and FRAX486, small chemical inhibitors of mammalian PAK, were discovered to limit fungal mycelial proliferation. According to our findings, fungal PAKs are interesting therapeutic targets for the development of new antifungal medicines.

Design, synthesis and anticancer activity evaluation of 4-(3-1H-indazolyl)amino quinazoline derivatives as PAK4 inhibitors

A novel series of 4-(3-1H-indazolyl)amino quinazoline derivatives were developed as PAK4 inhibitors based on a scaffold hopping strategy. Compounds 27e, 27g, 27i and 27j were found to exhibit potent inhibitory activity against PAK4 (IC50 = 10, 13, 11 and 9 nM, respectively). Subsequent cellular assay demonstrated that compound 27e possessed the strongest antiproliferative activity against A549 cells with an IC50 value of 0.61 μM, a little bit better than PF-3758309. Further anticancer mechanistic investigation revealed that compound 27e significantly induced apoptosis of A549 cells in a concentration-dependent manner and blocked the cell cycle at phase G0/G1. A docking model between compound 27e and PAK4 was proposed to elucidate its possible binding modes. As a promising PAK4 inhibitor, compound 27e may serve as a candidate for the development of novel PAK4-targeted anticancer drug.

Scoparone induces autophagic cell death via the PAK1/AKT axis in colorectal cancer

Colorectal cancer (CRC) is one of most common malignancies worldwide, yet curative therapy remains a clinical challenge. Here, we demonstrate that scoparone (Scop), a traditional Chinese medicine monomer, inhibits the growth of CRC cells both in vitro and in vivo. Further studies found that Scop treatment induces complete autophagic flux in CRC cells, while inhibition of autophagy markedly represses the antiproliferative activities of Scop, suggesting an antitumour property of Scop-induced autophagy in CRC. Mechanistically, Scop induced autophagy initiation by reducing P21-activated kinase 1 (PAK1) expression and subsequently repressing the AKT/mTOR signaling pathway. Collectively, our study suggests that Scop is a potential anti-CRC therapeutic option and provides an underlying molecular mechanism for its antitumour effect in CRC.

Activation of oncogenic signaling kinase PAK1 by ionising radiation confers an aggressive phenotype in head and neck squamous cell carcinoma

Head and neck squamous cancers are very aggressive tumors often diagnosed in late stages with poor prognosis. HNSCCs are usually treated by a course of radiation (IR) therapy and followed by surgery. These treatment regimens fail to bring a complete response. Molecular signatures in tumors are attributed to this response and an improved understanding of the signaling events could offer new avenues for therapy. Here, we show that P21 activated kinase-1 (PAK1) - an oncogenic signaling serine/threonine kinase, is activated upon exposure to IR and this leads to an accelerated tumorigenic character in HNSCC cells. Our results show that PAK1 is highly expressed in HNSCC cell lines, as compared to normal buccal mucosa cells and when HNSCC cells were exposed to IR, they show activated PAK1 and an aggressive phenotype as determined by in vitro functional assays. PAK1 levels were elevated in HNSCC as compared to adjacent normal oral tissues and our results also show convincing evidence of activated PAK1 in patient tumor samples of post- IR treatment as compared to pre-IR treatment and is associated with poor survival. Pak1 Knockout (KO) clones in HNSCC cells showed that they were more sensitive to IR as compared to wild type (wt) cells. This altered sensitivity to IR was attributed to enhanced DNA damage response modulated by PAK1 in cells. Overall, our results suggest that PAK1 expression in HNSCC could be a critical determinant in IR therapy response and silencing PAK1 is likely to be a treatment modality to improve clinical outcomes.

Affimer-mediated locking of p21-activated kinase 5 in an intermediate activation state results in kinase inhibition

Kinases are important therapeutic targets, and their inhibitors are classified according to their mechanism of action, which range from blocking ATP binding to covalent inhibition. Here, a mechanism of inhibition is highlighted by capturing p21-activated kinase 5 (PAK5) in an intermediate state of activation using an Affimer reagent that binds in the P+1 pocket. PAK5 was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells. Silencing of PAK5 resulted in reduced cell number, G1/S arrest, and enlargement of cells, suggesting it to be important in urothelial cancer cell line survival and proliferation. Affimer reagents were isolated to identify mechanisms of inhibition. The Affimer PAK5-Af17 recapitulated the phenotype seen with siRNA. Co-crystallization revealed that PAK5-Af17 bound in the P+1 pocket of PAK5, locking the kinase into a partial activation state. This mechanism of inhibition indicates that another class of kinase inhibitors is possible.