P21-activated Kinase Research Articles

Spatio-temporal localization of P21-activated kinase in endometrial cancer.

Endometrial cancer is the sixth most common gynecologic cancer, and has been reported as a malignancy arising due to the idiopathic effects of certain anticancer agents. Tamoxifen is the drug of choice in ER-positive breast cancer, and several studies have shown better disease-free survival in these patients. However, the long-term usage of tamoxifen has been associated with resistance and risk for endometrial malignancy. A direct mechanistic basis for tamoxifen-induced endometrial tumorigenesis is still unclear. Hyperactivation of PAK1 in endometrial cancer correlates with poor overall survival. The present study demonstrates that tamoxifen treatment induces nuclear localization of PAK1 in endometrial carcinoma cells. This nuclear transit is mediated through JAK2 phosphorylation of PAK1 and binding of β-PIX. In addition, a computational approach involving molecular modeling and simulation of phosphorylated and unphosphorylated forms of PAK1 was used to elucidate the dynamics of nuclear localization. Thus, PAK1 phosphorylation by JAK2 is a prerequisite for its nuclear localization and its tumorigenic effects on endometrial cancer cells.

Leptin on the apical surface inhibits casein production and STAT5 phosphorylation in mammary epithelial cells

Leptin is a peptide hormone present in both the blood and milk. A close relationship between leptin and milk production in lactating mammary glands has been previously reported. However, how leptin influences milk production in lactating mammary glands remains unclear. Also, whether leptin in milk or blood influences mammary epithelial cells (MECs) during lactation needs further investigation. This study investigated the effects of leptin on mouse MECs using a culture model in which MECs produced milk components and formed less permeable tight junctions. Our results showed that β-casein production in MEC was inhibited by leptin in a concentration-dependent manner. Leptin also inactivated the signal transducer and activator of transcription 5 (STAT5), a transcription factor that facilitates milk production in MECs. Leptin treatment induced the activation of p38 and c-Jun N-terminal kinase (JNK) in MEC before STAT5 inactivation, and anisomycin, an activator of p38 and JNK, induced the inactivation of STAT5. Furthermore, leptin exposure on the apical surface of MECs inhibited β-casein production and inactivated STAT5. However, leptin exposure on the basolateral surface hardly caused these effects. These findings suggested that milk leptin, but not plasma leptin, inhibited milk production in MECs.

PAK4 is Required for Meiotic Resumption, Spindle Assembly, and Cortical Migration in Mouse Oocytes During Meiotic Maturation.

Oocyte meiotic errors can cause infertility, miscarriage, and birth defects. Here the role and the underlying mechanism of p21 activated kinase 4 (PAK4) in mouse oocyte meiosis is evaluated. It is found that PAK4 expression and its phosphorylation are detected in high level at germinal vesicle (GV) stage, and gradually decreased after meiotic resumption in oocytes. PAK4 has direct physical interaction with both mitogen-activated protein kinases 1/2 (MEK1/2) and Paxillin, they are colocalized on the spindle structure during metaphases I and II. Phospho-PAK4 is distributed beneath the cytoplasmic membrane and on the chromosomes, and colocalized with the microtubule organizing center (MTOC) proteins, Pericentrin and γ-tubulin, as well as phosphor-MEK1/2 and phosphor-Paxillin on spindle poles. PAK4 inhibition by chemical inhibitor LCH-7749944, specific Pak4 morpholino oligo or the dominant negative mutant Pak4K350, 351M influence the meiotic resumption, spindle assembly and its cortical migration, and associated with the downregulation in the dephosphorylation of cyclin dependent kinase 1 (CDK1) and the levels of Cyclin B1, MEK1/2, Paxillin, g-tubulin, acetylated a-tubulin, Arp3, and Cofilin phosphorylation in oocytes. In sum, PAK4 functions to sustain the rational levels of Cyclin B1, MEK1/2, Paxillin, y-tubulin, acetylated a-tubulin, Arp3, and phosphor-Cofilin in mouse oocytes, thereby promotes the meiotic resumption, spindle assembly, and migration during meiotic maturation.

Network analysis of α-synuclein pathology progression reveals p21-activated kinases as regulators of vulnerability.

Longitudinal α-synuclein pathology assessment in 1046 brain regions over 9 monthsLinear diffusion modeling derivation of network vulnerability to α-synuclein pathologyPANGEA: assessment of over 19,000 genes in 302 brain regionsGroup II PAK inhibitor prevents α-synuclein pathology and neuron death.

PAK6 rescues pathogenic LRRK2-mediated ciliogenesis and centrosomal cohesion defects in a mutation-specific manner

P21 activated kinase 6 (PAK6) is a serine-threonine kinase with physiological expression enriched in the brain and overexpressed in a number of human tumors. While the role of PAK6 in cancer cells has been extensively investigated, the physiological function of the kinase in the context of brain cells is poorly understood. Our previous work uncovered a link between PAK6 and the Parkinson’s disease (PD)-associated kinase LRRK2, with PAK6 controlling LRRK2 activity and subcellular localization via phosphorylation of 14–3–3 proteins. Here, to gain more insights into PAK6 physiological function, we performed protein-protein interaction arrays and identified a subgroup of PAK6 binders related to ciliogenesis. We confirmed that endogenous PAK6 localizes at both the centrosome and the cilium, and positively regulates ciliogenesis not only in tumor cells but also in neurons and astrocytes. Notably, PAK6 rescues ciliogenesis and centrosomal cohesion defects associated with the G2019S but not the R1441C LRRK2 PD mutation. Since PAK6 binds LRRK2 via its GTPase/Roc-COR domain and the R1441C mutation is located in the Roc domain, we used microscale thermophoresis and AlphaFold2-based computational analysis to demonstrate that PD mutations in LRRK2 affecting the Roc-COR structure substantially decrease PAK6 affinity, providing a rationale for the differential protective effect of PAK6 toward the distinct forms of mutant LRRK2. Altogether, our study discloses a novel role of PAK6 in ciliogenesis and points to PAK6 as the first LRRK2 modifier with PD mutation-specificity.

FYCO1 regulates autophagy and senescence via PAK1/p21 in cataract

ARC (Age-related cataract) is one of the leading causes of vision impairment and blindness; however, its pathogenesis remains unclear. FYCO1 (FYVE and coiled-coil domain containing 1) serves as an autophagy adaptor. The present study investigated the role of FYCO1 in cataract. Ultraviolet-B (UVB) irradiation was used to establish a cataract mice model. Hematoxylin and eosin (H&E) assay were used to observe lens morphology. Cell models were constructed by cultivating SRA 01/04 cells with H2O2 and UVB. Cell counting kit-8 (CCK8) and Senescence-associated β-galactosidase (SA-β-Gal) assay were performed to explore proliferation and senescence. The gene and protein expression were assessed by quantitative real-time PCR (qRT-PCR), western blot and immunofluorescence staining. We demonstrated lens structural damage and downregulation of FYCO1 in mice with UVB-induced cataracts. In vitro results revealed a deletion in autophagy levels along with the decrease of FYCO1 expression in human lens epithelial cells (HLECs) after H2O2 treatment, which was confirmed in vivo. The knockout of FYCO1 in the HLECs did not change basal autophagy and senescence but suppressed HLECs response in the induction of both. Further investigation indicated that FYCO1 knockout inhibited senescence and p21 levels by suppressing the expression of p21 activated kinase 1 (PAK1) in cataract cell models. This study has newly characterized the role of FYCO1 in UVB-induced cataracts and in oxidative stress, both of which are associated with ARCs. A novel association between FYCO1 and PAK1/p21 in lens epithelial cell autophagy, senescence, and cataractogenesis also appears to have been established.

RAC2 gain-of-function variants causing inborn error of immunity drive NLRP3 inflammasome activation.

A growing number of patients presenting severe combined immunodeficiencies attributed to monoallelic RAC2 variants have been identified. The expression of the RHO GTPase RAC2 is restricted to the hematopoietic lineage. RAC2 variants have been described to cause immunodeficiencies associated with high frequency of infection, leukopenia, and autoinflammatory features. Here, we show that specific RAC2 activating mutations induce the NLRP3 inflammasome activation leading to the secretion of IL-1β and IL-18 from macrophages. This activation depends on the activation state of the RAC2 variant and is mediated by the downstream kinase PAK1. Inhibiting the RAC2-PAK1-NLRP3 inflammasome pathway might be considered as a potential treatment for these patients.

Effects of Down-Regulation of PAK1 on Differentiation and Apoptosis of MPN Cells with MPLW515L Gene Mutation and Survival of 6133/MPL Mice

To investigate the effects of down-regulation of p21 activated kinase 1 (PAK1) on the proliferation, differentiation, and apoptosis of myeloproliferative neoplasm (MPN) cells (6133/MPL) with thrombopoietin receptor MPL mutation at codon 515 (MPLW515L) and survival of 6133/MPL mice. Interference with the protein level of PAK1 in 6133/MPL cells was assessed using lentivirus-mediated shRNA transfection technology. CCK-8 assay was used to detect the effect of down-regulation of PAK1 on the proliferation viability of 6133/MPL cells, and colony-forming ability was measured by cell counting. Flow cytometry was used to detect the PAK1 kinase activity on the ability of polyploid DNA formation and cell apoptosis in 6133/MPL cells. The expression of cyclin D1, cyclin D3 and apoptosis-related protein Bax was detected by Western blot. The infiltration of tumor cells in spleen and bone marrow of 6133/MPL mice were detected by HE staining. Down-regulation of PAK1 inhibited the proliferation and reduced the ability of cell colony formation of 6133/MPL cells. After knocking down PAK1, the content of polyploid DNA in 6133/MPL cells increased from 31.8 to 57.5% and 48.0%, and the proportion of apoptosis increased approximately to 10.8%. Down-regulation of PAK1 led to a reduction of infiltration of tumor cells in liver and bone marrow of 6133/MPL mice, thereby prolonging survival time. Down-regulation of PAK1 can significantly inhibit the growth of 6133/MPL cells, promote the formation of polyploid DNA, induce 6133/MPL cell apoptosis, and prolong the survival time of 6133/MPL mice.

Design, synthesis and pharmacological evaluation of 1,2,3,4-tetrahydrobenzofuro[2,3-c]pyridine derivatives as p21-activated kinase 4 inhibitors for treatment of pancreatic cancer

The p21-activated kinase 4 (PAK4), a key regulator of malignancy, is negatively correlated with immune infiltration and has become an emergent drug target of cancer therapy. Given the lack of high efficacy PAK4 inhibitors, we herein reported the identification of a novel inhibitor 13 bearing a tetrahydrobenzofuro[2,3-c]pyridine tricyclic core and possessing high potency against MIA PaCa-2 and Pan02 cell lines with IC50 values of 0.38 and 0.50 μmol/L, respectively. This compound directly binds to PAK4 in a non-ATP competitive manner. In the mouse Pan02 model, compound 13 exhibited significant tumor growth inhibition at a dose of 100 mg/kg, accompanied by reduced levels of PAK4 and its phosphorylation together with immune infiltration in mice tumor tissue. Overall, compound 13 is a novel allosteric PAK4 inhibitor with a unique tricyclic structural feature and high potency both in vitro and in vivo, thus making it worthy of further exploration.

Spatiotemporal regulation of organelle transport by spindle position checkpoint kinase Kin4.

Asymmetric cell division in Saccharomyces cerevisiae involves Class V myosin-dependent transport of organelles along the polarised actin cytoskeleton to the emerging bud. Vac17 is the vacuole/lysosome-specific myosin receptor. Its timely breakdown terminates transport and results in the proper positioning of vacuoles in the bud. Vac17 breakdown is controlled by the bud-concentrated p21-activated kinase, Cla4, and the E3-Ubiquitin ligase, Dma1. We found that the spindle position checkpoint kinase, Kin4, and to a lesser extent its paralog Frk1, contribute to successful vacuole transport by preventing the premature breakdown of Vac17 by Cla4 and Dma1. Furthermore, Kin4 and Cla4 contribute to the regulation of peroxisome transport. We conclude that Kin4 acts antagonistically to the Cla4-/Dma1-pathway to coordinate spatiotemporal regulation of organelle transport.

Remifentanil-induced inflammation in microglial cells: Activation of the PAK4-mediated NF-κB/NLRP3 pathway and onset of hyperalgesia

BackgroundThe perioperative use of remifentanil is associated with postoperative hyperalgesia, which can impair recovery and extend hospitalization. Recent studies have revealed that microglia-mediated activation of the NLRP3 inflammasome plays a critical role in opioid-induced hyperalgesia, with NF-κB acting as a pivotal activation point for NLRP3. Despite these findings, the specific molecular mechanisms underlying remifentanil-induced postoperative hyperalgesia remain unclear. This study aims to develop a model of remifentanil-induced hyperalgesia and investigate the molecular mechanisms, focusing on the NF-κB/NLRP3 pathway, using both in vitro and in vivo approaches. MethodWe established a remifentanil-induced hyperalgesia model and performed proteomic analysis to identify differential protein expression in the spinal cord tissue of rats. NLRP3 or PAK4 antagonists were administered intrathecally in vivo, and mechanical pain thresholds in the hind paws were measured using Von Frey testing. In vitro, we applied NLRP3 or PAK4 inhibitors or used lentivirus infection to silence PAK4, NF-κB, and NLRP3 genes. Protein expression was assessed through immunohistochemistry, immunofluorescence, and Western blotting. Additionally, ELISA was performed to measure IL-1β and IL-18 levels, and RT-qPCR was conducted to evaluate the transcription of target genes. ResultsProteomic analysis revealed that remifentanil upregulates PAK4 protein in spinal cord tissue two hours after the surgery. In addition, remifentanil induces morphological changes in the spinal cord dorsal horn, characterized by increased expression of PAK4, p-p65, NLRP3 and Iba-1 proteins, which in turn leads to elevated IL-1β and IL-18 levels and an inflammatory response. Intrathecal injection of NLRP3 or PAK4 inhibitors mitigates remifentanil-induced hyperalgesia and associated changes. In vitro, downregulation of PAK4 inhibits the increase in PAK4, p-p65, NLRP3 and Caspase-1 induced by LPS. Conversely, the downregulation of NLRP3 does not impact the levels of PAK4 and p-p65 proteins, aligning with the in vivo results and suggesting that PAK4 acts as an upstream signaling molecule of NLRP3. ConclusionRemifentanil can increase PAK4 expression in spinal cord dorsal horn cells by activating the NF-κB/NLRP3 pathway and mediating microglial activation, thereby contributing to postoperative hyperalgesia.

Inhibition of NAMPT by PAK4 Inhibitors.

The serine/threonine kinase PAK4 plays a crucial role in regulating cell proliferation, survival, migration, and invasion. Overexpression of PAK4 correlates with poor prognosis in some cancers. KPT-9274, a PAK4 inhibitor, significantly reduces the growth of triple-negative breast cancer cells and mammary tumors in mouse models, and it also inhibits the growth of several other types of cancer cells. Interestingly, although it was first identified as a PAK4 inhibitor, KPT-9274 was also found to inhibit the enzyme NAMPT (nicotinamide phosphoribosyltransferase), which is crucial for NAD (nicotinamide adenine dinucleotide) synthesis and vital for cellular energy and growth. These results made us question whether growth inhibition in response to KPT-9274 was due to PAK4 inhibition, NAMPT inhibition, or both. To address this, we tested several other PAK4 inhibitors that also inhibit cell growth, to determine whether they also inhibit NAMPT activity. Our findings confirm that multiple PAK4 inhibitors also inhibit NAMPT activity. This was assessed both in cell-free assays and in a breast cancer cell line. Molecular docking studies were also used to help us better understand the mechanism by which PAK4 inhibitors block PAK4 and NAMPT activity, and we identified specific residues on the PAK4 inhibitors that interact with NAMPT and PAK4. Our results suggest that PAK4 inhibitors may have a more complex mechanism of action than previously understood, necessitating further exploration of how they influence cancer cell growth.

Carotid artery vascular stenosis causes the blood-CSF barrier damage and neuroinflammation

BackgroundThe choroid plexus (ChP) helps maintain the homeostasis of the brain by forming the blood-CSF barrier via tight junctions (TJ) at the choroid plexus epithelial cells, and subsequently preventing neuroinflammation by restricting immune cells infiltration into the central nervous system. However, whether chronic cerebral hypoperfusion causes ChP structural damage and blood-CSF barrier impairment remains understudied.MethodsThe bilateral carotid stenosis (BCAS) model in adult male C57BL/6 J mice was used to induce cerebral hypoperfusion, a model for vascular contributions to cognitive impairment and dementia (VCID). BCAS-mediated changes of the blood-CSF barrier TJ proteins, apical secretory Na+-K+-Cl− cotransporter isoform 1 (NKCC1) protein and regulatory serine-threonine kinases SPAK, and brain infiltration of myeloid-derived immune cells were assessed.ResultsBCAS triggered dynamic changes of TJ proteins (claudin 1, claudin 5) accompanied with stimulation of SPAK-NKCC1 complex and NF-κB in the ChP epithelial cells. These changes impacted the integrity of the blood-CSF barrier, as evidenced by ChP infiltration of macrophages/microglia, neutrophils and T cells. Importantly, pharmacological blockade of SPAK with its potent inhibitor ZT1a in BCAS mice attenuated brain immune cell infiltration and improved cognitive neurological function.ConclusionsBCAS causes chronic ChP blood-CSF damage and immune cell infiltration. Our study sheds light on the SPAK-NKCC1 complex as a therapeutic target in neuroinflammation.

Targeting RAC1 reactivates pyroptosis to reverse paclitaxel resistance in ovarian cancer by suppressing P21-activated kinase 4.

Pyroptosis may play an important role in the resistance of ovarian cancer (OC) to chemotherapy. However, the mechanism by which pyroptosis modulation can attenuate chemotherapy resistance has not been comprehensively studied in OC. Here, we demonstrated that RAS-associated C3 botulinum toxin substrate 1 (RAC1) is highly expressed in OC and is negatively correlated with patient outcomes. Through cell function tests and in vivo tumor formation tests, we found that RAC1 can promote tumor growth by mediating paclitaxel (PTX) resistance. RAC1 can mediate OC progression by inhibiting pyroptosis, as evidenced by high-throughput automated confocal imaging, the release of lactate dehydrogenase (LDH), the expression of the inflammatory cytokines IL-1β/IL-18 and the nucleotide oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. Mechanically, RNA-seq, gene set enrichment analysis (GSEA), coimmunoprecipitation (Co-IP), mass spectrometry (MS), and ubiquitination tests further confirmed that RAC1 inhibits caspase-1/gasdermin D (GSDMD)-mediated canonical pyroptosis through the P21-activated kinase 4 (PAK4)/mitogen-activated protein kinase (MAPK) pathway, thereby promoting PTX resistance in OC cells. Finally, the whole molecular pathway was verified by the results of in vivo drug combination tests, clinical specimen detection and the prognosis. In summary, our results suggest that the combination of RAC1 inhibitors with PTX can reverse PTX resistance by inducing pyroptosis through the PAK4/MAPK pathway.

Pentraxin 3 mediates inflammation and adipogenesis in Graves' orbitopathy pathogenesis.

Pentraxin 3 (PTX3) is a prototypic humoral soluble pattern-recognition molecule known to function in immunity-related inflammation. Given the lack of information on the precise functions of PTX3 in the pathogenesis of Graves' orbitopathy (GO), this study investigated the role of PTX3 in the inflammation and adipogenesis mechanisms of GO. We first compared the PTX3 expression between orbital tissues from patients with GO and normal controls using real-time PCR, which estimated significantly higher PTX3 transcript levels in the GO tissues than in the normal tissues. In addition, PTX3 production was markedly increased upon interleukin (IL)-1β and adipogenic stimulation. We then evaluated the effects of silencing PTX3 in primary orbital fibroblast cultures by analyzing the expression levels of pro-inflammatory cytokines, adipogenesis-related proteins, and downstream transcription factors in cells transfected with or without small interfering RNA against PTX3, using western blot. Silencing PTX3 attenuated the IL-1β-induced secretion of pro-inflammatory cytokines, including IL-6, IL-8, monocyte chemotactic protein-1, intercellular adhesion molecule-1, and cyclooxygenase-2, and suppressed the IL-1β-mediated activation of p38 kinase, nuclear factor-κB, and extracellular signal-regulated kinase. Moreover, PTX3 knockdown suppressed adipogenic differentiation, as assessed using Oil Red O staining, as well as the expression of adipogenesis-associated transcription factors including peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding proteins α and β, adipocyte protein 2, adiponectin, and leptin. Thus, this study suggests that PTX3 plays a significant role in the pathogenesis of GO and may serve as a novel therapeutic target for the condition.

FGFR1-mediated enhancement of foot-and-mouth disease virus entry

Foot-and-mouth disease virus (FMDV), a member of picornavirus, can enter into host cell via macropinocytosis. Although it is known that receptor tyrosine kinases (RTKs) play a crucial role in FMDV macropinocytic entry, the specific RTK responsible for regulating this process and the intricacies of RTK-mediated downstream signaling remain to be elucidated. Here, we conducted a screening of RTK inhibitors to assess their efficacy against FMDV. Our findings revealed that two compounds specifically targeting fibroblast growth factor receptor 1 (FGFR1) and FMS-like tyrosine kinase 3 (FLT3) significantly disrupted FMDV entry. Furthermore, additional evaluation through gene knockdown and overexpression confirmed the promotion effect of FGFR1 and FLT3 on FMDV entry. Interestingly, we discovered that the increasement of FMDV entry facilitated by FGFR1 and FLT3 can be ascribed to increased macropinocytic uptake. Additionally, in-depth mechanistic study demonstrated that FGFR1 interacts with FMDV VP3 and undergoes phosphorylation during FMDV entry. Furthermore, the FGFR1 inhibitor inhibited FMDV-induced activation of p21-activated kinase 1 (PAK1) on Thr212 and Thr423 sites. Consistent with these findings, the ectopic expression of FGFR1 resulted in a concomitant increase in phosphorylation level of PAK1 on Thr212 and Thr423 sites. Taken together, our findings represent the initial exploration of FGFR1's involvement in FMDV macropinocytic entry, providing novel insights with potential implications for the development of antiviral strategies.

Design, synthesis and biological evaluation of a novel PAK1 degrader for the treatment of triple negative breast cancer

Triple-negative breast cancer is one of the most malignant subtypes in clinical practice, and it is urgent to find new therapies. The p21-activated kinase I (PAK1) has been considered to be an attractive therapeutic target for TNBC. In this study, we designed and synthesized a series of novel PROTAC PAK1 degraders by conjugating VHL or CRBN ligase ligands to PAK1 inhibitors which are connected by alkyl chains or PEG chains. The most promising compound, 19s, can significantly degrade PAK1 protein at concentrations as low as 0.1 μM, and achieves potent anti-proliferative activity with an IC50 value of 1.27 μM in MDA-MB-231 cells. Additionally, 19s exhibits potent anti-migration activity in vitro and induces rapid tumor regression in vivo. Collectively, these findings document that 19s is a potent and novel PAK1 degrader with promising potential for TNBC treatment.

PAK3 Exacerbates Cardiac Lipotoxicity via SREBP1c in Obesity Cardiomyopathy.

Obesity-induced lipid overload in cardiomyocytes contributes to profound oxidative stress and cardiomyopathy, culminating in heart failure. In this study, we investigate a novel mechanism whereby lipids accumulate in cardiomyocytes and seek the relevant treatment strategies. P21-activated kinase 3 (PAK3) was elevated in obese human myocardium, and the murine hearts and cardiomyocytes upon diet- or fatty acid-induced stress, respectively. Mice with cardiac-specific overexpression of PAK3 were more susceptible to the development of cardiac dysfunction upon diet stress, at least partially, due to increased deposition of toxic lipids within the myocardium. Mechanistically, PAK3 promoted the nuclear expression of sterol regulatory element binding protein 1c (SREBP1c) through activation of mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase beta-1 (S6K1) pathway in cardiomyocytes, resulting in abnormal lipid genes profile, accumulation of excessive lipids, and oxidative stress. More importantly, PAK3 knockdown attenuated fatty acid-induced lipotoxicity and cell death in rat and human cardiomyocytes. More importantly, the S6K1 or SREBP1c inhibitor alleviated PAK3-triggered intracellular lipid overload and cardiac dysfunction under obese stress. Collectively, we have demonstrated that PAK3 impairs myocardial lipid homeostasis, while inhibition of cardiac lipotoxicity mitigates cardiac dysfunction. Our study provides a promising therapeutic strategy for ameliorating obesity cardiomyopathy.

PAK4 phosphorylates and inhibits AMPKα to control glucose uptake

Our recent studies have identified p21-activated kinase 4 (PAK4) as a key regulator of lipid catabolism in the liver and adipose tissue, but its role in glucose homeostasis in skeletal muscle remains to be explored. In this study, we find that PAK4 levels are highly upregulated in the skeletal muscles of diabetic humans and mice. Skeletal muscle-specific Pak4 ablation or administering the PAK4 inhibitor in diet-induced obese mice retains insulin sensitivity, accompanied by AMPK activation and GLUT4 upregulation. We demonstrate that PAK4 promotes insulin resistance by phosphorylating AMPKα2 at Ser491, thereby inhibiting AMPK activity. We additionally show that skeletal muscle-specific expression of a phospho-mimetic mutant AMPKα2S491D impairs glucose tolerance, while the phospho-inactive mutant AMPKα2S491A improves it. In summary, our findings suggest that targeting skeletal muscle PAK4 may offer a therapeutic avenue for type 2 diabetes.

Abstract Tu090: P21-activated kinase 1 safeguards cardiac function from impairment during fasting

Excessive calorie intake poses a heightened risk for cardiovascular disease, while caloric restriction and intermittent fasting have demonstrated benefits in enhancing cardiovascular health and improving cardiometabolic risk profiles. Despite this knowledge, the precise mechanisms underlying the cardioprotective effects of these dietary regimens remain inadequately understood. P21-activated kinase 1 (PAK1) is a serine/threonine protein kinase known to play a crucial role in promoting cardiomyocyte survival in response to various stressors. In this study, we aimed to elucidate the role of PAK1 in maintaining cardiac function during fasting, utilizing mice with modified PAK1 activity in the heart. Under normal feeding conditions, heart function exhibited no discernible differences among wild type (WT), transgenic (TG), and knockout (KO) mice, as evidenced by consistent fractional shortening (FS), ejection fraction (EF), E/A ratio, and isovolumic relaxation time (IVRT). After a one-day fast, systolic contractile function remained robust across all groups of mice. However, the E/A ratio decreased in KO mice, and IVRT was prolonged in KO mice, while shortened in TG mice, highlighting the crucial role of PAK1 in maintaining diastolic cardiac function. Upon a two-day fast, systolic function was compromised in both WT and KO mice, whereas TG mice exhibited preserved function, as evidenced by FS and EF measurements, emphasizing PAK1's equal significance in sustaining systolic cardiac function. Furthermore, fasting accelerated mitophagy, a process eliminating dysfunctional mitochondria through lysosomal degradation, as observed by mitochondria-associated LC3-II and a dual fluorescent mitophagy reporter. Additionally, we established PAK1 as a positive regulator of mitophagy, suggesting its potential protective role in the heart during fasting via mitophagy modulation. In summary, PAK1 emerges as a critical mediator of cardiac function during fasting, presenting promising avenues for therapeutic interventions aimed at improving cardiovascular health.