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The repurposing of existing drugs has emerged as an attractive additional strategy to the development of novel compounds in the fight against cancerous diseases. Inhibition of phosphodiesterase 5 (PDE5) has been claimed as a potential approach to target various cancer subtypes in recent years. However, data on the treatment of tumors with PDE5 inhibitors as well as the underlying mechanisms are as yet very scarce. Here, we report that treatment of tumor cells with low concentrations of Sildenafil was associated with decreased cancer cell proliferation and augmented apoptosis in vitro and resulted in impaired tumor growth in vivo. Notably, incubation of cancer cells with Sildenafil was associated with altered expression of HSP90 chaperone followed by degradation of protein kinase D2, a client protein previously reported to be involved in tumor growth. Furthermore, the involvement of low doses of PU-H71, an HSP90 inhibitor currently under clinical evaluation, in combination with low concentrations of Sildenafil, synergistically and negatively impacted on the viability of cancer cells in vivo. Taken together, our study suggests that repurposing of already approved drugs, alone or in combination with oncology dedicated compounds, may represent a novel cancer therapeutic strategy.

Background: N6-Methyladenosine (m6A) is involved in many of pathological processes including insulin resistance (IR). Quercetin (Que), a bioactive compound with strong antioxidant activity, has potential therapeutic effects on IR-related metabolic diseases. The aim of this study is to investigate the roles of m6A and Que in hyperinsulinemia.Methods: Male C57Bl/6 mice received a high fat diet (HFD) for 8 weeks to establish an IR model. Biochemical analyzes, oral glucose tolerance test and homeostasis model assessment of IR (HOMA-IR) were conducted. C2C12 cells were transfected with methyltransferase-like 3 (METTL3) siRNA before treatment with palmitate (PA). 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose (2-NBDG) was used to detect glucose uptake. Malondialdehyde (MDA) level, superoxide dismutase (SOD) activity, and glutathione (GSH) level were used to evaluate the levels of oxidative stress. SRAMP, a predicted site of m6A modification, was used to predict the relationship between METTL3 and erine-threonine kinase protein kinase D2 (PRKD2). Then, cells were treated with Que or/and transfected with pcDNA-METTL3 or PRKD2 siRNA. indirect calorimetry, Western blotting and qPCR were used to analyze protein and mRNA expression.Results: Que treatment reduced the body weight, blood glucose, plasma TG and serum insulin, ameliorated IR, and decreased oxidative stress in HFD-fed mice. Que at noncytotoxic doses promoted PA-induced glucose uptake and inhibited oxidative stress in C2C12 cells. Moreover, METTL3 and PRKD2 was downregulated in PA-induced C2C12 cells, and SRAMP revealed multiple m6A modification sites in the PRKD2 mRNA sequence. Downregulation of METTL3 enhanced PRKD2 expression by reducing m6A level and mRNA stability in PRKD2 mRNA transcript. C2C12 cells treatment with Que after inducing with PA inhibited m6A, METTL3 and phosphorylated insulin receptor substrate 1 (p-IRS1) levels, increased the protein expression of PRKD2, glucose transporter type 4 (GLUT4) and p-AKT and glucose uptake, and reduced oxidative stress. However, METTL3 overexpression or PRKD2 silence reversed the inhibitory effects of Que on the levels of MDA, GLUT4 and p-AKT, and the promotive effects on glucose uptake, SOD, GSH and p-IRS1 levels.Conclusion: Que promoted glucose uptake, repressed oxidative stress and improved IR through METTL3-mediated N6-methylation of PRKD2 mRNA.

Kidneys are critical target organs of COVID-19, but susceptibility and responses to infection remain poorly understood. Here, we combine SARS-CoV-2 variants with genome edited kidney organoids and clinical data to investigate tropism, mechanism, and therapeutics. SARS-CoV-2 specifically infects organoid proximal tubules amongst diverse cell types. Infections produce replicating virus, apoptosis, and disrupted cell morphology, features of which are revealed in the context of polycystic kidney disease. Cross-validation of gene expression patterns in organoids reflect proteomic signatures of COVID-19 in the urine of critically ill patients indicating interferon pathway upregulation. SARS-CoV-2 viral variants Alpha, Beta, Gamma, Kappa, and Delta exhibit comparable levels of replication in organoids. Infection is ameliorated in ACE2-/- organoids and blocked via treatment with de novo designed spike binder peptides. Collectively, these studies clarify the impact of kidney infection in COVID-19 as reflected in organoids and clinical populations, enabling assessment of viral fitness and emerging therapies.

B cells can act as potent suppressors of anti-tumor T cell immunity, presenting a mechanism of resistance to immunotherapy. In pancreatic ductal adenocarcinoma, B cells can display a T cell-suppressive or regulatory phenotype centered on the expression of the cytokine Interleukin 35 (IL-35). While B cell-mediated immunosuppression presents a barrier to anti-tumorigenic T cell function, it is not clear how regulatory B cell function could be targeted, and the signals that promote this suppressive phenotype in B cells are not well understood. Here we use a novel IL-35 reporter model to understand which signaling pathways are important for immunosuppressive properties in B cells. In vitro analysis of IL-35 reporter B cells revealed a synergy between the BCR and TLR4 signaling pathways is sufficient to induce IL-35 expression. However, in vivo, B cell receptor activation, as opposed to MyD88 signaling in B cells, is central to B cell-mediated suppression and promotion of pancreatic cancer growth. Further analysis identified protein kinase D2 (PKD2) as being a key downstream regulator of IL-35 expression in B cells. Regulatory B cells with an inactivating mutation in PKD2 failed to produce IL-35 or fully suppress effector T cell function in vitro. Furthermore, inhibition of PKD in B cells decreased tumor growth and promoted effector T cell function upon adoptive transfer into B cell-deficient mice. Collectively, these data provide insight into how regulatory B cell function is promoted in pancreatic cancer and identify potential therapeutic targets to restrain this function.

Recent findings have shown that dysregulation of circular RNAs (circRNAs) is implicated in various cancers. However, the contribution of circRNAs in oral squamous cell carcinoma (OSCC) remains largely unexplored. We screened circRNA expression profiles using a circRNA microarray in paired OSCC and normal tissues and explored the clinical significance of a downregulated circRNA, circ-PKD2. Moreover, the biological function of circ-PKD2 in OSCC was investigated both in vitro and in vivo. We found that downregulation of circ-PKD2 in OSCC correlated significantly with aggressive characteristics. Further analysis revealed that overexpression of circ-PKD2 inhibited OSCC cell proliferation, migration and invasion, induced apoptosis and cell cycle arrest, which were promoted by knockdown of circ-PKD2. In addition, circ-PKD2 was identified as a sponge for miR-204-3p and upregulated the expression of adenomatous polyposis coli 2 (APC2), which was the functional target of miR-204-3p. Moreover, circ-PKD2 attenuated the oncogenic effects of miR-204-3p-mediated APC2 on OSCC progression via multiple signaling pathways. These results demonstrate that the circ-PKD2/miR-204-3p/APC2 axis represents a novel pathway involved in the pathogenesis of OSCC and may serve as a novel therapeutic target of OSCC.

Protein kinase D2 (PKD2) has been reported to be related with progression and invasion in various cancers. However, its prognostic value and the underlying mechanism in lung cancer remains unclear. Herein we evaluated the expression of PKD2 in lung adenocarcinoma and investigated its relationship with EMT. GSEA, TCGA and K-M plotter database were applied and revealed that high PKD2 expression predicted poor outcome and related with lymph nodes metastasis in lung cancer. IHC and qRT-PCR were performed and found PKD2 was elevated in lung adenocarcinoma and negatively related with OS (p = 0.015), PFS (p = 0.006) and the level of E-cadherin (p = 0.021). Experiment in lung adenocarcinoma cell lines demonstrated up-regulation of PKD2 led to high expression of mesenchymal markers (N-cadherin, vim, mmp9 et al.) and EMT transcription factors(zeb1, twist, snail), and the results were reversed when PKD2 was knocked down. Further investigation showed that abrogation of PKD2 inhibited A549 cell migration, invasion, proliferation and induced cell arrest in G2/M phase. We concluded that high expression of PKD2 was associated with poor prognosis and cancer progression in lung adenocarcinoma patients by promoting EMT.

Protein Kinase D (PKD) family contains PKD1, PKD2, and PKD3 in human. Compared to consistent tumor‐suppressive functions of PKD1 in breast cancer, how PKD2/3 functions in breast cancer are not fully understood. In the current study, we found that PKD2 and PKD3 but not PKD1 were preferentially overexpressed in breast cancer and involved in regulating cell proliferation and metastasis. Integrated phosphoproteome, transcriptome, and interactome showed that PKD2 was associated with multiple cancer‐related pathways, including adherent junction, regulation of actin cytoskeleton, and cell cycle‐related pathways. ELAVL1 was identified as a common hub‐node in networks of PKD2/3‐regulated phosphoproteins and genes. Silencing ELAVL1 inhibited breast cancer growth in vitro and in vivo. Direct interaction between ELAVL1 and PKD2 or PKD3 was demonstrated. Suppression of PKD2 led to ELAVL1 translocation from the cytoplasm to the nucleus without significant affecting ELAVL1 expression. Taken together, we characterized the oncogenic functions of PKD2/3 in breast cancer and their association with cancer‐related pathways, which shed lights on the oncogenic roles and mechanisms of PKDs in breast cancer.

To detect the mutation site in a pedigree affected with autosomal dominant polycystic kidney disease (ADPKD) and verify its impact on the protein function.Peripheral blood samples were collected from the proband and his pedigree members for the extraction of genomic DNA. Mutational analysis was performed on the proband through whole-exome sequencing. Suspected variant was verified by Sanger sequencing. A series of molecular methods including PCR amplification, restriction enzyme digestion, ligation and transformation were also used to construct wild-type and mutant eukaryotic expression vectors of the PKD2 gene, which were transfected into HEK293T and HeLa cells for the observation of protein expression and cell localization.The proband was found to harbor a c.2051dupA (p. Tyr684Ter) frame shift mutation of the PKD2 gene, which caused repeat of the 2051st nucleotide of its cDNA sequence and a truncated protein. Immunofluorescence experiment showed that the localization of the mutant protein within the cell was altered compared with the wild-type, which may be due to deletion of the C-terminus of the PKD2 gene.The c.2051dupA (p. Tyr684Ter) mutation of the PKD2 gene probably underlay the pathogenesis of ADPKD in this pedigree.

Lipids are the most energy‐dense components of the diet, and their overconsumption promotes obesity and diabetes. Dietary fat content has been linked to the lipid processing activity by the intestine and its overall capacity to absorb triglycerides (TG). However, the signaling cascades driving intestinal lipid absorption in response to elevated dietary fat are largely unknown. Here, we describe an unexpected role of the protein kinase D2 (PKD2) in lipid homeostasis. We demonstrate that PKD2 activity promotes chylomicron‐mediated TG transfer in enterocytes. PKD2 increases chylomicron size to enhance the TG secretion on the basolateral side of the mouse and human enterocytes, which is associated with decreased abundance of APOA4. PKD2 activation in intestine also correlates positively with circulating TG in obese human patients. Importantly, deletion, inactivation, or inhibition of PKD2 ameliorates high‐fat diet‐induced obesity and diabetes and improves gut microbiota profile in mice. Taken together, our findings suggest that PKD2 represents a key signaling node promoting dietary fat absorption and may serve as an attractive target for the treatment of obesity., We show that upon fat ingestion, Protein Kinase D2 stimulates chylomicron‐mediated triglyceride absorption in the intestine. Targeting PKD2, genetically or with small molecule inhibitors, reduces triglycerides absorption and prevents the development of obesity in mice and presumably in humans.

Consistent with the gene dosage effect hypothesis, renal cysts can arise in transgenic murine models overexpressing either PKD1 or PKD2, which are causal genes for autosomal dominant polycystic kidney disease (ADPKD). To determine whether PKD gene overexpression is a universal mechanism driving cystogenesis or is merely restricted to rodents, other animal models are required. Previously, we failed to observe any renal cysts in a transgenic porcine model of PKD2 overexpression partially due to epigenetic silencing of the transgene. Thus, to explore the feasibility of porcine models and identify potential genes/pathways affected in ADPKD, LLC-PK1 cells with high PKD2 expression were generated. mRNA sequencing (RNA-seq) was performed, and MYC, IER3, and ADM were found to be upregulated genes common to the different PKD2 overexpression cell models. MYC is a well-characterized factor contributing to cystogenesis, and ADM is a biomarker for chronic kidney disease. Thus, these genes might be indicators of disease progression. Additionally, some ADPKD-associated pathways, e.g., the mitogen-activated protein kinase (MAPK) pathway, were enriched in the cells. Moreover, gene ontology (GO) analysis demonstrated that proliferation, apoptosis, and cell cycle regulation, which are hallmarks of ADPKD, were altered. Therefore, our experiment identified some biomarkers or indicators of ADPKD, indicating that high PKD2 expression would likely drive cystogenesis in future porcine models.

The Protein Kinase D (PKD) family is set up by three highly conserved members, the isoenzymes PKD1, 2 and 3. PKDs are diacylglyerol-stimulated serine/ threonine protein kinases and form a subgroup of the Calcium/ Calmodulin Protein Kinase (CAMK) superfamily. Various stimuli lead to a Protein Kinase C (PKC)- dependent PKD-activation and the performance of multiple functions in diverse biological systems. A major role in physiological as well as tumour angiogenesis is attributed to PKD2. Angiogenesis is referred to as formation of new blood vessels from pre-existing vascular structures by endothelial cell differentiation. In contrast, vasculogenesis describes de novo blood vessel formation by endothelial cell differentiation from mesoderm during development. PKCs, the prominent upstream activators of PKDs, are known to drive lineage commitment in mouse embryonic stem cells (mESCs). Moreover, in the developing embryo, vasculogenesis and angiogenesis are closely linked. However, the role of PKDs during early embryonic cell fate decision and especially vasculogenesis largely remains elusive. To address this question, this study uses a combined gain- and loss-of-function approach in mouse embryonic stem cells for in vitro experiments, complemented with chicken chorioallantoic membrane (CAM) xenografts for an in vivo approach. This study demonstrates PKD2 to be strongly expressed in mESCs, followed by PKD3 and PKD1 only presenting marginal expression levels. For the first time, we describe time-restricted PKD2-activity influencing early cell fate decision and angiogenesis. During early differentiation (days 0-4), PKD2-activity decreases mesendoderm formation and subsequent cardiovasculogenesis in favour of ectoderm, while promoting sprouting angiogenesis during late differentiation (days 4-14). Consistently, PKD2-loss-of-function analyses demonstrate opposed effects. These findings are confirmed in vivo, as embryoid bodies (EBs), transplanted on CAMs, induce an angiogenic response upon PKD2-overexpression from day 4 of differentiation onwards. Summing up, this thesis presents novel and time-dependent aspects of PKD2- activity during early embryonic development. Our data provide further insight into the complex regulation of angiogenesis, that might even be of clinical interest in the fields of cancer research and regenerative medicine.

Highlights • The AMAP1-PRKD2 pathway regulates the intracellular trafficking of CAIX. • The AMAP1-PIAS3 pathway stabilizes HIF-1α and promotes the transcription of CAIX. • High ASAP1 together with high PIAS3 correlates with high CA9 in breast cancer. • High ASAP1 with high PIAS3 predicts the unfavorable prognosis of breast cancer. • Regulation of CAIX transcription and trafficking may be interconnected via AMAP1., Background The cell-surface enzyme carbonic anhydrase IX (CAIX/CA9) promotes tumor growth, survival, invasion, and metastasis, mainly via its pH-regulating functions. Owing to its tumor-specific expression, CAIX-targeting antibodies/chemicals are utilized for therapeutic and diagnostic purposes. However, mechanisms of CAIX trafficking, which affects such CAIX-targeting modalities remain unclear. In this study, roles of the AMAP1-PRKD2 pathway, which mediates integrin recycling of invasive cancer cells, in CAIX trafficking were investigated. Methods Using highly invasive MDA-MB-231 breast cancer cells, the physical association and colocalization of endogenous proteins were analyzed by immunoprecipitation and immunofluorescence, protein/mRNA levels were quantified by western blotting/qPCR, and cell-surface transport and intracellular/extracellular pH regulation were measured by biotin-labeling and fluorescent dye-based assays, respectively. The correlation between mRNA levels and patients’ prognoses was analyzed using a TCGA breast cancer dataset. Results AMAP1 associated with the CAIX protein complex, and they colocalized at the plasma membrane and tubulovesicular structures. AMAP1 knockdown reduced total/surface CAIX, induced its lysosomal accumulation and degradation, and affected intracellular/extracellular pH. PRKD2 knockdown excluded AMAP1 from the CAIX complex and reduced total CAIX in a lysosome-dependent manner. Unexpectedly, AMAP1 knockdown also reduced CAIX mRNA. AMAP1 interacted with PIAS3, which stabilizes HIF-1α, a transcriptional regulator of CA9. AMAP1 knockdown inhibited the PIAS3-HIF-1α interaction and destabilized the HIF-1α protein. High-ASAP1 (AMAP1-encoding gene) together with high-PIAS3 correlated with high-CA9 and an unfavorable prognosis in breast cancer. Conclusion The AMAP1-PRKD2 pathway regulates CAIX trafficking, and modulates its total/surface expression. The AMAP1-PIAS3 interaction augments CA9 transcription by stabilizing HIF-1α, presumably contributing to an unfavorable prognosis.

Polarity complexes, including the PAR (Partitioning-defective), CRB (Crumbs) and SCRIB (Scribble) complexes, are required for the physiologic establishment, stabilization, and maintenance of a functional apical-basolateral polarity. Inactivation of some of the polarity complexes results in cystic kidneys, and apical-basolateral polarity defects are commonly observed in autosomal-dominant polycystic kidney disease (ADPKD); however, little is known about the role that polarity complexes play in ADPKD. Here, we demonstrate that Scribble, a core protein of the SCRIB complex, is down-regulated in ADPKD cell lines and the zebrafish model of this disease ( pkd2 morphants). Overexpression of Scribble could reduce cyst formation in pkd2 morphants, and loss of scrib led to a dilated pronephric duct in zebrafish. Furthermore, the Hippo signaling pathway was inactivated in scrib mutants and pkd2 morphants in which Yes-associated protein (YAP), which is physiologically located in the cytoplasm, was translocated to the nucleus. Of note, overexpression of cytoplasmic YAP, instead of nuclear YAP, could reduce cyst formation in pkd2 morphants. Consistently, knockout of yap resulted in cystic kidneys in zebrafish, which was rescued by the overexpression of cytoplasmic YAP, but not nuclear YAP. Finally, scrib and yap had a genetic interaction with pkd2 in cyst formation, and the overexpression of Scribble attenuated the down-regulation of cytoplasmic YAP in ADPKD. Altogether, our data indicate that Scribble induces the phosphorylation of YAP and, consequently, influences cyst formation in ADPKD by mediating YAP nucleocytoplasmic shuttling.-Xu, D., Lv, J., He, L., Fu, L., Hu, R., Cao, Y., Mei, C. Scribble influences cyst formation in autosomal-dominant polycystic kidney disease by regulating Hippo signaling pathway.

T follicular helper cells (TFH) participate in germinal center (GC) development and are necessary for B cell production of high-affinity, isotype-switched antibodies. In a forward genetic screen, we identified a missense mutation in Prkd2, encoding the serine/threonine kinase protein kinase D2, which caused elevated titers of immunoglobulin E (IgE) in the serum. Subsequent analysis of serum antibodies in mice with a targeted null mutation of Prkd2 demonstrated polyclonal hypergammaglobulinemia of IgE, IgG1, and IgA isotypes, which was exacerbated by the T cell-dependent humoral response to immunization. GC formation and GC B cells were increased in Prkd2-/- spleens. These effects were the result of excessive cell-autonomous TFH development caused by unrestricted Bcl6 nuclear translocation in Prkd2-/- CD4+ T cells. Prkd2 directly binds to Bcl6, and Prkd2-dependent phosphorylation of Bcl6 is necessary to constrain Bcl6 to the cytoplasm, thereby limiting TFH development. In response to immunization, Bcl6 repressed Prkd2 expression in CD4+ T cells, thereby committing them to TFH development. Thus, Prkd2 and Bcl6 form a mutually inhibitory positive feedback loop that controls the stable transition from naive CD4+ T cells to TFH during the adaptive immune response.

Protein kinase D2 (PKD2) is a serine/threonine protein kinase which plays an important role in vesicle fission at the trans-Golgi network (TGN) to coordinate subcellular trafficking with gene expression. We found that in the rat kidney, PKD2 is specifically expressed in collecting duct principal cells predominantly at the apical membrane and with lower basal expression in cytosolic compartments. When rats were maintained on a Na+ depleted diet (

HSP90 is a ubiquitously expressed molecular chaperone implicated in the correct folding and maturation of a plethora of proteins including protein kinases and transcription factors. While disruption of chaperone activity was associated with augmented cancer cell death and decreased tumor growth both in vitro and in vivo, the regulation of HSP90 is not clearly understood. Here we report that treatment of cancer cells with cold physical plasma, an emerging and less aggressive tumor therapy, resulted in ROS generation which subsequently triggered the cleavage of HSP90. Notably, cleavage of HSP90 was followed by the degradation of PKD2, a crucial regulator of tumor growth and angiogenesis. Pre-sensitization of cancer cells with subliminal doses of PU-H71, an HSP90 inhibitor currently under clinical evaluation, followed by treatment with cold-plasma, synergistically and negatively impacted on the viability of cancer cells. Taken together, cold-plasma can be used in conjunction with pharmacologic treatment in order to target the expression and activity of HSP90 and the downstream client proteins implicated in various cancer cell capabilities.

Aims Polymorphous adenocarcinoma (PAC) usually follows an indolent course, but some cases may show recurrences and high-grade features. The genetic events associated with recurrences and high-grade versions are yet to be defined. Our aim was to determine the genetic underpinning of recurrent PACs of the salivary gland and the repertoire of somatic genetic alterations in cases with high-grade histology. Methods and results Four PACs from three patients, including one case with matching primary and recurrent tumours, one de-novo high-grade PAC, and a PAC that transformed to a high-grade tumour following multiple recurrences, were subjected to targeted sequencing (Memorial Sloan Kettering Mutation Profiling of Actionable Cancer Targets assay) or whole-exome sequencing. Both matching primary and recurrent tumours, and the de-novo high-grade PAC, harboured clonal PRKD1 E710D hotspot mutations, whereas the PAC that underwent high-grade transformation upon recurrence, which was wild-type for PRKD1, harboured a PRKD2 rearrangement. The PACs analysed here also harboured mutations targeting cancer genes such as PIK3CA, SETD2, ARID1A, and NOTCH2. A clonal decomposition analysis of the matching primary and recurrent PACs revealed that a minor subclone from the primary tumour became dominant in the recurrent tumour following a clonal selection evolutionary pattern. Conclusions Our findings demonstrate that recurrent and high-grade PACs are underpinned by PRKD1 E710D hotspot mutations or PRKD2 rearrangements, and that recurrences of PACs may stem from the selection of pre-existing subclones in the primary tumour.

Protein kinase D (PKD) consists of a family of three structurally related enzymes that are co-expressed in the heart and have important roles in many biological responses. PKD1 is activated by pro-hypertrophic stimuli and has been implicated in adverse cardiac remodeling. Efforts to define the cardiac actions of PKD2 and PKD3 have been less successful at least in part because conventional methods provide a general screen for PKD activation but are poorly suited to resolve activation patterns for PKD2 or PKD3. This study uses Phos-tag SDS-PAGE, a method that exaggerates phosphorylation-dependent mobility shifts, to overcome this technical limitation. Phos-tag SDS-PAGE resolves PKD1 as distinct molecular species (indicative of pools of enzyme with distinct phosphorylation profiles) in unstimulated cardiac fibroblasts and cardiomyocytes; as a result, attempts to track PKD1 mobility shifts that result from agonist activation were only moderately successful. In contrast, PKD2 and PKD3 are recovered from resting cardiac fibroblasts and cardiomyocytes as single molecular species; both enzymes display robust mobility shifts in Phos-tag SDS-PAGE in response to treatment with sphingosine-1-phosphate, thrombin, PDGF, or H2O2. Studies with GF109203X implicate protein kinase C activity in the stimulus-dependent pathways that activate PKD2/PKD3 in both cardiac fibroblasts and cardiomyocytes. Studies with C3 toxin identify a novel role for Rho in the sphingosine-1-phosphate and thrombin receptor-dependent pathways that lead to the phosphorylation of PKD2/3 and the downstream substrate CREB in cardiomyocytes. In conclusion, Phos-tag SDS-PAGE provides a general screen for stimulus-specific changes in PKD2 and PKD3 phosphorylation and exposes a novel role for these enzymes in specific stress-dependent pathways that influence cardiac remodeling.

We investigated the interaction network of human PKD2 in the cytosol and in Golgi-enriched subcellular protein fractions by an affinity enrichment strategy combined with chemical cross-linking/mass spectrometry (MS). Analysis of the subproteomes revealed the presence of distinct proteins in the cytosolic and Golgi fractions. The covalent fixation of transient or weak interactors by chemical cross-linking allowed capturing interaction partners that might otherwise disappear during conventional pull-down experiments. In total, 31 interaction partners were identified for PKD2, including glycogen synthase kinase-3 beta (GSK3B), 14-3-3 protein gamma (YWHAG), and the alpha isoform of 55 kDa regulatory subunit B of protein phosphatase 2A (PPP2R2A). Remarkably, the entire seven-subunit Arp2/3 complex (ARPC1B, ARPC2, ARPC3, ARPC4, ARPC5, ACTR3, ACTR2) as well as ARPC1A and ARPC5L, which are putative substitutes of ARPC1B and ARPC5, were identified. We provide evidence of a direct protein-protein interaction between PKD2 and Arp2/3. Our findings will pave the way for further structural and functional studies of PKD2 complexes, especially the PKD2/Arp2/3 interaction, to elucidate the role of PKD2 for transport processes at the trans-Golgi network. Data are available via ProteomeXchange with identifiers PXD003909 (enrichment from cytosolic fractions), PXD003913 (enrichment from Golgi fractions), and PXD003917 (subcellular fractionation).

Matrix metalloproteinas-9 (MMP-9) is a glycosylated endopeptidase associated with host reaction to microbial endotoxins and also characterizes gastric mucosal inflammatory response to H. pylori infection. Here, we report on the factors involved in gastric mucosal MMP-9 secretion in response to H. pylori LPS, and the effect of hormone, ghrelin. We show that both the LPS-elicited induction in MMP-9 secretion and also the modulatory influence of ghrelin occur at the level of MMP-9 processing between the endoplasmic reticulum (ER) and Golgi. Further, we demonstrate that the LPS effect is associated with up-regulation in the activation of Arf1, a small GTPase of the ADP-ribosylation factor family, and the recruitment and phosphorylation of protein kinase D2 (PKD2), involved in the secretory cargo processing in the Golgi. Moreover, we reveal that the LPS-induced up-regulation in MMP-9 secretion is reflected in a marked increase in PKCδ-mediated PKD2 phosphorylation on Ser, while the modulatory effect of ghrelin is manifested by the SFK-PTKs-dependent phosphorylation of PKD2 on Tyr. Thus, our findings demonstrate the role of Arf1/PKD2 in mediation of H. pylori LPS-induced up-regulation in gastric mucosal MMP-9 secretion and suggest the modulatory mechanism of ghrelin action.

Deficiency in polycystin 1 triggers specific changes in energy metabolism. To determine whether defects in other human cystoproteins have similar effects, we studied extracellular acidification and glucose metabolism in human embryonic kidney (HEK-293) cell lines with polycystic kidney and hepatic disease 1 ( PKHD1) and polycystic kidney disease (PKD) 2 ( PKD2) truncating defects along multiple sites of truncating mutations found in patients with autosomal recessive and dominant PKDs. While neither the PKHD1 or PKD2 gene mutations nor their position enhanced cell proliferation rate in our cell line models, truncating mutations in these genes progressively increased overall extracellular acidification over time ( P < 0.001 for PKHD1 and PKD2 mutations). PKHD1 mutations increased nonglycolytic acidification rate (1.19 vs. 1.03, P = 0.002), consistent with an increase in tricarboxylic acid cycle activity or breakdown of intracellular glycogen. In addition, they increased basal and ATP-linked oxygen consumption rates [7.59 vs. 5.42 ( P = 0.015) and 4.55 vs. 2.98 ( P = 0.004)]. The PKHD1 and PKD2 mutations also altered mitochondrial morphology, resembling the effects of polycystin 1 deficiency. Together, these data suggest that defects in major PKD genes trigger changes in mitochondrial energy metabolism. After validation in in vivo models, these initial observations would indicate potential benefits of targeting energy metabolism in the treatment of PKDs.

Background Studies have identified many common genetic associations that influence renal function and all-cause CKD, but these explain only a small fraction of variance in these traits. The contribution of rare variants has not been systematically examined. Methods We performed exome sequencing of 3150 individuals, who collectively encompassed diverse CKD subtypes, and 9563 controls. To detect causal genes and evaluate the contribution of rare variants we used collapsing analysis, in which we compared the proportion of cases and controls carrying rare variants per gene. Results The analyses captured five established monogenic causes of CKD: variants in PKD1 , PKD2 , and COL4A5 achieved study-wide significance, and we observed suggestive case enrichment for COL4A4 and COL4A3 . Beyond known disease-associated genes, collapsing analyses incorporating regional variant intolerance identified suggestive dominant signals in CPT2 and several other candidate genes. Biallelic mutations in CPT2 cause carnitine palmitoyltransferase II deficiency, sometimes associated with rhabdomyolysis and acute renal injury. Genetic modifier analysis among cases with APOL1 risk genotypes identified a suggestive signal in AHDC1 , implicated in Xia–Gibbs syndrome, which involves intellectual disability and other features. On the basis of the observed distribution of rare variants, we estimate that a two- to three-fold larger cohort would provide 80% power to implicate new genes for all-cause CKD. Conclusions This study demonstrates that rare-variant collapsing analyses can validate known genes and identify candidate genes and modifiers for kidney disease. In so doing, these findings provide a motivation for larger-scale investigation of rare-variant risk contributions across major clinical CKD categories.

Aurora A kinase (AURKA) is a master cell-cycle regulator that is often dysregulated in human cancers. Its overexpression has been associated with genome instability and oncogenic transformation. The protein kinase D (PKD) family is an emerging therapeutic target of cancer. Aberrant PKD activation has been implicated in tumor growth and survival, yet the underlying mechanisms remain to be elucidated. This study identified, for the first time, a functional crosstalk between PKD2 and Aurora A kinase in cancer cells. The data demonstrate that PKD2 is catalytically active during the G2–M phases of the cell cycle, and inactivation or depletion of PKD2 causes delay in mitotic entry due to downregulation of Aurora A, an effect that can be rescued by overexpression of Aurora A. Moreover, PKD2 localizes in the centrosome with Aurora A by binding to γ-tubulin. Knockdown of PKD2 caused defects in centrosome separation, elongated G2 phase, mitotic catastrophe, and eventually cell death via apoptosis. Mechanistically, PKD2 interferes with Fbxw7 function to protect Aurora A from ubiquitin- and proteasome-dependent degradation. Taken together, these results identify PKD as a cell-cycle checkpoint kinase that positively modulates G2–M transition through Aurora A kinase in mammalian cells. Implications: PKD2 is a novel cell-cycle regulator that promotes G2–M transition by modulating Aurora A kinase stability in cancer cells and suggests the PKD2/Aurora A kinase regulatory axis as new therapeutic targets for cancer treatment. Mol Cancer Res; 16(11); 1785–97. ©2018 AACR.

Although influenza A virus (IAV) evades cellular defense systems to effectively propagate in the host, the viral immune-evasive mechanisms are incompletely understood. Our recent data showed that hemagglutinin (HA) of IAV induces degradation of type I IFN receptor 1 (IFNAR1). Here, we demonstrate that IAV HA induces degradation of type II IFN (IFN-γ) receptor 1 (IFNGR1), as well as IFNAR1, via casein kinase 1α (CK1α), resulting in the impairment of cellular responsiveness to both type I and II IFNs. IAV infection or transient HA expression induced degradation of both IFNGR1 and IFNAR1, whereas HA gene-deficient IAV failed to downregulate the receptors. IAV HA caused the phosphorylation and ubiquitination of IFNGR1, leading to the lysosome-dependent degradation of IFNGR1. Influenza viral HA strongly decreased cellular sensitivity to type II IFNs, as it suppressed the activation of STAT1 and the induction of IFN-γ-stimulated genes in response to exogenously supplied recombinant IFN-γ. Importantly, CK1α, but not p38 MAP kinase or protein kinase D2, was proven to be critical for HA-induced degradation of both IFNGR1 and IFNAR1. Pharmacologic inhibition of CK1α or small interfering RNA (siRNA)-based knockdown of CK1α repressed the degradation processes of both IFNGR1 and IFNAR1 triggered by IAV infection. Further, CK1α was shown to be pivotal for proficient replication of IAV. Collectively, the results suggest that IAV HA induces degradation of IFN receptors via CK1α, creating conditions favorable for viral propagation. Therefore, the study uncovers a new immune-evasive pathway of influenza virus. IMPORTANCE Influenza A virus (IAV) remains a grave threat to humans, causing seasonal and pandemic influenza. Upon infection, innate and adaptive immunity, such as the interferon (IFN) response, is induced to protect hosts against IAV infection. However, IAV seems to be equipped with tactics to evade the IFN-mediated antiviral responses, although the detailed mechanisms need to be elucidated. In the present study, we show that IAV HA induces the degradation of the type II IFN receptor IFNGR1 and thereby substantially attenuates cellular responses to IFN-γ. Of note, a cellular kinase, casein kinase 1α (CK1α), is crucial for IAV HA-induced degradation of both IFNGR1 and IFNAR1. Accordingly, CK1α is proven to positively regulate IAV propagation. Thus, this study unveils a novel strategy employed by IAV to evade IFN-mediated antiviral activities. These findings may provide new insights into the interplay between IAV and host immunity to impact influenza virus pathogenicity.

Protein kinase D2 (PKD2) is a serine/threonine kinase that belongs to the PKD family of calcium-calmodulin kinases, which comprises three isoforms: PKD1, PKD2, and PKD3. PKD2 is activated by many stimuli including growth factors, phorbol esters, and G-protein-coupled receptor agonists. PKD2 participation to uncontrolled growth, survival, neovascularization, metastasis, and invasion has been documented in various tumor types including pancreatic, colorectal, gastric, hepatic, lung, prostate, and breast cancer, as well as glioma multiforme and leukemia. This review discusses the versatile functions of PKD2 from the perspective of cancer hallmarks as described by Hanahan and Weinberg. The PKD2 status, signaling pathways affected in different tumor types and the molecular mechanisms that lead to tumorigenesis and tumor progression are presented. The latest developments of small-molecule inhibitors selective for PKD/PKD2, as well as the need for further chemotherapies that prevent, slow down, or eliminate tumors are also discussed in this review. ispartof: Oncogene vol:37 issue:10 pages:1263-1278 ispartof: location:England status: published

Although protein kinase D (PKD) has been shown to contribute to invasion and metastasis in several types of cancer, the role of PKD in the epithelial mesenchymal transition (EMT) of hepatocellular carcinoma (HCC) has remained unclear. We found that PKD2 is up-regulated in HCC and is correlated with the metastasis of HCC. PKD2 positively regulated TNF-α-induced EMT and metastasis of HCC. Mechanistic studies revealed TNF-α-induced PKD2 activation is mediated by the formation of a TNFR1/TRAF2 complex. PKD2 bound directly to the p110α and p85 subunits of PI3K and promoted the PI3K/Akt/GSK-3β signaling cascade to stimulate EMT. In conclusion, our results have uncovered a novel role for the regulation of EMT and suggest inhibition of PKD2 as a potential therapeutic strategy for HCC.