Serine-threonine Research Articles

Abstract P1155: P62 Overexpression Rescues Defective Autophagy Flux In Cardiomyocytes Deficient In P21 Activated Kinase 1

P21-Activated Kinase 1 (PAK1) is a serine-threonine kinase that plays a critical role in cardiomyocyte survival under numerous stressful conditions. However, the underlying mechanisms for PAK1’s cardioprotective effects remain unclear. Autophagy is a lysosome-dependent degradation pathway for eliminating and recycling long-lived proteins and unwanted cellular components, which is essential for cellular homeostasis. In this study, we investigated whether and how PAK1 regulates autophagy in cardiomyocytes. PAK1 was either silenced by siRNA knockdown or overexpressed by adenovirus-mediated gene transfer in H9c2 cardiac myoblast cells. Autophagy flux was determined by measuring LC3-II protein levels or by a fluorescent autophagy reporter with and without the lysosomal protease inhibitors pepstatin A (pepA) and Aloxistatin (E64d). PAK1 downregulation reduced LC3-II levels in the total cell lysates, which were not affected by pepA and E64d, suggesting that knockdown of PAK1 inhibited autophagy flux. Inversely, PAK1 overexpression increased LC3-II levels, which were further elevated by pepA and E64d, suggesting that PAK1 was sufficient to accelerate autophagy flux. Interestingly, the expression of p62, a ubiquitin-binding autophagic adaptor, was concurrently upregulated by PAK1 overexpression or downregulated by PAK1 knockdown, suggesting that p62 may mediates PAK1-dependent autophagy. Indeed, overexpression of p62 restored autophagy flux in the absence of PAK1 (Figure 1). Together, these results suggest that PAK1 is both sufficient and necessary for maintaining normal autophagy function in cardiomyocytes, which is likely mediated by p62.

SPI1 involvement in malignant melanoma pathogenesis by regulation of HK2 through the AKT1/mTOR pathway.

Spi-1 proto-oncogene (SPI1) plays a vital role in carcinogenesis. Our work aimed to investigate the potential regulatory mechanism of SPI1 in melanoma. The mRNA and protein levels were measured via qRT-PCR and Western blotting. Cell viability was assessed by CCK-8 assay. The target relationship between SPI1 and hexokinase 2 (HK2) was determined using dual-luciferase reporter detection. ChIP was conducted to confirm the targeted relationship between SPI1 and the HK2 promoter. Immunohistochemistry analysis was conducted to measure the positive cell number of SPI1 and HK2 in melanoma tissues. The cell migration abilities were determined using a wound healing assay. Glucose consumption, pyruvate dehydrogenase activity, lactate production and ATP levels were measured to assess glycolysis. SPI1 transcription in melanoma cells and tissues was dramatically higher than that in adjacent normal tissues and epidermal melanocyte HEMa-LP, respectively. Knockdown of SPI1 restrained cell viability, metastasis and glycolysis in melanoma cells. SPI1 directly targeted HK2, and knockdown of SPI1 repressed HK2 expression. Overexpression of HK2 weakened the inhibitory effects of SPI1 knockdown on the viability, metastasis and glycolysis of melanoma cells. The serine-threonine kinase 1 (AKT1)/mammalian target of rapamycin (mTOR) axis is involved in melanoma progression. SPI1 knockdown restrained melanoma cell proliferation, metastasis and glycolysis by regulating the AKT1/mTOR pathway.

Abstract P2128: Insight Into Glycogen Synthase Kinase 3 Beta Localization Within The Heart: Role On Ischemia Reperfusion Injury

Glycogen synthase kinase 3 beta (GSK3β) is a cytosolic serine-threonine kinase first described for inhibiting glycogen synthase. GSK3β structure shows a small sequence that can work as a mitochondrial target sequence, however mitochondrial GSK3β (mtGSK3β) in the heart, and its role have not been investigated. It is known the enzyme can be activated under ischemia-reperfusion (I/R) injury and modulating mitochondrial permeability transition pore (MPTP) opening. In the nervous system, the truncation of GSK3β activates the enzyme, but in the heart, the truncation function remains unknown. We hypothesized that a small subset of GSK3β is truncated and translocated to the mitochondrial matrix, which might modulate MPTP opening . To test our hypothesis, we studied GSK3β expression by Western blot in mitochondrial fractions isolated from differential centrifugation. We found a percentage of GSK3β present in purified mitochondria (Pmito) truncated at the C-terminal side. To evaluate if mtGSK3β is involved in I/R injury, we study its expression in the fractions isolated from hearts subjected to I/R protocol. We found GSK3β is being truncated and its expression was increased in Pmito from I/R-hearts in comparison to normoxia-hearts (controls). To study the role of mtGSK3β in MPTP regulation we measure the sensitivity of the pore under a GSK3β inhibitor, SB216763 (SB21) by fluorescente of Fura-2-FF in a spectrofluorometer. MPTP Ca 2+ -sensitivity was reduced in crude mitochondria fraction (Cm, mitochondria with other organelles) and increased in Pmito under SB21 treatment. This data suggests mtGSK3β is inactive in Pmito and SB21 is inducing MPTP opening by another mechanism. Since GSK3β can be activated and inhibited by phosphorylation at T390 site (pT390) and S9 site (pS9), respectively, we measure it on mtGSK3β. We found pT390 is not present in Pmito likely missing during truncation, while pS9 is in a low amount. Moreover, pS9 increases on Pmito from hearts subjected to I/R protocol. All the data together showed that a small percentage of GSK3β is truncated and translocated within the mitochondrial matrix. However, this mtGSK3β is not involved in the modulation of MPTP opening, since the lack of a C-terminal side causes its inactivation.

Discovery and characterization of a specific inhibitor of serine-threonine kinase cyclin-dependent kinase-like 5 (CDKL5) demonstrates role in hippocampal CA1 physiology.

Pathological loss-of-function mutations in cyclin-dependent kinase-like 5 (CDKL5) cause CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental disorder associated with severe and medically refractory early-life epilepsy, motor, cognitive, visual, and autonomic disturbances in the absence of any structural brain pathology. Analysis of genetic variants in CDD has indicated that CDKL5 kinase function is central to disease pathology. CDKL5 encodes a serine-threonine kinase with significant homology to GSK3β, which has also been linked to synaptic function. Further, Cdkl5 knock-out rodents have increased GSK3β activity and often increased long-term potentiation (LTP). Thus, development of a specific CDKL5 inhibitor must be careful to exclude cross-talk with GSK3β activity. We synthesized and characterized specific, high-affinity inhibitors of CDKL5 that do not have detectable activity for GSK3β. These compounds are very soluble in water but blood-brain barrier penetration is low. In rat hippocampal brain slices, acute inhibition of CDKL5 selectively reduces postsynaptic function of AMPA-type glutamate receptors in a dose-dependent manner. Acute inhibition of CDKL5 reduces hippocampal LTP. These studies provide new tools and insights into the role of CDKL5 as a newly appreciated key kinase necessary for synaptic plasticity. Comparisons to rodent knock-out studies suggest that compensatory changes have limited the understanding of the roles of CDKL5 in synaptic physiology, plasticity, and human neuropathology.

An updated literature on BRAF inhibitors (2018-2023).

BRAF is the most common serine-threonine protein kinase and regulates signal transduction from RAS to MEK inside the cell. The BRAF is a highly active isoform of RAF kinase. BRAF has two domains such as regulatory and kinase domains. The BRAF inhibitors bind in the c-terminus of the kinase domain and inhibit the downstream pathways. The mutation occurs mainly in the A-loop of the kinase domain. The mutation occurs due to a conversion of valine to glutamate/lysine/arginine/aspartic acid at 600th position. Among the diverse mutations, BRAFV600E is the most common and responsible for numerous cancer such as melanoma, colorectal, ovarian, and thyroid cancer. Due to mutations in RAC1, loss of PTEN, NF1, CCND1, USP28-FBW7 complex, COT overexpression, and CCND1 amplification, the BRAF kinase enzyme developed resistance over the commercially available BRAF inhibitors. There is still unmute urgence for the development of BRAF inhibitors to overcome the persistent limitation such as resistance, mutation, and adverse effects of drugs. In the current study, we described the structure, activation, downstream signaling pathway, and mutation of BRAF. Our group also provided a detailed review of BRAF inhibitors from the last five years (2018-2023) highlighting the structure-activity relationship, mechanistic study, and molecular docking studies. We hope that the current analysis will be a useful resource for researchers and provide chemists a glimpse into the future as design and development of more effective and secure BRAF kinase inhibitors.

Discovery of potent and selective HPK1 inhibitors based on the 2,4-disubstituted pyrimidine scaffold with immune modulatory properties for ameliorating T cell exhaustion

Hematopoietic progenitor kinase 1 (HPK1), a member of the mitogen-activated protein kinase (MAP4K) family, is a serine/threonine (SER/THR) kinase and has been demonstrated as a negative regulator of T cell receptor signaling. Targeting HPK1 has been considered as an attractive therapeutic strategy for immune-oncology. Here, we describe the discovery and structure–activity relationship (SAR) of potent HPK1 inhibitors based on the 2,4-disubstituted pyrimidine scaffold. Systematically SAR exploration afforded the desired compound HMC-H8 (F1) with potent HPK1 inhibition (IC50 = 1.11 nM) and highly selectivity profile. Compound HMC-H8 also exhibited robust inhibition of p-SLP 76 (IC50 = 283.0 nM) and promotion IL-2 release (EC50 = 157.08 nM), and INF-γ production in a dose-dependent manner in vitro assays. Strikingly, HMC-H8 shown effective immune reversal response in immunesuppressive condition. Moreover, Compound HMC-H8 displayed acceptable metabolic stability (T1/2 = 56.87 min), along with low CYP450 inhibition in human liver microsomes and good oral bioavailability (F = 15.05%) in rat. Furthermore, HMC-H8 was found to modulate the expression of c-Myc in Western blotting experiments. Taken together, this study provides new potent HPK1 inhibitors for further anticancer drug discovery based on immuno-oncology.

HM-chromanone isolated from Portulaca oleracea L. alleviates insulin resistance and inhibits gluconeogenesis by regulating palmitate-induced activation of ROS/JNK in HepG2 cells.

Oxidative stress is a major cause of hepatic insulin resistance. This study investigated whether (E)-5-hydroxy-7-methoxy-3-(2-hydroxybenzyl)-4-chromanone (HM-chromanone), a homoisoflavonoid compound isolated from Portulaca oleracea L., alleviates insulin resistance and inhibits gluconeogenesis by reducing palmitate (PA)-induced reactive oxygen species (ROS)/c-Jun NH2-terminal kinase (JNK) activation in HepG2 cells. PA treatment (0.5mM) for 16h resulted in the highest production of ROS and induced insulin resistance in HepG2 cells. HM-chromanone, like N-acetyl-1-cysteine, significantly decreased PA-induced ROS production in the cells. HM-chromanone also significantly inhibited PA-induced JNK activation, showing a significant reduction in tumor necrosis factor and interleukin expression levels. Thus, HM-chromanone decreased the phosphorylation of Ser307 in insulin receptor substrate 1, while increasing phosphorylation of serine-threonine kinase (AKT), thereby restoring the insulin signaling pathway impaired by PA. HM-chromanone also significantly increased the phosphorylation of forkhead box protein O, thereby inhibiting the expression of gluconeogenic enzymes and reducing glucose production in PA-treated HepG2 cells. HM-chromanone also increased glycogen synthesis by phosphorylating glycogen synthase kinase-3β. Therefore, HM-chromanone may alleviate insulin resistance and inhibit gluconeogenesis by regulating PA-induced ROS/JNK activation in HepG2 cells.

DYRK1A-mediated PLK2 phosphorylation regulates the proliferation and invasion of glioblastoma cells.

Polo-like kinases (PLKs) are a family of serine-threonine kinases that exert regulatory effects on diverse cellular processes. Dysregulation of PLKs has been implicated in multiple cancers, including glioblastoma (GBM). Notably, PLK2 expression in GBM tumor tissue is lower than that in normal brains. Notably, high PLK2 expression is significantly correlated with poor prognosis. Thus, it can be inferred that PLK2 expression alone may not be sufficient for accurate prognosis evaluation, and there are unknown mechanisms underlying PLK2 regulation. In the present study, it was demonstrated that dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) interacts with and phosphorylates PLK2 at Ser358. DYRK1A-mediated phosphorylation of PLK2 increases its protein stability. Moreover, PLK2 kinase activity was markedly induced by DYRK1A, which was exemplified by the upregulation of alpha-synuclein S129 phosphorylation. Furthermore, it was found that phosphorylation of PLK2 by DYRK1A contributes to the proliferation, migration and invasion of GBM cells. DYRK1A further enhances the inhibition of the malignancy of GBM cells already induced by PLK2. The findings of the present study indicate that PLK2 may play a crucial role in GBM pathogenesis partially in a DYRK1A-dependent manner, suggesting that PLK2 Ser358 may serve as a therapeutic target for GBM.

Loss of miR-144/451 alleviates β-thalassemia by stimulating ULK1-mediated autophagy of free α-globin

AbstractMost cells can eliminate unstable or misfolded proteins through quality control mechanisms. In the inherited red blood cell disorder β-thalassemia, mutations in the β-globin gene (HBB) lead to a reduction in the corresponding protein and the accumulation of cytotoxic free α-globin, which causes maturation arrest and apoptosis of erythroid precursors and reductions in the lifespan of circulating red blood cells. We showed previously that excess α-globin is eliminated by Unc-51–like autophagy activating kinase 1 (ULK1)-dependent autophagy and that stimulating this pathway by systemic mammalian target of rapamycin complex 1 (mTORC1) inhibition alleviates β-thalassemia pathologies. We show here that disrupting the bicistronic microRNA gene miR-144/451 alleviates β-thalassemia by reducing mTORC1 activity and stimulating ULK1-mediated autophagy of free α-globin through 2 mechanisms. Loss of miR-451 upregulated its target messenger RNA, Cab39, which encodes a cofactor for LKB1, a serine-threonine kinase that phosphorylates and activates the central metabolic sensor adenosine monophosphate–activated protein kinase (AMPK). The resultant enhancement of LKB1 activity stimulated AMPK and its downstream effects, including repression of mTORC1 and direct activation of ULK1. In addition, loss of miR-144/451 inhibited the expression of erythroblast transferrin receptor 1, causing intracellular iron restriction, which has been shown to inhibit mTORC1, reduce free α-globin precipitates, and improve hematological indices in β-thalassemia. The beneficial effects of miR-144/451 loss in β-thalassemia were inhibited by the disruption of Cab39 or Ulk1 genes. Together, our findings link the severity of β-thalassemia to a highly expressed erythroid microRNA locus and a fundamental, metabolically regulated protein quality control pathway that is amenable to therapeutic manipulation.

Calcium/Calmodulin-Dependent Serine Protein Kinase (CASK) Gene Polymorphisms in Pigeons

Calcium/calmodulin-dependent serine protein kinase (CASK) is an multidomain protein involved in tissue development and cell signalling. In skeletal muscle, it is involved in the development of neuromuscular junctions. The participation of a pigeon in racing is a great physical effort that causes many changes in the skeletal muscles. Thus, the purpose of the study was to detect the nucleotide sequence variability in the calcium/calmodulin-dependent serine kinase (CASK) gene in domestic pigeons (Columba livia domestica) and assess the potential impact of DNA polymorphisms on the flight performance of pigeons. The research included a total of 517 individuals. DNA was extracted from the blood. A DNA fragment from nucleotides 8689 to 9049 of the CASK (NW_004973256.1 sequence) of six unrelated pigeons were sequenced. One of the detected polymorphic sites (g.8893G > A), located a very close to the start codon, was selected for genotyping in all individuals. The association studies included a total of 311 young homing pigeons that participated in racing competitions. The homing pigeons showed higher frequencies of the AA genotype than non-homing ones (p < 0.05). In rock pigeons only the GG genotype was found. Further research could confirm the functionality of the CASK g.8893G > A SNP in shaping the racing phenotype of pigeons, and the AA genotype could be useful as a selection criterion in pigeon breeding.

Capturing conformational transitions of full-length PDK1 that dictate kinase substrate selectivity.

PDK1 is a constitutively active master kinase that can phosphorylate and activate as many as 24 enzymes, all belonging to the AGC family of serine-threonine protein kinases. In this issue of Science Signaling, Sacerdoti etal. uncover how allosteric communication between different functional domains directs the selectivity of PDK1 toward particular subsets of substrates.

STRAP binds to and promotes the repair of N1-methyldeoxyadenosine in DNA

N1-methyladenosine (m1A) is an important RNA modification that functions in various biological processes by interacting with cellular proteins. However, the binding proteins of N1-methyldeoxyadenosine (1mdA) in DNA remain largely unknown. Herein, we employed a quantitative proteomics strategy to identify the potential binding proteins of 1mdA in human cells. Our results revealed that serine‑threonine kinase receptor-associated protein (STRAP) can bind to 1mdA-carrying DNA. We further demonstrated that STRAP participates in alkylating agent-induced DNA damage response and can promote the repair of 1mdA embedded in DNA. Moreover, we investigated the effects of STRAP on 1mdA-induced perturbation in transcription using a shuttle vector- and next-generation sequencing-based assay, and found that STRAP is involved in the transcriptional bypass of 1mdA in human cells. Together, our study revealed STRAP as a novel 1mdA-binding protein in human cells and provided new insight into the biological implications of STRAP and 1mdA modification in human diseases.

Adipose tissue insulin resistance exacerbates liver inflammation and fibrosis in a diet-induced NASH model.

Insulin regulates various biological processes in adipocytes, and adipose tissue dysfunction due to insulin resistance in this tissue plays a central role in the development of metabolic diseases, including NAFLD and NASH. However, the combined impact of adipose tissue insulin resistance and dietary factors on the pathogenesis of NAFLD-NASH has remained unknown. 3'-phosphoinositide-dependent kinase 1 (PDK1) is a serine-threonine protein kinase that mediates the metabolic actions of insulin. We recently showed that adipocyte-specific PDK1 knockout (A-PDK1KO) mice maintained on normal chow exhibit metabolic disorders, including progressive liver disease leading to NASH, in addition to reduced adipose tissue mass. We here show that maintenance of A-PDK1KO mice on the Gubra amylin NASH (GAN) diet rich in saturated fat, cholesterol, and fructose exacerbates inflammation and fibrosis in the liver. Consistent with these histological findings, RNA-sequencing analysis of the liver showed that the expression of genes related to inflammation and fibrosis was additively upregulated by adipocyte-specific PDK1 ablation and the GAN diet. Of note, the reduced adipose tissue mass of A-PDK1KO mice was not affected by the GAN diet. Our results thus indicate that adipose tissue insulin resistance and the GAN diet additively promote inflammation and fibrosis in the liver of mice. A-PDK1KO mice fed with the GAN diet, constitute a new mouse model for studies of the pathogenesis of NAFLD-NASH, especially that in lean individuals, as well as for the development of potential therapeutic strategies for this disease.

Structure-guided discovery of adenosine triphosphate-competitive casein kinase 2 inhibitors.

Casein kinase 2 (CK2) is a ubiquitous, highly pleiotropic serine-threonine kinase. CK2 has been identified as a potential drug target for the treatment of cancer and related disorders. Several adenosine triphosphate-competitive CK2 inhibitors have been identified and have progressed at different levels of clinical trials. This review presents details of CK2 protein, structural insights into adenosine triphosphate binding pocket, current clinical trial candidates and their analogues. Further, it includes the emerging structure-based drug design approaches, chemistry, structure-activity relationship and biological screening of potent and selective CK2 inhibitors. The authors tabulated the details of CK2 co-crystal structures because these co-crystal structures facilitated the structure-guided discovery of CK2 inhibitors. The narrow hinge pocket compared with related kinases provides useful insights into the discovery of CK2 inhibitors.

STK11 alterations as predictive biomarkers of resistance to immune checkpoint inhibitors in multiple cancers with mismatch repair gene alterations.

2619 Background: STK11 is an important tumor suppressor gene that encodes for a serine-threonine kinase involved in a variety of metabolic functions. STK11 alterations are associated with an inert or cold tumor microenvironment in KRAS-mutant lung adenocarcinoma, and are a major driver of primary resistance to immune checkpoint inhibitors (ICIs). Here, we explored STK11 alterations as predictive biomarkers of resistance to ICIs in multiple solid tumors with mismatch repair (MMR) gene alterations. Methods: The cBioCancer Genomics Portal (cBioportal, http://www.cbioportal.org ) was used to obtain genomic data. The association between STK11 alterations and median overall survival (mOS) was explored in an ICI-treatment cohort comprising a total of 1572 evaluable patients. This cohort includes patients with cancers of the lung, colon, unknown primary, head and neck, gastroesophageal, bladder, skin (melanoma), brain, breast and kidney that had received at least one dose of ICI. These were then cross-referenced with patients who had MSK-IMPACT testing done in the context of routine clinical care. Kaplan-Meier survival curves were generated and compared using log-rank testing. Results: In the ICI-treated patients cohort (N = 1572), 96 patients (6.1%) had STK11 alterations. Patients with STK11 alterations had higher median tumor mutation burden (TMB) than patients without STK11 alterations (8.81 mut/Mb vs. 5.87 mut/Mb, p < 0.0001). Yet, they had worse mOS (7 m, 95% CI: 6-13 m vs. 20 m, 95% CI: 18-22 m, p < 0.001). MMR alterations (not including gene promoter methylation) were identified in 103 (6.6%) and co-occurrence of STK11 with MMR alterations was identified in 9 (0.6%) patients. Compared to patients without MMR or STK11 alterations (N = 1382), the mOS was better in patients with MMR alterations without STK11 alterations (N = 94) (59 m vs. 19 m, p = 0.0004) and trending for worse in patients with MMR and STK11 alterations (N = 9) (5 m vs. 19 m, p = 0.117). The mOS was better in patients with MMR alterations without STK11 alterations compared to patients with MMR and STK11 alterations (59 m vs 5 m, p = 0.016). Controlling for KRAS mutation status, a multivariable Cox regression model shows that besides MMR and STK11 alteration status (p = 0.0008), BRAF mutant status (p = 0.0001, HR 0.598; 95% CI, 0.459-0.779) was another biomarker associated with significantly better OS. Lung cancer diagnosis was associated with worse OS (p = 0.0016, HR 1.340; 95% CI, 1.117-1.607). Conclusions: In a large cohort of ICI-treated patients with multiple cancers, STK11 alterations were associated with worse mOS despite having higher TMB. MMR alterations were only predictive of response to ICIs in patients without STK11 alterations. Our findings suggest that STK11 alterations in patients with MMR alterations could potentially identify patients who are less likely to respond to ICIs but more data are needed.

The photosensory function of Zmphot1 differs from that of Atphot1 due to the C-terminus of Zmphot1 during phototropic response

The role of phot1 in triggering hypocotyl phototropism and optimizing growth orientation has been well-characterized in Arabidopsis, whereas the role of Zmphot1 in maize remains largely unclear. Here, we show that Zmphot1 is involved in blue light-induced phototropism. Compared with Atphot1, Zmphot1 exhibited a weaker phototropic response to very low-fluence rates of blue light (< 0.01 μmol m−2 s−1), but stronger phototropic response to high-fluence rates of blue light (> 10 μmol m−2 s−1) than Atphot1. Notably, blue light exposure induced Zmphot1-green fluorescent protein (GFP), but not Atphot1-GFP, to form the aggregates in the cytoplasm of Nicotiana benthamiana cells. Furthermore, by generating the chimeric phot1 proteins, we found that the serine-threonine kinase (STK) domain at the C-terminus is responsible for a more volatile membrane association of Zmphot1. Consistently, the chimeric phot1 protein fusing the STK domain of Zmphot1 with other domains of Atphot1 responded similarly as Zmphot1 to both low and high fluence rates of blue light. Interestingly, although both Zmphot1 and Atphot1 interact with AtNPH3, Zmphot1 induced weaker dephosphorylation of NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) than Atphot1. Together, our findings indicate that Zmphot1 and Atphot1 exhibit different photosensory function during phototropic response and that the STK domain may play a key role in determining their properties.

TMEM16A partners with mTOR to influence pathways of cell survival, proliferation, and migration in cholangiocarcinoma.

Expression of transmembrane protein 16 A (TMEM16A), a calcium activated chloride channel, is elevated in some human cancers and impacts tumor cell proliferation, metastasis, and patient outcome. Evidence presented here uncovers a molecular synergy between TMEM16A and mechanistic/mammalian target of rapamycin (mTOR), a serine-threonine kinase that is known to promote cell survival and proliferation in cholangiocarcinoma (CCA), a lethal cancer of the secretory cells of bile ducts. Analysis of gene and protein expression in human CCA tissue and CCA cell line detected elevated TMEM16A expression and Cl- channel activity. The Cl- channel activity of TMEM16A impacted the actin cytoskeleton and the ability of cells to survive, proliferate, and migrate as revealed by pharmacological inhibition studies. The basal activity of mTOR, too, was elevated in the CCA cell line compared with the normal cholangiocytes. Molecular inhibition studies provided further evidence that TMEM16A and mTOR were each able to influence the regulation of the other's activity or expression respectively. Consistent with this reciprocal regulation, combined TMEM16A and mTOR inhibition produced a greater loss of CCA cell survival and migration than their individual inhibition alone. Together these data reveal that the aberrant TMEM16A expression and cooperation with mTOR contribute to a certain advantage in CCA.NEW & NOTEWORTHY This study points to the dysregulation of transmembrane protein 16 A (TMEM16A) expression and activity in cholangiocarcinoma (CCA), the inhibition of which has functional consequences. Dysregulated TMEM16A exerts an influence on the regulation of mechanistic/mammalian target of rapamycin (mTOR) activity. Moreover, the reciprocal regulation of TMEM16A by mTOR demonstrates a novel connection between these two protein families. These findings support a model in which TMEM16A intersects the mTOR pathway to regulate cell cytoskeleton, survival, proliferation, and migration in CCA.

SpkH (Sll0005) from Synechocystis sp. PCC 6803 is an active Mn2+-dependent Ser kinase

Twelve genes for the potential serine-threonine protein kinases (STPKs) have been annotated in the genome of Synechocystis sp. PCC 6803. Based on similarities and distinctive domain organization, they were divided into two clusters: serine/threonine-protein N2-like kinases (PKN2-type) and “activity of bc1 complex” kinases (ABC1-type). While the activity of the PKN2-type kinases have been demonstrated, no ABC1-type kinases activity have hitherto been reported. In this study, a recombinant protein previously annotated as a potential STPK of ABC1-type (SpkH, Sll0005) was expressed and purified to homogeneity. We demonstrated SpkH phosphorylating activity and substrate preference for casein in in vitro assays using [γ-32P]ATP. Detailed analyses of activity showed that Mn2+ had the strongest activation effect. The activity of SpkH was significantly inhibited by heparin and spermine, but not by staurosporine. By means of semi-quantitative mass-spectrometric detection of phosphopeptides, we identified a consensus motif recognized by this kinase – X1X2pSX3E. Thus, we first report here that SpkH of Synechocystis represents a true active serine protein kinase, which shares the properties of casein kinases according to its substrate specificity and sensitivity to some activity effectors.

Inhibition of extranodal NK/T-cell lymphoma by Chiauranib through an AIF-dependent pathway and its synergy with L-asparaginase

Extranodal NK/T-cell lymphoma (NKTL) is a rare and aggressive form of extranodal lymphoma with a poor prognosis. Currently, there are very limited treatment options for patients with advanced-stage disease or those with relapsed/recurrent disease. Here we show that Chiauranib, an orally small molecule inhibitor of select serine-threonine kinases (aurora B, VEGFRs, PDGFR, CSF1R, c-Kit), inhibited NKTL cell proliferation, induced cell cycle arrest, as well as suppressed the microvessel density in vitro and in vivo similar as in other types of cancer cells. Surprisingly, Chiauranib unfolded a new effect to induce apoptosis of NKTL cells by triggering AIF-dependent apoptosis other than the traditional cyt-c/caspase mitochondrial apoptosis pathway. The knockdown of AIF in vitro and in vivo dramatically blocked the efficacy of Chiauranib on NKTL. Mechanistically, the release of AIF from mitochondria is due to the upregulation of VDAC1 by the AKT-GSK3β pathway and activation of calcium-dependent m-calpain, which promotes the cleavage of VDAC1 and therefore permits the release of AIF. Notably, the low expression of Bax in both NKTL cells and patient tissues restrained the cyt-c release. It resulted in the inhibition of cyt-c/caspase mitochondrial pathway, suggesting that drugs targeting this traditional pathway may not be effective in NKTL. Furthermore, we found that L-asparaginase triggered CD95 (Fas/Apo-1)-caspase 8-caspase 3 apoptotic pathway in NKTL cells, and combination of Chiauranib and L-asparaginase exhibited a synergistic effect, suggesting a feasibility to combine these two drugs for effective treatment of NKTL. This study demonstrates Chiauranib’s positive efficacy toward NKTL through the activation of the AIF-dependent apoptosis pathway for the first time. The novel and multi-targets of Chiauranib and the synergistic effect with L-asparaginase may provide a promising therapy for NKTL patients.

The dark kinase STK32A regulates hair cell planar polarity opposite of EMX2 in the developing mouse inner ear.

The vestibular maculae of the inner ear contain sensory receptor hair cells that detect linear acceleration and contribute to equilibrioception to coordinate posture and ambulatory movements. These hair cells are divided between two groups, separated by a line of polarity reversal (LPR), with oppositely oriented planar-polarized stereociliary bundles that detect motion in opposite directions. The transcription factor EMX2 is known to establish this planar polarized organization in mouse by regulating the distribution of the transmembrane receptor GPR156 at hair cell boundaries in one group of cells. However, the genes regulated by EMX2 in this context were previously not known. Using mouse as a model, we have identified the serine threonine kinase STK32A as a downstream effector negatively regulated by EMX2. Stk32a is expressed in hair cells on one side of the LPR in a pattern complementary to Emx2 expression in hair cells on the opposite side. Stk32a is necessary to align the intrinsic polarity of the bundle with the core planar cell polarity (PCP) proteins in EMX2-negative regions, and is sufficient to reorient bundles when ectopically expressed in neighboring EMX2-positive regions. We demonstrate that STK32A reinforces LPR formation by regulating the apical localization of GPR156. These observations support a model in which bundle orientation is determined through separate mechanisms in hair cells on opposite sides of the maculae, with EMX2-mediated repression of Stk32a determining the final position of the LPR.