Protein Kinase Family Research Articles

Testis-specific serine kinase protein family in male fertility and as targets for non-hormonal male contraception†.

Male contraception is a very active area of research. Several hormonal agents have entered clinical trials, while potential non-hormonal targets have been brought to light more recently and are at earlier stages of development. The general strategy is to target genes along the molecular pathways of sperm production, maturation, or function, and it is predicted that these novel approaches will hopefully lead to more selective male contraceptive compounds with a decreased side effect burden. Protein kinases are known to play a major role in signaling events associated with sperm differentiation and function. In this review, we focus our analysis on the testis-specific serine kinase (TSSK) protein family. We have previously shown that members of the family of TSSKs are postmeiotically expressed in male germ cells and in mature mammalian sperm. The restricted postmeiotic expression of TSSKs as well as the importance of phosphorylation in signaling processes strongly suggests that TSSKs have an important role in germ cell differentiation and/or sperm function. This prediction has been supported by the reported sterile phenotype of the Tssk6 knockout (KO) mice and of the double Tssk1 and Tssk2 KO mice and by the male subfertile phenotype observed in a Tssk4 KO mouse model.

Lack of skeletal muscle liver kinase B1 alters gene expression, mitochondrial content, inflammation and oxidative stress without affecting high-fat diet-induced obesity or insulin resistance

Ad libitum high-fat diet (HFD) induces obesity and skeletal muscle metabolic dysfunction. Liver kinase B1 (LKB1) regulates skeletal muscle metabolism by controlling the AMP-activated protein kinase family, but its importance in regulating muscle gene expression and glucose tolerance in obese mice has not been established. The purpose of this study was to determine how the lack of LKB1 in skeletal muscle (KO) affects gene expression and glucose tolerance in HFD-fed, obese mice.KO and littermate control wild-type (WT) mice were fed a standard diet or HFD for 14 weeks. RNA sequencing, and subsequent analysis were performed to assess mitochondrial content and respiration, inflammatory status, glucose and insulin tolerance, and muscle anabolic signaling.KO did not affect body weight gain on HFD, but heavily impacted mitochondria-, oxidative stress-, and inflammation-related gene expression. Accordingly, mitochondrial protein content and respiration were suppressed while inflammatory signaling and markers of oxidative stress were elevated in obese KO muscles. KO did not affect glucose or insulin tolerance. However, fasting serum insulin and skeletal muscle insulin signaling were higher in the KO mice. Furthermore, decreased muscle fiber size in skmLKB1-KO mice was associated with increased general protein ubiquitination and increased expression of several ubiquitin ligases, but not muscle ring finger 1 or atrogin-1. Taken together, these data suggest that the lack of LKB1 in skeletal muscle does not exacerbate obesity or insulin resistance in mice on a HFD, despite impaired mitochondrial content and function and elevated inflammatory signaling and oxidative stress.

Screening and analysis of bioactive food compounds for modulating the CDK2 protein for cell cycle arrest: Multi-cheminformatics approaches for anticancer therapeutics

The cyclin-dependent kinase-2 (CDK2) belongs to the protein kinase family and its overexpression leads to an unusual regulation of cell-cycle which directly linked with hyperproliferation in many cancer cell types. CDK2 activation spontaneously promotes the cell cycle progression and also involved in a large number of cellular processes including cell cycle regulation, DNA replication, DNA damage response and apoptotic pathways, therefore targeting the CDK2 can be reemerged as a therapeutic boulevard to restrain cancer cell proliferation. For the last two decades, emerging evidences suggested that CDK2 inhibition draws out some antitumor/anticancer activity, which has driven the research possibility for developing next-generation newer or cost-effective inhibitors with greater specificity to CDK2. In the current work, compounds from the FooDB - a world’s largest food constituents database was retrieved and curated and followed by multi-pharmacoinformatics approaches adopted to find out potential CDK2 inhibitors. The curated dataset was considered for screening through “Virtual Screening Workflow” (VSW) employed in Schrödinger suite. The numbers of cost-effective food constituents were reduced by removing low potential molecules in terms of interaction affinity and further explored for pharmacokinetics analysis. Based on strong binding interaction profiles with the lowest binding interactions affinity and energy values, four food compounds were proposed as CDK2 inhibitors. A number of key analyzing parameters from molecular dynamics (MD) simulations studies were successfully substantiated that all four proposed food compounds can act as CDK2 inhibitors based on their proficient structural and molecular interactions integrity with CDK2 protein following in the active site cavity. Furthermore, the binding free energy was calculated using the MM-PBSA (Molecular Mechanics Poisson-Boltzmann Surface Area) approach from the entire trajectory frames derived in MD simulation revealed strong interaction affinity. The binding free energy was found to be in the range of −991.831 to −210.452 kJ/mol. High binding free energy was undoubtedly explained that all molecules possess strong affection towards CDK2. Hence, proposed molecules may be crucial to stop the hyperproliferation in cancer cells subjected to experimental validation.

De Novo Variants in CDK19 Are Associated with a Syndrome Involving Intellectual Disability and Epileptic Encephalopathy

We identified three unrelated individuals with de novo missense variants in CDK19, encoding a cyclin-dependent kinase protein family member that predominantly regulates gene transcription. These individuals presented with hypotonia, global developmental delay, epileptic encephalopathy, and dysmorphic features. CDK19 is conserved between vertebrate and invertebrate model organisms, but currently abnormalities in CDK19 are not known to be associated with a human disorder. Loss of Cdk8, the fly homolog of CDK19, causes larval lethality, which is suppressed by expression of human CDK19 reference cDNA. In contrast, the CDK19 p.Tyr32His and p.Thr196Ala variants identified in the affected individuals fail to rescue the loss of Cdk8 and behave as null alleles. Additionally, neuronal RNAi-mediated knockdown of Cdk8 in flies results in semi-lethality. The few eclosing flies exhibit severe seizures and a reduced lifespan. Both phenotypes are fully suppressed by moderate expression of the CDK19 reference cDNA but not by expression of the two variants. Finally, loss of Cdk8 causes an obvious loss of boutons and synapses at larval neuromuscular junctions (NMJs). Together, our findings demonstrate that human CDK19 fully replaces the function of Cdk8 in the fly, the human disease-associated CDK19 variants behave as strong loss-of-function variants, and deleterious CDK19 variants underlie a syndromic neurodevelopmental disorder.

Knockdown of ALPK2 blocks development and progression of renal cell carcinoma

Renal cell carcinoma (RCC) is one of the most common malignant tumors in the urinary system, whose molecular mechanism is still not clear. ALPK2 is a member of alpha protein kinase family, and its relationship with RCC is never reported. In this study, expression of ALPK2 in tumor tissues or cells of RCC was detected by qPCR, western blotting and immunohistochemical analysis. The effects of ALPK2 knockdown on cell proliferation, colony formation, cell migration and apoptosis were assessed by MTT, colony formation assay, wound-healing assay, Transwell assay and flow cytometry, respectively. The influence of ALPK2 knockdown on tumor growth in vivo was evaluated by mice xenograft models. The results demonstrated that ALPK2 was upregulated in tumor tissues of RCC and its high expression was significantly associated with advanced stage and poor prognosis. Knockdown of ALPK2 could inhibited cell proliferation, colony formation and cell migration of RCC cells, while promoting cell apoptosis. The suppression of tumor growth in vivo by ALPK2 knockdown was also showed by using mice xenograft models. Moreover, the regulation of RCC by ALPK2 may involve Akt, CDK6, Cyclin D1 and PIK3CA signaling. Therefore, our studies suggested that ALPK2 may act as a tumor promotor in the development and progression of RCC, and could be considered as a novel therapeutic target for RCC treatment.

Molecular cloning and expression of AmCDPK from mangrove Avicennia marina under elevated temperature.

Considered as an essential calcium sensor, the calcium-dependent protein kinase (CDPK) family plays a critical part in terrestrial plants' responses to both biotic and abiotic stresses. In the study, Avicennia marina was proved to have better heat tolerance than other species. A CDPK gene was cloned from mangrove species A. marina using RACE-PCR and designated as AmCDPK. By predicting and analyzing its properties, structures and expression patterns, we found that the amino acid sequence, containing a kinase domain and four EF-hand Ca2+-binding sites, shared high identity with Handroanthus impetiginosus and Sesamum indicum. Quantitative real-time PCR data analysis suggested that AmCDPK demonstrated significant up-regulation under heat stress. It is likely that AmCDPK is a versatile gene involved in various stresses, including dehydration, cold, light, defense and ABA stress responses by analyzing cis-elements. It is the first time that CDPKs from mangroves have been cloned and our results brought evidence to the effect of AmCDPK on heat stress, which is particularly important under the background of global warming.

JNK Signaling in Stem Cell Self-Renewal and Differentiation.

C-JUN N-terminal kinases (JNKs), which belong to the mitogen-activated protein kinase (MAPK) family, are evolutionarily conserved kinases that mediate cell responses to various types of extracellular stress insults. They regulate physiological processes such as embryonic development and tissue regeneration, playing roles in cell proliferation and programmed cell death. JNK signaling is also involved in tumorigenesis and progression of several types of malignancies. Recent studies have shown that JNK signaling has crucial roles in regulating the traits of cancer stem cells (CSCs). Here we describe the functions of the JNK signaling pathway in self-renewal and differentiation, which are essential features of various types of stem cells, such as embryonic, induced pluripotent, and adult tissue-specific stem cells. We also review current knowledge of JNK signaling in CSCs and discuss its role in maintaining the CSC phenotype. A better understanding of JNK signaling as an essential regulator of stemness may provide a basis for the development of regenerative medicine and new therapeutic strategies against malignant tumors.

Protective role of endothelial calpain knockout in lipopolysaccharide-induced acute kidney injury via attenuation of the p38-iNOS pathway and NO/ROS production

To explore the role of calpain and its signaling pathway in lipopolysaccharide (LPS)-induced acute kidney injury (AKI), animal models of endotoxemia were established by administration of LPS to mice with endothelial-specific Capn4 knockout (TEK/Capn4−/−), mice with calpastatin (an endogenous calpain inhibitor) overexpression (Tg-CAST) and mice with myeloid-specific Capn4 knockout (LYZ/Capn4−/−). Mouse pulmonary microvascular endothelial cells (PMECs) were used as a model of the microvascular endothelium and were stimulated with LPS. Renal function, renal inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) expression, cellular apoptosis, plasma and renal levels of NO and reactive oxygen species (ROS), and phosphorylation of mitogen-activated protein kinase (MAPK) family members (p38, ERK1/2, and JNK1/2) were examined. Moreover, a calpain inhibitor, calpastatin overexpression adenoviruses and MAPK inhibitors were used. Significant renal dysfunction was induced by LPS stimulation, and recovery was observed in TEK/Capn4−/− and Tg-CAST mice but not in LYZ/Capn4−/− mice. Endothelial Capn4 knockout also abrogated the LPS-induced increases in renal iNOS expression, caspase-3 activity and apoptosis and plasma and renal NO and ROS levels but did not obviously affect renal eNOS expression. Moreover, LPS increased both calpain and caspase-3 activity, and only the expression of iNOS in PMECs was accompanied by increased phosphorylation of p38 and JNK. Inhibiting calpain activity or p38 phosphorylation alleviated the increased iNOS expression, NO/ROS production, and cellular apoptosis induced by LPS. These results suggest that endothelial calpain plays a protective role in LPS-induced AKI by inhibiting p38 phosphorylation, thus attenuating iNOS expression and further decreasing NO and ROS overproduction-induced endothelial apoptosis.

Catalytic Subunit of PKA as a Prototype of the Eukaryotic Protein Kinase Family.

The catalytic subunit of protein kinase A (PKAc) is conserved in all eukaryotic protein kinases. PKAc consists of two lobes that form the catalytic cleft containing the ATP-binding, peptide-binding site, and catalytic sites. During folding, PKAc secondary structures organize so that the non-polar regions form a globular core, while mobile loops and tails are exposed and can act as regulatory elements. De novo synthesized PKAc is phosphorylated at the T-loop, resulting in the formation of the active center capable of high-affinity binding of co-substrates. The ATP-molecule "sticks" the two lobes together, whereas the binding of peptide substrate completes the active center formation. The resulting catalytic triad (γ-phosphate of ATP, hydroxyl of Ser/Thr residue of the protein substrate, and Asp166 carboxyl) occupies a position optimal for catalysis. During the catalytic cycle, dynamic reorganization of polar and hydrophobic interactions ensures PKAc transition from the open to the closed conformation and vice versa. Understanding the structural basis of functioning of eukaryotic protein kinases (ePKs) is essential for successful design of ePK modulators.

An atypical DYRK kinase connects quorum-sensing with posttranscriptional gene regulation in Trypanosoma brucei.

The sleeping sickness parasite, Trypanosoma brucei, uses quorum sensing (QS) to balance proliferation and transmission potential in the mammal bloodstream. A signal transduction cascade regulates this process, a component of which is a divergent member of the DYRK family of protein kinases, TbDYRK. Phylogenetic and mutational analysis in combination with activity and phenotypic assays revealed that TbDYRK exhibits a pre-activated conformation and an atypical HxY activation loop motif, unlike DYRK kinases in other eukaryotes. Phosphoproteomic comparison of TbDYRK null mutants with wild-type parasites identified molecules that operate on both the inhibitory 'slender retainer' and activatory 'stumpy inducer' arms of the QS control pathway. One of these molecules, the RNA-regulator TbZC3H20, regulates parasite QS, this being dependent on the integrity of its TbDYRK phosphorylation site. This analysis reveals fundamental differences to conventional DYRK family regulation and links trypanosome environmental sensing, signal transduction and developmental gene expression in a coherent pathway.

The CBL-CIPK Calcium Signaling Network: Unified Paradigm from 20 Years of Discoveries.

Calcium (Ca2+) serves as an essential nutrient as well as a signaling agent in all eukaryotes. In plants, calcineurin B-like proteins (CBLs) are a unique group of Ca2+ sensors that decode Ca2+ signals by activating a family of plant-specific protein kinases known as CBL-interacting protein kinases (CIPKs). Interactions between CBLs and CIPKs constitute a signaling network that enables information integration and physiological coordination in response to a variety of extracellular cues such as nutrient deprivation and abiotic stresses. Studies in the past two decades have established a unified paradigm that illustrates the functions of CBL-CIPK complexes in controlling membrane transport through targeting transporters and channels in the plasma membrane and tonoplast.

Genome-wide identification of the mitogen-activated protein kinase (MAPK) family in cotton (Gossypium hirsutum) reveals GhMPK6 involved in fiber elongation.

Mitogen-activated protein kinases (MAPKs) are important in regulating plant development as well as stress response. In this study, we genome-widely identified 56 MAPK genes in upland cotton. These MAPK genes unequally distribute on 22 chromosomes of cotton genome, but no MAPK gene is located on At_Chr6, Dt_Chr6, At_Chr13 and Dt_Chr13. The exons and introns in GhMAPK gene family vary widely at the position, number and length. Furthermore, GhMAPK family can be divided into 4 groups (A, B, C and D), and the TEY type of T-loop exists in three groups (A, B and C), but the TDY type of T-loop is only in group D. Further study revealed that some GhMAPK genes (including GhMPK6) are preferentially expressed in elongating fibers. GhMPK6 maintains a high phosphorylation level in elongating fibers, and its phosphorylation was enhanced in fibers by phytohormones brassinosteroid (BR), ethylene and indole-3-acetic acid (IAA). Additionally, GhMPK6 could interact with GhMKK2-2 and GhMKK4, suggesting that GhMKK2-2/4-GhMPK6 module may be involved in phosphorylation of its downstream proteins for regulating fiber elongation of cotton.

Emerging roles of the αC-β4 loop in protein kinase structure, function, evolution, and disease.

The faithful propagation of cellular signals in most organisms relies on the coordinated functions of a large family of protein kinases that share a conserved catalytic domain. The catalytic domain is a dynamic scaffold that undergoes large conformational changes upon activation. Most of these conformational changes, such as movement of the regulatory αC-helix from an "out" to "in" conformation, hinge on a conserved, but understudied, loop termed the αC-β4 loop, which mediates conserved interactions to tether flexible structural elements to the kinase core. We previously showed that the αC-β4 loop is a unique feature of eukaryotic protein kinases. Here, we review the emerging roles of this loop in kinase structure, function, regulation, and diseases. Through a kinome-wide analysis, we define the boundaries of the loop for the first time and show that sequence and structural variation in the loop correlate with conformational and regulatory variation. Many recurrent disease mutations map to the αC-β4 loop and contribute to drug resistance and abnormal kinase activation by relieving key auto-inhibitory interactions associated with αC-helix and inter-lobe movement. The αC-β4 loop is a hotspot for post-translational modifications, protein-protein interaction, and Hsp90 mediated folding. Our kinome-wide analysis provides insights for hypothesis-driven characterization of understudied kinases and the development of allosteric protein kinase inhibitors.

P90RSK Inhibition Ameliorates TGF-β1 Signaling and Pulmonary Fibrosis by Inhibiting Smad3 Transcriptional Activity.

Idiopathic pulmonary fibrosis (IPF) is a specific form of progressive and chronic interstitial lung disease of unknown cause. IPF is characterized by excessive deposition of extracellular matrix (ECM) and destructive pathological remodeling due to epithelial-to-mesenchymal transition (EMT). Eventually, lung interstitium thickens and stiffens and breathing becomes difficult. It has been well established that the transforming growth factor-β1 (TGF-β1)/Smad signaling pathway plays a critical role in the pathogenesis of pulmonary fibrosis. TGF-β1-mediated activation of mitogen activated protein kinase (MAPK) family affects Smad signaling. p90RSK is a serine/threonine kinase and is activated by the extracellular signal-regulated kinase (ERK) signaling pathway. However, the roles played by p90RSK in TGF-β1 signaling and the pathogenesis of pulmonary fibrosis remain unknown. We investigated whether p90RSK regulates the pathogenesis of pulmonary fibrosis using in vitro and in vivo systems and Western blotting, real-time quantitative PCR, transcriptional activity assays and immunofluorescence studies. Pharmacological inhibition of p90RSK by FMK or inhibition of p90RSK with adenoviral vector encoding a dominant negative form of p90RSK suppressed TGF-β1-induced ECM accumulation and EMT in lung epithelial cells and fibroblasts. Interestingly, FMK significantly inhibited TGF-β1-induced Smad3 nuclear translocation and smad binding element-dependent transcriptional activity, but not Smad3 phosphorylation. Furthermore, in a mouse model of bleomycin-induced lung fibrosis, FMK ameliorated pulmonary fibrosis. These findings indicate that p90RSK plays critical roles in pulmonary fibrosis, which suggests it be viewed as a novel therapeutic target for the treatment of lung fibrosis.

Kinome Profiling to Predict Sensitivity to MAPK Inhibition in Melanoma and to Provide New Insights into Intrinsic and Acquired Mechanism of Resistance.

Mitogen-activated protein kinase (MAPK) inhibition with the combination of BRAF (Rapidly Accelerated Fibrosarcoma) and MEK (Mitogen-activated protein kinase kinase) inhibitors has become the standard of first-line therapy of metastatic melanoma harbouring BRAF V600 mutations. However, about half of the patients present with primary resistance while the remaining develop secondary resistance under prolonged treatment. Thus, there is a need for predictive biomarkers for sensitivity and/or resistance to further refine the patient population likely to benefit from MAPK inhibitors. In this study, we explored a top-down approach using a multiplex kinase assay, first, to discover a kinome signature predicting sensitivity, intrinsic and acquired resistance to MAPK inhibitors in melanoma, and second, to understand the mechanism of resistance using cell lines. Pre-dose tissues from patients (four responders and three non-responders to BRAFi monotherapy) were profiled for phosphotyrosine kinase (PTK) and serine-threonine kinase (STK) activities on a PamChip® peptide microarray in the presence and absence of ex vivo BRAFi. In addition, molecular studies were conducted on four sensitive parental lines, their offspring with acquired resistance to BRAFi and two lines with intrinsic resistance. PTK and STK activities in cell lysates were measured in the presence and absence of ex vivo BRAFi and/or MEKi. In tissue lysates, concentration-dependent ex vivo inhibition of STK and PTK activities with dabrafenib was stronger in responders than in non-responders. This difference was confirmed in cell lines comparing sensitive and resistant ones. Interestingly, common features of resistance were increased activity of receptor tyrosine kinases, Proto-oncogene tyrosine-protein kinase Src (Src) family kinases and protein kinase B (PKB, AKT) signalling. These latter results were confirmed by Western blots. While dabrafenib alone showed an inhibition of STK and PTK activities in both tissues and cell lines, the combination of dabrafenib and trametinib showed an antagonism on the STK activities and a synergism on PTK activities, resulting in stronger inhibitions of overall tyrosine kinase activities. Altogether; these data reveal that resistance of tumours and cell lines to MAPK inhibitors can be predicted using a multiplex kinase assay and is associated with an increase in specific tyrosine kinase activities and globally to AKT signalling in the patient’s tissue. Thus, such a predictive kinome signature would help to identify patients with innate resistance to MAPK double inhibition in order to propose other therapies.

Autophagy-based unconventional secretion of HMGB1 by keratinocytes plays a pivotal role in psoriatic skin inflammation

ABSTRACT The precise mechanism through which macroautophagy/autophagy affects psoriasis is poorly understood. Here, we found that keratinocyte (KC) autophagy, which was positively correlated with psoriatic severity in patients and mouse models and could be inhibited by mitogen-activated protein kinase (MAPK) family inactivation. The impairment of autophagic flux alleviated psoriasisform inflammation. We also found that an autophagy-based unconventional secretory pathway (autosecretion) dependent on ATG5 (autophagy related 5) and GORASP2 (golgi reassembly stacking protein 2) promoted psoriasiform KC inflammation. Moreover, the alarmin HMGB1 (high mobility group box 1) was more effective than other autosecretory proteins in regulating psoriasiform cutaneous inflammation. HMGB1 neutralization in autophagy-efficient KCs eliminated the differences in psoriasiform inflammation between Krt14+/+-Atg5f/f KCs and Krt14Cre/+-atg5f/f KCs, and conversely, recombinant HMGB1 almost completely restored psoriasiform inflammation in Krt14Cre/+-atg5f/f KCs in vivo. These results suggest that HMGB1-associated autosecretion plays a pivotal role in cutaneous inflammation. Finally, we demonstrated that Krt14Cre/+-hmgb1f/f mice displayed attenuated psoriatic inflammation due to the essential crosstalk between KC-specific HMGB1-associated autosecretion and γδT cells. Thus, this study uncovered a novel autophagy mechanism in psoriasis pathogenesis, and the findings imply the clinical significance of investigating and treating psoriasis. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AGER: advanced glycosylation end-product specific receptor; Anti-HMGB1: anti-HMGB1 neutralizing antibody; Anti-IL18: anti-IL18 neutralizing antibody; Anti-IL1B: anti-IL1B neutralizing antibody; ATG5: autophagy related 5; BAF: bafilomycin A1; BECN1: beclin 1; CASP1: caspase 1; CCL: C-C motif chemokine ligand; CsA: cyclosporine A; ctrl shRNA: lentivirus harboring shRNA against control; CXCL: C-X-C motif chemokine ligand; DCs: dendritic cells; DMEM: dulbecco’s modified Eagle’s medium; ELISA: enzyme-linked immunosorbent assay; EM: electron microscopy; FBS: fetal bovine serum; GORASP2 shRNA: lentivirus harboring shRNA against GORASP2; GORASP2/GRASP55: golgi reassembly stacking protein 2; GR1: a composite epitope between LY6 (lymphocyte antigen 6 complex) locus C1 and LY6 locus G6D antigens; H&E: hematoxylin and eosin; HMGB1: high mobility group box 1; HMGB1 shRNA: lentivirus harboring shRNA against HMGB1; IFNG/IFN-γ: interferon gamma; IL17A: interleukin 17A; IL18: interleukin 18; IL1A/IL-1α: interleukin 1 alpha; IL1B/IL-1β: interleukin 1 beta; IL22/IL-22: interleukin 22; IL23A: interleukin 23 subunit alpha; IL23R: interleukin 23 receptor; IMQ: imiquimod; ITGAM/CD11B: integrin subunit alpha M; ITGAX/CD11C: integrin subunit alpha X; IVL: involucrin; KC: keratinocyte; KD: knockdown; KO: knockout; Krt14+/+-Atg5f/f mice: mice bearing an Atg5 flox allele, in which exon 3 of the Atg5 gene is flanked by two loxP sites; Krt14+/+-Hmgb1f/f : mice bearing an Hmgb1 flox allele, in which exon 2 to 4 of the Hmgb1 gene is flanked by two loxP sites; Krt14Cre/+-atg5f/f mice: keratinocyte-specific atg5 knockout mice generated by mating Atg5-floxed mice with mice expressing Cre recombinase under the control of the promoter of Krt4; Krt14Cre/+-hmgb1f/f mice: keratinocyte-specific hmgb1 knockout mice generated by mating Hmgb1-floxed mice with mice expressing Cre recombinase under the control of the promoter of Krt14; Krt14-Vegfa mice: mice expressing 164-amino acid Vegfa splice variant recombinase under the control of promoter of Krt14; LAMP1: lysosomal associated membrane protein 1; LDH: lactate dehydrogenase; LORICRIN: loricrin cornified envelope precursor protein; M5: TNF, IL1A, IL17A, IL22 and OSM in combination; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MKI67: marker of proliferation Ki-67; MTT: thiazolyl blue tetrazolium bromide; NFKB/NF-κB: nuclear factor kappa B; NHEKs: primary normal human epidermal keratinocytes; NS: not significant; OSM: oncostatin M; PASI: psoriasis area and severity index; PtdIns3K: class III phosphatidylinositol 3-kinase; qRT-PCR: quantitative RT-PCR; RELA/p65: RELA proto-oncogene, NF-kB subunit; rHMGB1: recombinant HMGB1; rIL18: recombinant interleukin 18; rIL1B: recombinant interleukin 1 beta; S100A: S100 calcium binding protein A; SQSTM1/p62: sequestosome 1; T17: IL17A-producing T; TCR: T-cell receptor; tcrd KO mice: tcrd (T cell receptor delta chain) knockout mice, which show deficient receptor expression in all adult lymphoid and epithelial organs; TLR: toll-like receptor; TNF/TNF-α: tumor necrosis factor; WOR: wortmannin; WT: wild-type; γδT17 cells: IL17A-producing γδ T cells.

Abstract P6-04-06: Protein kinase C beta 1 (PKCβ1), a novel drug target of Z-endoxifen, inhibits growth of estrogen receptor positive (ER+) breast cancer via activation of the NF-kB transcription factor RelA

Abstract Background: The first in-human phase-I clinical trial of the tamoxifen metabolite, Z-endoxifen, demonstrated promising antitumor activity in both aromatase inhibitor (AI) and tamoxifen resistant ER+ breast cancer patients (Goetz M.P., et al; JCO, 2017). By utilizing a letrozole-sensitive MCF7 aromatase expressing (MCF7AC1) cell line, and its corresponding letrozole-resistant (MCF7LR) cell line, we have shown superior antitumor and antiestrogenic activity of Z-endoxifen over tamoxifen in the AI-sensitive and resistant settings in vivo. This study also revealed that Z-endoxifen may target protein kinases in addition to estrogen receptor alpha. Follow-up studies demonstrated that Z-endoxifen, but not tamoxifen, binds to protein kinase C beta 1 (PKCβ1), a member of the serine/threonine-specific protein kinase family that regulates signaling pathways involved in cell proliferation and tumorigenic transformation (Guo C., et al; SABCS, 2017). We have further shown that PKCβ1 silencing, similar to Z-endoxifen, potently inhibits ER+ cell proliferation and additionally causes ERα turnover. Here, we report a potential mechanism by which PKCβ1 may mediate its anti-proliferative effects in ER+ breast cancer cells. Methods: The effects of siRNA-mediated PKCβ1-silencing on gene transcription were evaluated by RNAseq in MCF7AC1 cells. RNAseq results were further validated by quantitative real-time polymerase chain reaction (qRT-PCR) in PKCβ1-silenced MCF7AC1 cells as well as in ER+ T47D cells. The effects of Z-endoxifen on the mRNA expression of genes altered by PKCβ1-silencing in MCF7AC1 cells were evaluated by qRT-PCR analysis. PKCβ1 silencing effects on protein expression in MCF7AC1 cells were evaluated by immunoblot analysis. Results: RNAseq analysis of PKCβ1-silenced MCF7AC1 cells compared to non-silenced cells revealed NF-κB signaling pathway as the top biological pathway significantly altered by PKCβ1 silencing (p=5.93e-08). Enhanced mRNA expression of multiple NF-κB downstream target genes including CXCL10, RELB, MMP1 and IL6 were observed following PKCβ1 silencing. qRT-PCR analyses validated increased mRNA expression of these genes in PKCβ1-silenced MCF7AC1 and T47D cells. Notably, Z-endoxifen treatment in MCF7AC1 cells mirrored PKCβ1 silencing effects, resulting in enhanced mRNA expression of the aforementioned genes. Evaluation of the protein expression of RelA and RelB, the canonical and noncanonical NF-κB transcription factors respectively, in PKCβ1-silenced MCF7AC1 cells revealed significant upregulation of RelA (p=0.0203) but not RelB (p=0.1360) expression. ERα silencing in MCF7AC1 cells did not affect RelA protein expression. Consistent with prior reports, treatment of MCF7 cells with TNFα inhibited cell proliferation. Finally, targeted siRNA-mediated silencing of RelA in PKCβ1-silenced MCF7AC1 cells significantly attenuated PKCβ1-mediated growth inhibition (p=0.0002). Conclusion: Our findings demonstrate that both Z-endoxifen treatment and PKCβ1-silencing commonly activates a NF-κB-mediated gene signature in ER+ breast cancer cells. Notably, PKCβ1-silencing induces the protein expression of the NF-κB transcription factor RelA in an ERα-independent manner, which appears to contribute to PKCβ1-mediated growth inhibition. Current studies are ongoing to evaluate the impact of RelA silencing on Z-endoxifen mediated growth inhibition. This information will foster our understanding of the estrogenic and non-estrogenic mechanisms by which Z-endoxifen mediates its antitumor activity in ER+ breast cancer. Citation Format: Swaathi Jayaraman, Mary J Kuffel, Krishna R Kalari, Kevin J Thompson, Xiaojia Tang, Elizabeth S Bruinsma, John R Hawse, Matthew P Goetz. Protein kinase C beta 1 (PKCβ1), a novel drug target of Z-endoxifen, inhibits growth of estrogen receptor positive (ER+) breast cancer via activation of the NF-kB transcription factor RelA [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-06.

Abstract P3-01-21: Functional duality of NUAK2 in breast cancer metastases

Abstract Background: Breast cancer patients with lymph nodes metastasis were proven to have more aggressive biologically phenotypes. This study was aimed to find different biomarker ectopic expression between breast cancer patients' metastatic lymph nodes and primary cancer, and to explore the potential molecular mechanisms. Method: From our previous research, we selected the pairing samples from primary breast cancer with their early stage lymph node metastasis to carry out differential gene expression profiling. Genes with high expression level in sentinel lymph node metastasis were obtained. Then according to the findings of CRISPR/Cas9 screening and the results in relevant literature and online public database, we decided to investigate the role of NUAK2 in our further research. In vitro and in vivo analyses were carried out to determine the potential mechanisms of NUAK2 in lymph node metastases and prognosis. Results NUAK2 belongs to the AMP-activated protein kinase (AMPK) family, with its members controling protein metabolism, polarity, and overall cellular homeostasis actin cytoskeleton organization. The effect of NUAK2 variant expression in breast cancer metastases remains unknown. In this study, it was found that the expression level of NUAK2 was significantly higher in 5 paired metastatic lymph node than that in primary cancer, using Affymetrix GeneChip® Human Transcriptome Array 2.0. From online public database, higher NUAK2 expression level was markedly associated with better metastasis-free survival in breast cancer patients. The effects of NUAK2 on proliferation were investigated by CCK-8 and IncuCyte ZOOM™ assay, respectively. The results demonstrated that knockdown NUAK2 was able to significantly inhibit the proliferation of breast cancer cells. However, for cancer metastases, it was demonstrated that knock-down of NUAK2 significantly accelerated the migration of breast cancer cells, while overexpression of NUAK2 notably inhibited breast cancer cell migration. When promoting proliferation of breast cancer cells, NUAK2 might involve signal pathways of cell cycle. As for the anti-metastasis function, it would regulate cell adhesion, cell motility and microenvironment. Conclusion: Our in vitro study demonstrated that NUAK2 has shown reverse effects of tumor cell proliferation and invasion. The interaction of NUAK2 and cancer cells could be an important role in cell proliferation involved in cell cycle signal pathway, and modulate cell mobility by inducing cell-cell detachment. Its molecular mechanism of dual effect in breast cancer need further investigation. Citation Format: Wang Jia, XUE Jingyan, Hou Jianjing, Xiu Bingqiu, Li Lun, Chi Yayun, Wu Jiong. Functional duality of NUAK2 in breast cancer metastases [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-21.

The calcineurin β-like interacting protein kinase CIPK25 regulates potassium homeostasis under low oxygen in Arabidopsis.

Hypoxic conditions often arise from waterlogging and flooding, affecting several aspects of plant metabolism, including the uptake of nutrients. We identified a member of the CALCINEURIN β-LIKE INTERACTING PROTEIN KINASE (CIPK) family in Arabidopsis, CIPK25, which is induced in the root endodermis under low-oxygen conditions. A cipk25 mutant exhibited higher sensitivity to anoxia in conditions of potassium limitation, suggesting that this kinase is involved in the regulation of potassium uptake. Interestingly, we found that CIPK25 interacts with AKT1, the major inward rectifying potassium channel in Arabidopsis. Under anoxic conditions, cipk25 mutant seedlings were unable to maintain potassium concentrations at wild-type levels, suggesting that CIPK25 likely plays a role in modulating potassium homeostasis under low-oxygen conditions. In addition, cipk25 and akt1 mutants share similar developmental defects under waterlogging, further supporting an interplay between CIPK25 and AKT1.

Calcium-Regulated Phosphorylation Systems Controlling Uptake and Balance of Plant Nutrients

Essential elements taken up from the soil and distributed throughout the whole plant play diverse roles in different tissues. Cations and anions contribute to maintenance of intracellular osmolarity and the formation of membrane potential, while nitrate, ammonium, and sulfate are incorporated into amino acids and other organic compounds. In contrast to these ion species, calcium concentrations are usually kept low in the cytosol and calcium displays unique behavior as a cytosolic signaling molecule. Various environmental stresses stimulate increases in the cytosolic calcium concentration, leading to activation of calcium-regulated protein kinases and downstream signaling pathways. In this review, we summarize the stress responsive regulation of nutrient uptake and balancing by two types of calcium-regulated phosphorylation systems: CPK and CBL-CIPK. CPK is a family of protein kinases activated by calcium. CBL is a group of calcium sensor proteins that interact with CIPK kinases, which phosphorylate their downstream targets. In Arabidopsis, quite a few ion transport systems are regulated by CPKs or CBL-CIPK complexes, including channels/transporters that mediate transport of potassium (KAT1, KAT2, GORK, AKT1, AKT2, HAK5, SPIK), sodium (SOS1), ammonium (AMT1;1, AMT1;2), nitrate and chloride (SLAC1, SLAH2, SLAH3, NRT1.1, NRT2.4, NRT2.5), and proton (AHA2, V-ATPase). CPKs and CBL-CIPKs also play a role in C/N nutrient response and in acquisition of magnesium and iron. This functional regulation by calcium-dependent phosphorylation systems ensures the growth of plants and enables them to acquire tolerance against various environmental stresses. Calcium serves as the key factor for the regulation of membrane transport systems.