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Non‐small cell lung cancer comprises up to 85% of lung cancer cases and has a poor prognosis. At present, there are still no effective treatments for this illness. Evidence suggests that the prostaglandin [cyclooxygenase‐2 (COX‐2)] and leukotriene [lipoxygenase‐5 (5‐LOX)] pathways are involved in lung cancer carcinogenesis. Therefore, novel agents that target COX‐2 and 5‐LOX may have therapeutic potential. In the present study, we examined the role of asiatic acid (AA), a triterpenoid saponin, in targeting the protein kinases responsible for lung cancer proliferation and mobility. The experimental data revealed that AA inhibited the growth of lung cancer cells (> 50%) and it significantly impeded the proliferation of lung cancer cells by inhibiting COX‐2, which results in downregulation of the phosphotidyl inositol‐3 kinase/protein kinase B/mammalian target of rapamycin signaling pathway, leading to an induction of cytotoxic autophagy‐mediated apoptosis. Mechanistically, the expression of mitogen‐activated protein kinase/extracellular signal‐regulated kinase, hypoxia‐inducible factor‐1 and vascular endothelial growth factor is downregulated by AA, thereby reducing cell mobility and invasion. It also shows negative osmotic fragility on healthy human erythrocytes. It is concluded that AA may be a viable therapeutic drug for non‐small cell lung cancer treatment, which opens new opportunities for synthesizing analogues. [ABSTRACT FROM AUTHOR]

P38 mitogen‐activated protein kinases are key in the regulation of the cellular response to stressors. P38 is known to regulate transcription, mRNA processing, stability, and translation. The transcriptional changes mediated by phosphorylated p38 (P‐p38) in response to extracellular stimuli have been thoroughly analyzed in many tissues and organisms. However, the genomic localization of chromatin‐associated P‐p38 remains poorly understood. Here, we analyze the chromatin binding of activated P‐p38 and its role in the response to reactive oxygen species (ROS) in Drosophila S2 cells. We found that P‐p38 is already bound to chromatin in basal conditions. After ROS exposure, chromatin‐associated P‐p38 relocates towards genes involved in the recovery process. Our findings highlight the role of P‐p38 dynamic chromatin binding in orchestrating gene expression responses to oxidative stress. [ABSTRACT FROM AUTHOR]

Microtubule affinity‐regulating kinase 2 (MARK2) is a Ser/Thr protein kinase that regulates cell polarity and immune responses. Here, we report that Orf9b, one of the accessory proteins encoded in the SARS‐CoV‐2 genome, increases MARK2 activity via interaction with the autoinhibitory KAI domain. We found that co‐expression of Orf9b enhances the kinase activity of MARK2 in HEK293 cells. Orf9b does not bind to or enhance the activity of the mutant form of MARK2 lacking the KA1 domain. Orf9b lowers inhibitory phosphorylation of MARK2 at T595 while mutation experiments indicate that this site is dispensable for Orf9b‐mediated enhancement of MARK2 activity. Our results suggest that Orf9b enhances MARK2 activity by binding the autoinhibitory KA1 domain, which closely interacts with the kinase domain. [ABSTRACT FROM AUTHOR]

Cilia are microtubule‐based sensory organelles present in a number of eukaryotic cells. Mutations in the genes encoding ciliary proteins cause ciliopathies in humans. A‐kinase anchoring proteins (AKAPs) tether ciliary signaling proteins such as protein kinase A (PKA). The dimerization and docking domain (D/D) on the RIIα subunit of PKA interacts with AKAPs. Here, we show that AKAP240 from the central‐pair microtubules of Chlamydomonas reinhardtii cilia uses two C‐terminal amphipathic helices to bind to its partner FAP174, an RIIα‐like protein with a D/D domain at the N‐terminus. Co‐immunoprecipitation using anti‐FAP174 antibody with an enriched central‐pair microtubule fraction isolated seven interactors whose mass spectrometry analysis revealed proteins from the C2a (FAP65, FAP70, and FAP147) and C1b (CPC1, HSP70A, and FAP42) microtubule projections and FAP75, a protein whose sub‐ciliary localization is unknown. Using RII D/D and FAP174 as baits, we identified two additional AKAPs (CPC1 and FAP297) in the central‐pair microtubules. [ABSTRACT FROM AUTHOR]

The liver kinase B1 (LKB1)/AMP‐activated protein kinase (AMPK) axis pivotally controls cell metabolism and suppresses abnormal growth in various cancers. Wnt/β‐catenin is a frequently dysregulated signaling pathway that drives oncogenesis. Here, we discovered a crosstalk mechanism between the LKB1/AMPK axis and Wnt/β‐catenin signaling. Activated AMPK phosphorylates the deubiquitinase USP10 to potentiate the deubiquitination and stabilization of the key scaffold protein Axin1. This phosphorylation also strengthens the binding between USP10 and β‐catenin and supports the phase transition of β‐catenin. Both processes suppress Wnt/β‐catenin amplitude in parallel and inhibit colorectal cancer growth in a clinically relevant manner. Collectively, we established a crosstalk route by which LKB1/AMPK regulates Wnt/β‐catenin signaling in cancer. USP10 acts as the hub in this process, thus enabling LKB1/AMPK to suppress tumor growth via regulation of both metabolism and cell proliferation. [ABSTRACT FROM AUTHOR]

The cAMP‐dependent protein kinase A (PKA) is the archetypical eukaryotic kinase. The catalytic subunit (PKA‐C) structure is highly conserved among the AGC‐kinase family. PKA‐C is a bilobal enzyme with a dynamic N‐lobe, harbouring the Adenosine‐5′‐triphosphate (ATP) binding site and a more rigid helical C‐lobe. The substrate‐binding groove resides at the interface of the two lobes. A distinct feature of PKA‐C is the positive binding cooperativity between nucleotide and substrate. Several PKA‐C mutations lead to the development of adenocarcinomas, myxomas, and other rare forms of liver tumours. Nuclear magnetic resonance (NMR) spectroscopy shows that these mutations disrupt the allosteric communication between the two lobes, causing a drastic decrease in binding cooperativity. The loss of cooperativity correlates with changes in substrate fidelity and reduced kinase affinity for the endogenous protein kinase inhibitor (PKI). The similarity between PKI and the inhibitory sequence of the kinase regulatory subunits suggests that the overall mechanism of regulation of the kinase may be disrupted. We surmise that a reduced or obliterated cooperativity may constitute a common trait for both orthosteric and allosteric mutations of PKA‐C that may lead to dysregulation and disease. [ABSTRACT FROM AUTHOR]

Stiffness of the extracellular matrix regulates various biological responses, but the response mechanisms are poorly understood. Here, we found that the nuclear diphosphorylated myosin regulatory light chain (2P‐MRLC) is a critical mechanomediator that suppresses apoptosis in response to substrate stiffness. Stiff substrates promoted the nuclear localization of 2P‐MRLC. Zipper‐interacting protein kinase [ZIPK; also known as death‐associated protein kinase 3 (DAPK3)], a kinase for MRLC, was localized in the nucleus in response to stiff substrates and promoted the nuclear localization of 2P‐MRLC. Moreover, actin fiber formation induced by substrate stiffness promoted the nuclear localization of 2P‐MRLC via ZIPK. 2P‐MRLC in response to substrate stiffness suppressed the expression of MAF bZIP transcription factor B (MafB) and repressed apoptosis. These findings reveal a newly identified role of MRLC in mechanotransduction. [ABSTRACT FROM AUTHOR]

In this study, we investigated the role of calcium/calmodulin‐dependent protein kinase II (CaMKII) in contraction‐stimulated glucose uptake in skeletal muscle. C2C12 myotubes were contracted by electrical pulse stimulation (EPS), and treadmill running was used to exercise mice. The activities of CaMKII, the small G protein Rac1, and the Rac1 effector kinase PAK1 were elevated in muscle by running exercise or EPS, while they were lowered by the CaMKII inhibitor KN‐93 and/or small interfering RNA (siRNA)‐mediated knockdown. EPS induced the mRNA and protein expression of the Rac1‐GEF Kalirin in a CaMKII‐dependent manner. EPS‐induced Rac1 activation was lowered by the Kalirin inhibitor ITX3 or siRNA‐mediated Kalirin knockdown. KN‐93, ITX3, and siRNA‐mediated Kalirin knockdown reduced EPS‐induced glucose uptake. These findings define a CaMKII‐Kalirin‐Rac1 signaling pathway that contributes to contraction‐stimulated glucose uptake in skeletal muscle myotubes and tissue. [ABSTRACT FROM AUTHOR]

Profilin regulates actin polymerization, and its balanced expression is required for cellular growth and development. Most tumours have compromised profilin expression, and its overexpression in MDA MB-231 breast cancer cells has been reported to activate AMP-activated protein kinase a (AMPKa), an energy-sensing molecule that affects various cellular processes including autophagy. This study aims to explore the role of profilin in autophagy induction. We employed all-trans retinoic acid (ATRA) as an inducer of profilin expression and showed that profilin induces autophagy through mTOR inhibition, autophagy-activating kinase ULK1 upregulation, and AMPK stabilization as well as its activation. Furthermore, evidence from our study indicates physical interaction between profilin and AMPK, which results in AMPK stabilization and induction of prolonged autophagy, thereby leading to apoptosis. This study uncovers a novel mechanism that induces autophagy in triple-negative breast cancer cells. [ABSTRACT FROM AUTHOR]

Factors that increase cAMP levels can induce lineage‐specific differentiation of glioma cells into astrocyte‐like cells. However, the differentiation pattern and underlying mechanisms remain unclear. Here, we find that cAMP/protein kinase A (PKA)/cAMP responsive element binding protein 1 (CREB1)‐induced miR‐221/222 suppression contributes to the neuron‐like differentiation of gliomas. cAMP agonists selectively induced neuron‐ and astrocyte‐like but not oligodendrocyte‐like differentiation of C6 glioma cells. PKA inhibitors and CREB1 knockout blocked neuron‐like differentiation of glioma cells. cAMP inhibited miR‐221/222 in a PKA/CREB1‐dependent manner. Importantly, both in vitro and in vivo assays demonstrated that transcriptional suppression of miR‐221/222 is required for neuronal differentiation of glioma cells. Our findings suggest that increasing cAMP levels can induce bidirectional differentiation of glioma cells. Furthermore, the miR‐221/222 cluster acts as an epigenetic brake during glioma differentiation. [ABSTRACT FROM AUTHOR]

The high‐affinity tyrosine kinase receptor MET plays a pivotal role in several facets of cell regulation. Although its mitogenic effect is well documented, some aspects of connection patterns between signaling pathways involved in cell cycle progression remain to be deciphered. We have used a tractable heterologous expression system, the Xenopus oocyte, to detect connections between distinct MET signaling cascades involved in G2/M progression. Our results reveal that Src acts as an adapter via its SH2 domain to recruit 3‐phosphoinositide‐dependent protein kinase 1 (PDK1) to the MET signaling complex leading to Akt phosphorylation. These data define an original crosstalk between Src and Akt signaling pathways that contributes to MET‐induced entry into the M phase, and deserves further investigation in pathologies harboring deregulation of this receptor. [ABSTRACT FROM AUTHOR]

Eukaryotes harbour a conserved signalling pathway, called General Amino Acid Control (GAAC) in Saccharomyces cerevisiae, for overcoming amino acid starvation. Upon starvation, the protein kinase Gcn2, which phosphorylates the eukaryotic translation initiation factor eIF2α, becomes stimulated to trigger the GAAC response. Genetic studies suggest that Yih1, which is the yeast homolog of mammalian IMPACT and which binds monomeric actin, inhibits Gcn2 when released from actin. Here, we found that D56A substitution in actin (the act1‐9 allele) leads to reduced eIF2α phosphorylation, suggesting that the Asp56 residue is required for full Gcn2 activation. In the act1‐9 mutant, Yih1 overexpression further enhanced the sensitivity to amino acid starvation‐inducing drugs and further impaired eIF2α phosphorylation, suggesting that Gcn2 inhibition was mediated via Yih1. The D56A substitution may impair the actin–Yih1 interaction, directly or indirectly, thereby increasing the amount of Yih1 available to inhibit Gcn2. [ABSTRACT FROM AUTHOR]

The OV20.0 virulence factor of orf virus antagonizes host antiviral responses. One mechanism through which it functions is by inhibiting activation of the dsRNA‐activated protein kinase R (PKR) by sequestering dsRNA and by physically interacting with PKR. Sequence alignment indicated that several key residues critical for dsRNA binding were conserved in OV20.0, and their contribution to OV20.O function was investigated in this study. We found that residues F141, K160, and R164 were responsible for the dsRNA‐binding ability of OV20.0. Interestingly, mutation at K160 (K160A) diminished the OV20.0–PKR interaction and further reduced the inhibitory effect of OV20.0 on PKR activation. Nevertheless, OV20.0 homodimerization was not influenced by K160A. The contribution of the dsRNA‐binding domain and K160 to the suppression of RNA interference by OV20.0 was further demonstrated in plants. In summary, K160 is essential for the function of OV20.0, particularly its interaction with dsRNA and PKR that ultimately contributes to the suppression of PKR activation. [ABSTRACT FROM AUTHOR]

Homeodomain‐interacting protein kinase 2 (HIPK2) is a highly conserved, constitutively active Ser/Thr protein kinase that is involved in various important biological processes. HIPK2 activates itself by auto‐phosphorylation during its synthesis, and its activity is mainly controlled through modulation of its expression by ubiquitin‐dependent degradation. By comparing the expression of wild‐type and kinase‐defective HIPK2, we have recently described a novel mechanism of HIPK2 regulation that is based on preferential co‐translational degradation of kinase‐defective versus wild‐type HIPK2. Here, we have addressed this novel regulatory mechanism in more detail by focusing on the possible involvement of chaperones. Our work shows that HIPK2 is a client of the CDC37‐HSP90 chaperone complex and points to a novel role of CDC37 in the co‐translational degradation of a client protein. [ABSTRACT FROM AUTHOR]

The molecular mechanisms generating the mania‐like abnormal behaviors caused by diacylglycerol (DG) kinase (DGK) η deficiency remain unclear. Here, we found that DGKη knockout markedly increased dopamine (DA) levels in the midbrain (DA‐producing region, 2.8‐fold) and cerebral cortex (DA projection region, 1.2‐fold). Moreover, DGKη deficiency significantly augmented phosphorylated DA transporter (DAT) levels (1.4‐fold increase), which induce DA efflux to the synaptic cleft, in the cerebral cortex. Moreover, phosphorylation levels of protein kinase C‐β, which is activated by DG and involved in DAT phosphorylation, were also increased. DAT expressed in Neuro‐2a cells recruited DGKη to the plasma membrane and colocalized with it. These results strongly suggest that dopaminergic hyperfunction caused by DGKη deficiency in the brain leads to mania‐like behaviors. [ABSTRACT FROM AUTHOR]

Yeast impact homolog 1 (Yih1), or IMPACT in mammals, is part of a conserved regulatory module controlling the activity of General Control Nonderepressible 2 (Gcn2), a protein kinase that regulates protein synthesis. Yih1/IMPACT is implicated not only in many essential cellular processes, such as neuronal development, immune system regulation and the cell cycle, but also in cancer. Gcn2 must bind to Gcn1 in order to impair the initiation of protein translation. Yih1 hinders this key Gcn1‐Gcn2 interaction by binding to Gcn1, thus preventing Gcn2‐mediated inhibition of protein synthesis. Here, we solved the structures of the two domains of Saccharomyces cerevisiae Yih1 separately using Nuclear Magnetic Resonance and determined the relative positions of the two domains using a range of biophysical methods. Our findings support a compact structural model of Yih1 in which the residues required for Gcn1 binding are buried in the interface. This model strongly implies that Yih1 undergoes a large conformational rearrangement from a latent closed state to a primed open state to bind Gcn1. Our study provides structural insight into the interactions of Yih1 with partner molecules. [ABSTRACT FROM AUTHOR]

Thermophilic proteins maintain their structure at high temperatures through a combination of various factors. Here, we report the ligand‐induced stabilization of a thermophilic Ser/Thr protein kinase. Thermus thermophilus TpkD unfolds completely at 55 °C despite the optimum growth temperature of 75 °C. Unexpectedly, we found that the TpkD structure is drastically stabilized by its natural ligands ATP and ADP, as evidenced by the increase in the melting temperature to 80 °C. Such a striking effect of a substrate on thermostability has not been reported for other protein kinases. Conformational changes upon ATP binding were observed in fluorescence quenching and limited proteolysis experiments. Urea denaturation of Trp mutants suggested that ATP binding affects not only the ATP‐binding site, but also the remote regions. Our findings shed light on thermoadaptation of thermophilic proteins. [ABSTRACT FROM AUTHOR]

We previously reported the involvement of protein arginine methyltransferase 1 (PRMT1) in adipocyte thermogenesis. Here, we investigate the effects of PRMT1 inhibitors on thermogenesis. Unexpectedly, we find that the PRMT1 inhibitor TC‐E 5003 (TC‐E) induces the thermogenic properties of primary murine and human subcutaneous adipocytes. TC‐E treatment upregulates the expression of Ucp1 and Fgf21 significantly and activates protein kinase A signaling and lipolysis in primary subcutaneous adipocytes from both mouse and humans. We further find that the thermogenic effects of TC‐E are independent of PRMT1 and beta‐adrenergic receptors. Our data indicate that TC‐E exerts strong effects on murine and human subcutaneous adipocytes by activating beige adipocytes via PKA signaling. [ABSTRACT FROM AUTHOR]

Autophagy is an intracellular process that regulates the degradation of cytosolic proteins and organelles. Dying cells often accumulate autophagosomes. However, the mechanisms by which necroptotic stimulation induces autophagosomes are not defined. Here, we demonstrate that the activation of necroptosis with TNF‐α plus the cell‐permeable pan‐caspase inhibitor Z‐VAD induces LC3‐II and LC3 puncta, markers of autophagosomes, via the receptor‐interacting protein kinase 3 (RIPK3) in intestinal epithelial cells. Surprisingly, necroptotic stimulation reduces autophagic activity, as evidenced by enlarged puncta of the autophagic substrate SQSTM1/p62 and its increased colocalization with LC3. However, necroptotic stimulation does not induce the lysosomal‐associated membrane protein 1 (LAMP1) nor syntaxin 17, which mediates autophagosome–lysosome fusion, to colocalize with LC3. These data indicate that necroptosis attenuates autophagic flux before the lysosome fusion step. Our findings may provide insights into human diseases involving necroptosis. [ABSTRACT FROM AUTHOR]

This study aimed to identify the intracellular binding partner of a unique class of staphylococcal secreted exotoxins called superantigen‐like proteins (SSL) from human macrophage and keratinocyte cell lysates. Here, we report that SSL1 specifically binds to human extracellular signal‐regulated kinase 2 (hERK2), an important stress‐activated kinase in mitogen‐activated protein kinase signaling pathways. Western blot and in vitro binding studies with recombinant hERK2 confirmed the binding interaction of SSL1, SSL7, and SSL10 with hERK2. Moreover, the SSLs‐hERK2 interaction was validated biochemically by ELISA. Our finding shows that SSLs play a novel role by binding with host cell MAP kinase signaling pathway protein. Understanding the SSL‐hERK2 interaction will also provide a basis for designing SSL‐based peptide inhibitors of hERK2 in cancer therapy. [ABSTRACT FROM AUTHOR]

Ciliogenesis associated kinase 1 (CILK1) was previously known as intestinal cell kinase because it was cloned from that origin. However, CILK1 is now recognized as a widely expressed and highly conserved serine/threonine protein kinase. Mutations in the human CILK1 gene have been associated with ciliopathies, a group of human genetic disorders with defects in the primary cilium. In mice, both Cilk1 knock‐out and Cilk1 knock‐in mutations have recapitulated human ciliopathies. Thus, CILK1 has a fundamental role in the function of the cilium. Several candidate substrates have been proposed for CILK1 and the challenge is to relate these to the mutant phenotypes. In this review, we summarize what is known about CILK1 functions and targets, and discuss gaps in current knowledge that motivate further experimentation to fully understand the role of CILK1 in organ development in humans. [ABSTRACT FROM AUTHOR]

Successful genome duplication is required for cell proliferation and demands extraordinary precision and accuracy. The mechanisms by which cells enter, progress through, and exit S phase are intense areas of focus in the cell cycle and genome stability fields. Key molecular events in the G1 phase of the cell division cycle, especially origin licensing, are essential for pre‐establishing conditions for efficient DNA replication during the subsequent S phase. If G1 events are poorly regulated or disordered, then DNA replication can be compromised leading to genome instability, a hallmark of tumorigenesis. Upon entry into S phase, coordinated origin firing and replication progression ensure complete, timely, and precise chromosome replication. Both G1 and S phase progressions are controlled by master cell cycle protein kinases and ubiquitin ligases that govern the activity and abundance of DNA replication factors. In this short review, we describe current understanding and recent developments related to G1 progression and S phase entrance and exit with a particular focus on origin licensing regulation in vertebrates. [ABSTRACT FROM AUTHOR]

The inter‐organelle transport of lipids must be regulated to ensure appropriate lipid composition of each organelle. In mammalian cells, ceramide synthesised in the endoplasmic reticulum (ER) is transported to the trans‐Golgi regions, where ceramide is converted to sphingomyelin (SM) with the concomitant production of diacylglycerol. Ceramide transport protein (CERT) transports ceramide from the ER to the trans‐Golgi regions at the ER–Golgi membrane contact sites (MCS). The function of CERT is down‐regulated by multisite phosphorylation of a serine‐repeat motif (SRM) and up‐regulated by phosphorylation of serine 315 in CERT. Multisite phosphorylation of the SRM is primed by protein kinase D, which is activated by diacylglycerol. The function of CERT is regulated by a phosphorylation‐dependent feedback mechanism in response to cellular requirements of SM. CERT‐dependent ceramide transport is also affected by the pool of phosphatidylinositol (PtdIns)‐4‐phosphate (PtdIns(4)P) in the trans‐Golgi regions, while the PtdIns(4)P pool is regulated by PtdIns‐4‐kinases and oxysterol‐binding protein. The ER‐Golgi MCS may serve as inter‐organelle communication zones, in which many factors work in concert to serve as an extensive rheostat of SM, diacylglycerol, cholesterol and PtdIns(4)P. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) regulates growth and developmental processes in response to limiting water conditions. ABA functions through a core signaling pathway consisting of PYR1/PYL/RCAR ABA receptors, type 2C protein phosphatases (PP2Cs), and SnRK2‐type protein kinases. Other signaling modules might converge with ABA signals through the modulation of core ABA signaling components. We have investigated the role of the protein kinase WNK8 in ABA signaling. WNK8 interacted with PP2CA and PYR1, phosphorylated PYR1 in vitro, and was dephosphorylated by PP2CA. A hypermorphic wnk8‐ct Arabidopsis mutant allele suppressed ABA and glucose hypersensitivities of pp2ca‐1 mutants during young seedling development, and WNK8 expression in protoplasts suppressed ABA‐induced reporter gene expression. We conclude that WNK8 functions as a negative modulator of ABA signaling. [ABSTRACT FROM AUTHOR]

Protein kinases phosphorylate specific amino acid residues of substrate proteins and regulate many cellular processes. Specificity for phosphorylation depends on the accessibility of these residues, and more importantly, kinases have preferences for certain residues flanking the phospho‐acceptor site. Translation initiation factor 2α [eukaryotic translation initiation factor 2α (eIF2α)] kinase phosphorylates serine51 (Ser51) of eIF2α and downregulates cellular protein synthesis. Structural information on eIF2α reveals that Ser51 is located within a flexible loop, referred to as the Ser51 loop. Recently, we have shown that conformational change of the Ser51 loop increases the accessibility of Ser51 to the kinase active site for phosphorylation. Here, we show that the specificity of Ser51 phosphorylation depends largely on its relative position in the Ser51 loop and minimally on the flanking residues. [ABSTRACT FROM AUTHOR]

The protein kinase D (PKD) family is regulated through multi‐site phosphorylation, including autophosphorylation. For example, PKD displays in vivo autophosphorylation on Ser‐742 (and Ser‐738 in vitro) in the activation loop and Ser‐910 in the C‐tail (hPKD1 numbering). In this paper, we describe the surprising observation that PKD also displays in vitro autocatalytic activity towards a Tyr residue in the P + 1 loop of the activation segment. We define the molecular determinants for this unusual activity and identify a Cys residue (C705 in PKD1) in the catalytic loop as of utmost importance. In cells, PKD Tyr autophosphorylation is suppressed through the association of an inhibitory factor. Our findings provide important novel insights into PKD (auto)regulation. [ABSTRACT FROM AUTHOR]

A plasma membrane receptor protein kinase, FERONIA (FER), regulates various aspects of plant reproductive and vegetative growth. In roots, binding of a peptide ligand to FER causes rapid suppression of cell elongation whereas in ovules, FER is involved in gametophyte interactions. Here, we examined the effect of a mutation that eliminates kinase activity, on both ovule fertilization and root growth, using the same batch of seeds containing a kinase‐dead mutation. The kinase‐dead mutation of FER reduced the ability to complement fer‐4 knockout phenotypes, compared with wild‐type sequence in root, but not in ovules. Our results support a model in which cell type‐specific regulatory mechanisms, such as different interacting partners and/or downstream signaling events, lead to cell type‐specific functions of FER. [ABSTRACT FROM AUTHOR]

The ribosomal protein S6 kinases (S6K) are among the major substrates and crucial effectors of the target of rapamycin (TOR) kinase, which is an evolutionarily conserved regulator of cell growth and proliferation. Recent research indicates that yeast Ypk3 is an ortholog of mammalian S6Ks. Here, we find that plant S6Ks restore ribosomal protein S6 phosphorylation in a rapamycin‐sensitive manner in yeast cells lacking Ypk3. However, phosphorylation of a hydrophobic motif, which is mediated through TOR signaling and essential for mammalian S6K activity, is not detected in plant S6Ks. Furthermore, deletion of the N‐terminal region of rice S6Ks shows phosphorylation of the hydrophobic motif and reduced rapamycin sensitivity. Our findings suggest a mechanism of plant S6K activation distinct from that of mammalian S6Ks. [ABSTRACT FROM AUTHOR]

Glucose uptake is crucial for providing both an energy source and a signal that regulates cell proliferation. Therefore, it is important to clarify the mechanisms underlying glucose uptake and its transmission to intracellular signaling pathways. In this study, we searched for a novel regulatory factor involved in glucose-induced signaling by using Saccharomyces cerevisiae as a eukaryotic model. Requirement of the extracellular protein Ecm33 in efficient glucose uptake and full activation of the nutrient-responsive TOR kinase complex 1 ( TORC1) signaling pathway is shown. Cells lacking Ecm33 elicit a series of starvation-induced pathways even in the presence of extracellular high glucose concentration. This results in delayed cell proliferation, reduced ATP, induction of autophagy, and dephosphorylation of the TORC1 substrates Atg13 and Sch9. [ABSTRACT FROM AUTHOR]

Protein S-acylation is important for many biological processes. It confers proteins with the ability to attach to the plasma membrane and the membranes confining the ER and Golgi compartments. Yet, the contribution of S-acylation to regulating and targeting lysosomal/vacuolar proteins remains largely enigmatic. Here, we report that vacuolar targeting of the calcium sensor calcineurin B-like protein 6 (CBL6) from Arabidopsis thaliana is brought about by S-acylation of N-terminal cysteine residues. Our results suggest distinctions in mechanisms and efficiency of targeting between CBL6 and the previously characterized vacuolar-targeted CBL2 protein. Moreover, we define which CBL-interacting protein kinases (CIPKs) could interact with CBL6 and observe a remarkable temperature dependence of CBL6/ CIPK complex formation. Collectively, these findings indicate a common S-acyla tion-dependent vacuolar membrane targeting pathway for proteins. [ABSTRACT FROM AUTHOR]

NDRG2, a newly identified tumor suppressor, is also responsive to various stresses, such as hypoxia and DNA damage. Here, we reported that in human hepatoma SK-Hep-1 and HepG2 cells, NDRG2 mRNA and protein levels were upregulated by different endoplasmic reticulum stress inducers including Tg, Tm, and DTT. Further, using NDRG2-overexpressing hepatoma cell lines and Ndrg2 KO mice liver tissues, we found that, among the three branches of unfolded protein response signaling, NDRG2 facilitates protein kinase RNA-like ER kinase ( PERK) pathway via interaction with PERK, enhancing its downstream ATF4 and CHOP. Functionally, NDRG2 promotes ERS-induced apoptosis partially through ATF4 or CHOP. Thus, NDRG2 is a novel ERS-responsive protein and acts as PERK co-factor to facilitate PERK branch, thereby contributing to ERS-induced apoptosis. [ABSTRACT FROM AUTHOR]

The evolutionarily highly conserved SNF1-related protein kinase (SnRK1) protein kinase is a metabolic master regulator in plants, balancing the critical energy consumption between growth- and stress response-related metabolic pathways. While the regulation of the mammalian [AMP-activated protein kinase ( AMPK)] and yeast (SNF1) orthologues of SnRK1 is well-characterised, the regulation of SnRK1 kinase activity in plants is still an open question. Here we report that the activity and T-loop phosphorylation of AKIN10, the kinase subunit of the SnRK1 complex, is regulated by the redox status. Although this regulation is dependent on a conserved cysteine residue, the underlying mechanism is different to the redox regulation of animal AMPK and has functional implications for the regulation of the kinase complex in plants under stress conditions. [ABSTRACT FROM AUTHOR]

Upon stopping metabolic processes, some tardigrades can undergo anhydrobiosis. Secretory abundant heat-soluble ( SAHS) proteins have been reported as candidates for anhydrobiosis-related proteins in tardigrades, which seem to protect extracellular components and/or secretory organelles. We determined structures of a SAHS protein from Ramazzottius varieornatus ( Rv SAHS1), which is one of the toughest tardigrades. Rv SAHS1 shows a β-barrel structure similar to fatty acid-binding proteins ( FABPs), in which hydrophilic residues form peculiar hydrogen bond networks, which would provide Rv SAHS1 with better tolerance against dehydration. We identified two putative ligand-binding sites: one that superimposes on those of some FABPs and the other, unique to and conserved in SAHS proteins. These results indicate that SAHS proteins constitute a new FABP family. [ABSTRACT FROM AUTHOR]

HMG-CoA reductase ( HMGR) is a key enzyme in the mevalonate pathway for sterols and cytosolic isoprenoid production. Although HMGR kinases from spinach, barley, and cauliflower tissues have been strongly suggested as members of SNF1-related protein kinases 1 (Sn RK1), the phosphorylation and inactivation of HMGR by plant Sn RK1s has not been demonstrated. In this study, we elucidated that AKIN10, an Arabidopsis Sn RK1, acts as an HMGR kinase. The recombinant AKIN10 phosphorylates and inactivates At HMGR1S using recombinant GRIK1 as the AKIN10 activator. In contrast, AKIN10- GRIK1 fails to inactivate At HMGR1S-S577A, suggesting that this is achieved through Ser577 phosphorylation. Moreover, phosphorylation is detected not only in At HMGR1S but also in At HMGR1S-S577A, suggesting the presence of a novel regulatory mechanism of plant HMGR. [ABSTRACT FROM AUTHOR]

Inositol-requiring enzyme 1 ( IRE1) is a conserved sensor of the unfolded protein response that has protein kinase and endoribonuclease ( RNase) enzymatic activities and thereby initiates HAC1/ XBP1 splicing. Previous studies demonstrated that human IRE1α (h IRE1α) does not cleave Saccharomyces cerevisiae HAC1 m RNA. Using an in vitro cleavage assay, we show that adenine to cytosine nucleotide substitution at the +1 position in the 3′ splice site of HAC1 RNA is required for specific cleavage by h IRE1α. A similar restricted nucleotide specificity in the RNA substrate was observed for XBP1 splicing in vivo. Together these findings underscore the essential role of cytosine nucleotide at +1 in the 3′ splice site for determining cleavage specificity of h IRE1α. [ABSTRACT FROM AUTHOR]

Activation of AMP-activated protein kinase ( AMPK) has been viewed as an important target for the treatment of insulin resistance. Here, by proteomic analysis, we found that expression of heat shock protein beta-1 ( HSPB1) was induced by the AMP analog 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside in palmitate-induced insulin-resistant cells. Overexpression of AMPKα2, or activation of AMPKα via acute/chronic exercise training, increased HSPB1 expression in the skeletal muscle. In AMPKα2−/− mice, HSPB1 expression was downregulated in the quadriceps muscles. Exercise did not increase HSPB1 expression in AMPKα2−/− mice. Moreover, overexpression of HSPB1 enhanced insulin sensitivity in palmitate-induced insulin-resistant cells and restored metabolic phenotypes associated with defective AMPK. Finally, HSPB1 was required for AMPK-mediated activation of the class IIa histone deacetylases and glucose uptake in the skeletal muscle. Our results demonstrate that AMPK-mediated HSPB1 expression enhanced insulin sensitivity in the skeletal muscle. [ABSTRACT FROM AUTHOR]

Various pathological conditions contribute to pH fluctuations and affect the functions of vital organs such as the heart. In this study, we show that in rat cardiomyocytes, acidic extracellular pH (pHe) inhibits autophagy, whereas alkaline pHe stimulates it. We also find that adenosine monophosphate-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR) and Unc-51-like kinase 1 (ULK1) are very sensitive to pHe changes. Furthermore, by interfering with AMPK, mTOR or ULK1 activity, we demonstrate that the AMPK-ULK1 pathway, but not the mTOR pathway, plays a crucial role on pHe-regulated autophagy and cardiomyocyte viability. These data provide a potential therapeutic strategy against cardiomyocyte injury triggered by pH fluctuations. [ABSTRACT FROM AUTHOR]

The cardiac ankyrin repeat protein (CARP) is up-regulated in the myocardium during cardiovascular disease and in response to mechanical or toxic stress. Stress-induced CARP interacts with the N2A spring region of the titin filament to modulate muscle compliance. We characterize the interaction between CARP and titin-N2A and show that the binding site in titin spans the dual domain UN2A-Ig81. We find that the unique sequence UN2A is not structurally disordered, but that it has a stable, elongated α-helical fold that possibly acts as a constant force spring. Our findings portray CARP/titin-N2A as a structured node and help to rationalize the molecular basis of CARP mechanosensing in the sarcomeric I-band. [ABSTRACT FROM AUTHOR]

The Z- DNA-binding domain of PKZ from zebrafish ( Danio rerio; drZα PKZ) contains the largest β-wing among known Z- DNA-binding domains. To elucidate the functional implication of the β-wing, we solved the crystal structure of apo-drZα PKZ. Structural comparison with its Z- DNA-bound form revealed a large conformational change within the β-wing during Z- DNA binding. Biochemical studies of protein mutants revealed that two basic residues in the β-wing are responsible for Z- DNA recognition as well as fast B-Z transition. Therefore, the extra basic residues in the β-wing of drZα PKZ are necessary for the fast B-Z transition activity. [ABSTRACT FROM AUTHOR]

In chloroplasts, protein phosphorylation regulates important processes, including metabolism, photosynthesis, gene expression, and signaling. Because the hitherto known plastid protein kinases represent only a fraction of existing kinases, we aimed at the identification of novel plastid-localized protein kinases that potentially phosphorylate enzymes of the tetrapyrrole biosynthesis (TBS) pathway. We screened publicly available databases for proteins annotated as putative protein kinase family proteins with predicted chloroplast localization. Additionally, we analyzed chloroplast fractions which were separated by sucrose density gradient centrifugation by mass spectrometry. We identified four new candidates for protein kinases, which were confirmed to be plastid localized by expression of GFP-fusion proteins in tobacco leaves. A phosphorylation assay with the purified kinases confirmed the protein kinase activity for two of them. [ABSTRACT FROM AUTHOR]

Luminous organisms use different protein-mediated strategies to modulate light emission color. Here, we report the transient-state kinetic studies of the interaction between photoprotein clytin from Clytia gregaria and its antenna protein, cgre GFP. We propose that cgre GFP forms a transient complex with Ca2+-bound clytin before the excited singlet state of the coelenteramide product is formed. From the spectral distribution and donor-acceptor separation distance, we infer that clytin reaction intermediates may interact only with the middle side part of cgre GFP. [ABSTRACT FROM AUTHOR]

Coronins constitute a family of conserved proteins expressed in all eukaryotes that have been implicated in the regulation of a wide variety of cellular activities. Recent work showed an essential role for coronin 1 in the modulation of the cAMP/ PKA pathway in neurons through the interaction of coronin 1 with the G protein subtype Gαs in a stimulus-dependent manner, but the molecular mechanism regulating coronin 1-Gαs interaction remains unclear. We here show that phosphorylation of coronin 1 on Thr418/424 by cyclin-dependent kinase ( CDK) 5 activity was responsible for coronin 1-Gαs association and the modulation of cAMP production. Together these results show an essential role for CDK5 activity in promoting the coronin 1-dependent cAMP/ PKA pathway. [ABSTRACT FROM AUTHOR]

Yak1 is a member of dual-specificity Tyr phosphorylation-regulated kinases (DYRKs) that are evolutionarily conserved. The downstream targets of Yak1 and their functions are largely unknown. Here, a homologous protein AtYAK1 was identified in Arabidopsis thaliana and the phosphoprotein profiles of the wild type and an atyak1 mutant were compared on two-dimensional gel following Pro-Q Diamond phosphoprotein gel staining. Annexin1, Annexin2 and RBD were phosphorylated at serine/threonine residues by the AtYak1 kinase. Annexin1, Annexin2 and Annexin4 were also phosphorylated at tyrosine residues. Our study demonstrated that AtYak1 is a dual specificity protein kinase in Arabidopsis that may regulate the phosphorylation status of the annexin family proteins. [ABSTRACT FROM AUTHOR]

REDD1 is induced by various cellular stresses; however, its expression in response to lipopolysaccharide (LPS) has not been clearly elucidated in immune cells. LPS stimulated CREB-dependent and NF-κB-independent REDD1 expression in macrophages. Early increases in CREB phosphorylation and REDD1 expression at 8 h following LPS treatment were blocked by inhibition of p38MAPK and mitogen- and stress-activated protein kinase 1 (MSK1), but not PKA. However, delayed CREB-mediated REDD1 expression at 16 h was suppressed by inhibition of cyclooxygenase-2 (COX-2) and PKA. It indicates that LPS induces REDD1 expression by two distinct CREB-mediated mechanisms, the early p38MAPK/MSK1 and the delayed COX-2/PGE 2 /PKA pathways. [ABSTRACT FROM AUTHOR]

PKCδ and p63 are respectively reported to play important roles in cell apoptosis. But there is no report on interaction between them in regulation of apoptosis. In the present study, we found that PKCδ can directly associate and up-regulate TA isoforms of p63 (TAp63) proteins via increasing their stability. PKCδ kinase activity and Thr157 site in TAp63 are crucial for this PKCδ-induced accumulation of TAp63. PKCδ can also enhance TAp63-mediated transcription and cell apoptosis. Taken together, our data indicate that PKCδ phosphorylates TAp63 proteins at Thr157 to stabilize them and promote cell apoptosis. [ABSTRACT FROM AUTHOR]

PKD-mediated phosphorylation of class IIa HDACs frees the MEF2 transcription factor to activate genes that govern muscle differentiation and growth. Studies of the regulation and function of this signaling axis have involved MC1568 and Gö-6976, which are small molecule inhibitors of class IIa HDAC and PKD catalytic activity, respectively. We describe unanticipated effects of these compounds. MC1568 failed to inhibit class IIa HDAC catalytic activity in vitro, and exerted divergent effects on skeletal muscle differentiation compared to a bona fide inhibitor of these HDACs. In cardiomyocytes, Gö-6976 triggered calcium signaling and activated stress-inducible kinases. Based on these findings, caution is warranted when employing MC1568 and Gö-6976 as pharmacological tool compounds to assess functions of class IIa HDACs and PKD. [ABSTRACT FROM AUTHOR]

Distorted mRNA metabolism contributes to amyotrophic lateral sclerosis (ALS). The human antigen R (HuR) is a major mRNA stabilizer. We report that abnormal localization of HuR was associated with enhanced AMP-activated protein kinase (AMPK) activity in the motor neurons of ALS patients. Activation of AMPK changed the location of HuR in mouse motor neurons and in a motor neuron cell line via phosphorylation of importin-α1. Stimulation of the A 2A adenosine receptor normalized the AMPK-evoked redistribution of HuR. This suggests that aberrant activation of AMPK in motor neurons disrupts the normal distribution of HuR, which might imbalance RNA metabolism and contribute to ALS pathogenesis. [ABSTRACT FROM AUTHOR]

As a well-characterized master player in epigenetic regulatory network, EZH2 is widely implicated in the development of many malignancies. We previously found that EZH2 promoted Wnt/β-catenin activation through downregulation of CXXC4 expression. In this report, we demonstrated that CXXC4 inhibited MAPK signaling through binding to ERK-1/2 and abrogating the interaction of ERK 1/2 with MEK1/2. L183, the critical residue in CXXC4 ERK D domain, was found to be essential for CXXC4–ERK 1/2 interaction and the growth inhibitory effect of CXXC4 in human cancer cells. In summary, CXXC4 directly disrupted MEK1/2–ERK 1/2 interaction to inactivate MAPK signaling. L183 site is indispensable for the binding of CXXC4 to ERK1/2 and growth inhibitory effect of CXXC4. Therefore, EZH2 can activate MAPK signaling by inhibiting CXXC4 expression. [ABSTRACT FROM AUTHOR]

AMPK is known to be activated by oxidative stress. Addition of glucose oxidase to cells generates H 2 O 2 at a constant rate that is opposed by enzymic degradation, providing a good model for physiological oxidative stress. AMPK activation by glucose oxidase correlated with increases in cellular AMP:ATP and was greatly reduced in cells expressing an AMP-insensitive AMPK mutant, although a small degree of activation remained. The effects of increased AMP were partly due to inhibition of Thr172 dephosphorylation. These results suggest that changes in adenine nucleotides, rather than direct oxidative modification, are the major drivers of AMPK activation during oxidative stress. [ABSTRACT FROM AUTHOR]