CAMTA3 repressor destabilization triggers TIR domain protein TN2-mediated autoimmunity in the Arabidopsis exo70B1 mutant.

Calcium-dependent protein kinases (CPKs) can decode and translate intracellular calcium signals to induce plant immunity. Mutation of the exocyst subunit gene EXO70B1 causes autoimmunity that depends on CPK5 and the Toll/interleukin-1 receptor (TIR) domain resistance protein TIR-NBS2 (TN2), where direct interaction with TN2 stabilizes CPK5 kinase activity. However, how the CPK5-TN2 interaction initiates downstream immune responses remains unclear. Here, we show that, besides CPK5 activity, the physical interaction between CPK5 and functional TN2 triggers immune activation in exo70B1 and may represent reciprocal regulation between CPK5 and the TIR domain functions of TN2 in Arabidopsis (Arabidopsis thaliana). Moreover, we detected differential phosphorylation of the calmodulin-binding transcription activator 3 (CAMTA3) in the cpk5 background. CPK5 directly phosphorylates CAMTA3 at S964, contributing to its destabilization. The gain-of-function CAMTA3A855V variant that resists CPK5-induced degradation rescues immunity activated through CPK5 overexpression or exo70B1 mutation. Thus, CPK5-mediated immunity is executed through CAMTA3 repressor degradation via phosphorylation-induced and/or calmodulin-regulated processes. Conversely, autoimmunity in camta3 also partially requires functional CPK5. While the TIR domain activity of TN2 remains to be tested, our study uncovers a TN2-CPK5-CAMTA3 signaling module for exo70B1-mediated autoimmunity, highlighting the direct embedding of a calcium-sensing decoder element within resistance signalosomes.

DAPK1 interaction with NMDA receptor NR2B subunits mediates brain damage in stroke.

N-methyl-D-aspartate (NMDA) receptors constitute a major subtype of glutamate receptors at extrasynaptic sites that link multiple intracellular catabolic processes responsible for irreversible neuronal death. Here, we report that cerebral ischemia recruits death-associated protein kinase 1 (DAPK1) into the NMDA receptor NR2B protein complex in the cortex of adult mice. DAPK1 directly binds with the NMDA receptor NR2B C-terminal tail consisting of amino acid 1292-1304 (NR2B(CT)). A constitutively active DAPK1 phosphorylates NR2B subunit at Ser-1303 and in turn enhances the NR1/NR2B receptor channel conductance. Genetic deletion of DAPK1 or administration of NR2B(CT) that uncouples an activated DAPK1 from an NMDA receptor NR2B subunit in vivo in mice blocks injurious Ca(2+) influx through NMDA receptor channels at extrasynaptic sites and protects neurons against cerebral ischemic insults. Thus, DAPK1 physically and functionally interacts with the NMDA receptor NR2B subunit at extrasynaptic sites and this interaction acts as a central mediator for stroke damage.

Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks.

Nutrient signalling integrates and coordinates gene expression, metabolism and growth. However, its primary molecular mechanisms remain incompletely understood in plants and animals. Here we report unique Ca2+ signalling triggered by nitrate with live imaging of an ultrasensitive biosensor in Arabidopsis leaves and roots. A nitrate-sensitized and targeted functional genomic screen identifies subgroup III Ca2+-sensor protein kinases (CPKs) as master regulators that orchestrate primary nitrate responses. A chemical switch with the engineered mutant CPK10(M141G) circumvents embryo lethality and enables conditional analyses of cpk10 cpk30 cpk32 triple mutants to define comprehensive nitrate-associated regulatory and developmental programs. Nitrate-coupled CPK signalling phosphorylates conserved NIN-LIKE PROTEIN (NLP) transcription factors to specify the reprogramming of gene sets for downstream transcription factors, transporters, nitrogen assimilation, carbon/nitrogen metabolism, redox, signalling, hormones and proliferation. Conditional cpk10 cpk30 cpk32 and nlp7 mutants similarly impair nitrate-stimulated system-wide shoot growth and root establishment. The nutrient-coupled Ca2+ signalling network integrates transcriptome and cellular metabolism with shoot-root coordination and developmental plasticity in shaping organ biomass and architecture.

Identification of a glioma functional network from gene fitness data using machine learning.

Glioblastoma multiforme (GBM) is an aggressive form of brain tumours that remains incurable despite recent advances in clinical treatments. Previous studies have focused on sub-categorizing patient samples based on clustering various transcriptomic data. While functional genomics data are rapidly accumulating, there exist opportunities to leverage these data to decipher glioma-associated biomarkers. We sought to implement a systematic approach to integrating data from high throughput CRISPR-Cas9 screening studies with machine learning algorithms to infer a glioma functional network. We demonstrated the network significantly enriched various biological pathways and may play roles in glioma tumorigenesis. From densely connected glioma functional modules, we further predicted 12 potential Wnt/ß-catenin signalling pathway targeted genes, including AARSD1, HOXB5, ITGA6, LRRC71, MED19, MED24, METTL11B, SMARCB1, SMARCE1, TAF6L, TENT5A and ZNF281. Cox regression modelling with these targets was significantly associated with glioma overall survival prognosis. Additionally, TRIB2 was identified as a glioma neoplastic cell marker in single-cell RNA-seq of GBM samples. This work establishes novel strategies for constructing functional networks to identify glioma biomarkers for the development of diagnosis and treatment in clinical practice.

Distinct signaling routes mediate intercellular and intracellular rhizobial infection in Lotus japonicus.

Rhizobial infection of legume roots during the development of nitrogen-fixing root nodules can occur intracellularly, through plant-derived infection threads traversing cells, or intercellularly, via bacterial entry between epidermal plant cells. Although it is estimated that around 25% of all legume genera are intercellularly infected, the pathways and mechanisms supporting this process have remained virtually unexplored due to a lack of genetically amenable legumes that exhibit this form of infection. In this study, we report that the model legume Lotus japonicus is infected intercellularly by the IRBG74 strain, recently proposed to belong to the Agrobacterium clade of the Rhizobiaceae. We demonstrate that the resources available for L. japonicus enable insight into the genetic requirements and fine-tuning of the pathway governing intercellular infection in this species. Inoculation of L. japonicus mutants shows that Ethylene-responsive factor required for nodulation 1 (Ern1) and Leu-rich Repeat Receptor-Like Kinase (RinRK1) are dispensable for intercellular infection in contrast to intracellular infection. Other symbiotic genes, including nod factor receptor 5 (NFR5), symbiosis receptor-like kinase (SymRK), Ca2+/calmodulin dependent kinase (CCaMK), exopolysaccharide receptor 3 (Epr3), Cyclops, nodule inception (Nin), nodulation signaling pathway 1 (Nsp1), nodulation signaling pathway 2 (Nsp2), cystathionine-ß-synthase (Cbs), and Vapyrin are equally important for both entry modes. Comparative RNAseq analysis of roots inoculated with IRBG74 revealed a distinctive transcriptome response compared with intracellular colonization. In particular, several cytokinin-related genes were differentially regulated. Corroborating this observation, cyp735A and ipt4 cytokinin biosynthesis mutants were significantly affected in their nodulation with IRBG74, whereas lhk1 cytokinin receptor mutants formed no nodules. These results indicate a differential requirement for cytokinin signaling during intercellular rhizobial entry and highlight distinct modalities of inter- and intracellular infection mechanisms in L. japonicus.

The Ectomycorrhizal Fungus Laccaria bicolor Produces Lipochitooligosaccharides and Uses the Common Symbiosis Pathway to Colonize Populus Roots.

Mycorrhizal fungi form mutualistic associations with the roots of most land plants and provide them with mineral nutrients from the soil in exchange for fixed carbon derived from photosynthesis. The common symbiosis pathway (CSP) is a conserved molecular signaling pathway in all plants capable of associating with arbuscular mycorrhizal fungi. It is required not only for arbuscular mycorrhizal symbiosis but also for rhizobia-legume and actinorhizal symbioses. Given its role in such diverse symbiotic associations, we hypothesized that the CSP also plays a role in ectomycorrhizal associations. We showed that the ectomycorrhizal fungus Laccaria bicolor produces an array of lipochitooligosaccharides (LCOs) that can trigger both root hair branching in legumes and, most importantly, calcium spiking in the host plant Populus in a CASTOR/POLLUX-dependent manner. Nonsulfated LCOs enhanced lateral root development in Populus in a calcium/calmodulin-dependent protein kinase (CCaMK)-dependent manner, and sulfated LCOs enhanced the colonization of Populus by L. bicolor Compared with the wild-type Populus, the colonization of CASTOR/POLLUX and CCaMK RNA interference lines by L. bicolor was reduced. Our work demonstrates that similar to other root symbioses, L. bicolor uses the CSP for the full establishment of its mutualistic association with Populus.

OsDMI3-mediated OsUXS3 phosphorylation improves oxidative stress tolerance by modulating OsCATB protein abundance in rice.

Calcium (Ca2+ )/calmodulin (CaM)-dependent protein kinase (CCaMK) is an important positive regulator of antioxidant defenses and tolerance against oxidative stress. However, the underlying molecular mechanisms are largely unknown. Here, we report that the rice (Oryza sativa) CCaMK (OsDMI3) physically interacts with and phosphorylates OsUXS3, a cytosol-localized UDP-xylose synthase. Genetic and biochemical evidence demonstrated that OsUXS3 acts downstream of OsDMI3 to enhance the oxidative stress tolerance conferred by higher catalase (CAT) activity. Indeed, OsUXS3 interacted with CAT isozyme B (OsCATB), and this interaction was required to increase OsCATB protein abundance under oxidative stress conditions. Furthermore, we showed that OsDMI3 phosphorylates OsUXS3 on residue Ser-245, thereby further promoting the interaction between OsUXS3 and OsCATB. Our results indicate that OsDMI3 promotes the association of OsUXS3 with OsCATB to enhance CAT activity under oxidative stress. These findings reveal OsUXS3 as a direct target of OsDMI3 and demonstrate its involvement in antioxidant defense.

Abscisic acid-induced degradation of Arabidopsis guanine nucleotide exchange factor requires calcium-dependent protein kinases.

Abscisic acid (ABA) plays essential roles in plant development and responses to environmental stress. ABA induces subcellular translocation and degradation of the guanine nucleotide exchange factor RopGEF1, thus facilitating ABA core signal transduction. However, the underlying mechanisms for ABA-triggered RopGEF1 trafficking/degradation remain unknown. Studies have revealed that RopGEFs associate with receptor-like kinases to convey developmental signals to small ROP GTPases. However, how the activities of RopGEFs are modulated is not well understood. Type 2C protein phosphatases stabilize the RopGEF1 protein, indicating that phosphorylation may trigger RopGEF1 trafficking and degradation. We have screened inhibitors followed by several protein kinase mutants and find that quadruple-mutant plants in the Arabidopsis calcium-dependent protein kinases (CPKs) cpk3/4/6/11 disrupt ABA-induced trafficking and degradation of RopGEF1. Moreover, cpk3/4/6/11 partially impairs ABA inhibition of cotyledon emergence. Several CPKs interact with RopGEF1. CPK4 binds to and phosphorylates RopGEF1 and promotes the degradation of RopGEF1. CPK-mediated phosphorylation of RopGEF1 at specific N-terminal serine residues causes the degradation of RopGEF1 and mutation of these sites also compromises the RopGEF1 overexpression phenotype in root hair development in Arabidopsis Our findings establish the physiological and molecular functions and relevance of CPKs in regulation of RopGEF1 and illuminate physiological roles of a CPK-GEF-ROP module in ABA signaling and plant development. We further discuss that CPK-dependent RopGEF degradation during abiotic stress could provide a mechanism for down-regulation of RopGEF-dependent growth responses.

Ca2+-Dependent Protein Kinase 6 Enhances KAT2 Shaker Channel Activity in Arabidopsis thaliana.

Post-translational regulations of Shaker-like voltage-gated K+ channels were reported to be essential for rapid responses to environmental stresses in plants. In particular, it has been shown that calcium-dependent protein kinases (CPKs) regulate Shaker channels in plants. Here, the focus was on KAT2, a Shaker channel cloned in the model plant Arabidopsis thaliana, where is it expressed namely in the vascular tissues of leaves. After co-expression of KAT2 with AtCPK6 in Xenopuslaevis oocytes, voltage-clamp recordings demonstrated that AtCPK6 stimulates the activity of KAT2 in a calcium-dependent manner. A physical interaction between these two proteins has also been shown by Förster resonance energy transfer by fluorescence lifetime imaging (FRET-FLIM). Peptide array assays support that AtCPK6 phosphorylates KAT2 at several positions, also in a calcium-dependent manner. Finally, K+ fluorescence imaging in planta suggests that K+ distribution is impaired in kat2 knock-out mutant leaves. We propose that the AtCPK6/KAT2 couple plays a role in the homeostasis of K+ distribution in leaves.

LncRNA ZEB1-AS1 promotes pancreatic cancer progression by regulating miR-505-3p/TRIB2 axis.

Long noncoding RNAs (lncRNAs) are crucial regulatory factors in the development and progression of human malignancies. The purpose of this study was to investigate the potential mechanism of ZEB1-AS1 in pancreatic cancer (PC). The expression of ZEB1-AS1 in PC tissues and cells was assessed by RT-qPCR. The overall survival rate was evaluated using the Kaplan-Meier analysis. The association between ZEB1-AS1 and miR-505 was verified by dual-luciferase reporter assay. CCK-8 assay was employed to analyze PC cell viability. Transwell assay was employed to detect the migration and invasion of PC cells. Our results revealed that ZEB1-AS1 expression was significantly upregulated in PC tissues and cells, and the high expression of ZEB1-AS1 indicated the low overall survival rate in PC patients. Loss-of-function and gain-of-function assays indicated that knockdown of ZEB1-AS1 inhibited the cell viability, migration and invasion of PC cells, while overexpression of ZEB1-AS1 promoted PC cell progression. Moreover, ZEB1-AS1 upregulated TRIB2 expression via sponging miR-505. Finally, rescue assays demonstrated that TRIB2 overexpression partially abrogated the inhibitory effect of ZEB1-AS1 knockdown on the viability, migration and invasion of PC cells. These results confirmed that ZEB1-AS1 promoted the tumorigenesis of PC through the miR-505/TRIB2 axis, which indicated that ZEB1-AS1 might function as a biomarker for PC treatment and provide a new therapeutic direction in PC.

CgCmk1 Activates CgRds2 To Resist Low-pH Stress in Candida glabrata.

In Candida glabrata, the transcription factor CgRds2 has been previously characterized as a regulator of glycerophospholipid metabolism, playing a crucial role in the response to osmotic stress. Here, we report that CgRds2 is also involved in the response to pH 2.0 stress. At pH 2.0, the deletion of CgRDS2 led to reduced cell growth and survival, by 33% and 57%, respectively, compared with those of the wild-type strain. These adverse phenotypes resulted from the downregulation of genes related to energy metabolism in the Cgrds2Δ strain at pH 2.0, which led to a 34% reduction of the intracellular ATP content and a 24% decrease in membrane permeability. In contrast, the overexpression of CgRDS2 rescued the growth defect of the Cgrds2Δ strain and increased cell survival at pH 2.0 by 17% compared with that of the wild-type strain, and this effect was accompanied by significant increases in ATP content and membrane permeability, by 42% and 19%, respectively. Furthermore, we found that the calcium/calmodulin-dependent protein kinase (CaMK) CgCmk1 physically interacts with the PAS domain of CgRds2, and CgCmk1 is required for CgRds2 activation and translocation from the cytoplasm to the nucleus under pH 2.0 stress. Moreover, CgCmk1 is critical for CgRds2 function in resistance to pH 2.0 stress, because cells of the Cgrds2-pas strain with a disrupted CgCmk1-CgRds2 interaction exhibited impaired energy metabolism and membrane permeability at pH 2.0. Therefore, our results indicate that CgCmk1 positively regulates CgRds2 and suggest that they promote resistance to low-pH stress by enhancing energy metabolism and membrane permeability in C. glabrataIMPORTANCE An acidic environment is the main problem in the production of organic acids in C. glabrata The present study reports that the calcium/calmodulin-dependent protein kinase CgCmk1 positively regulates CgRds2 to increase intracellular ATP content, membrane permeability, and resistance to low-pH stress. Hence, the transcription factor CgRds2 may be a potential target for improving the acid stress tolerance of C. glabrata during the fermentation of organic acids. The present study also establishes a new link between the calcium signaling pathway and energy metabolism.

Arabidopsis CPK6 positively regulates ABA signaling and drought tolerance through phosphorylating ABA-responsive element-binding factors.

Abscisic acid (ABA) regulates numerous developmental processes and drought tolerance in plants. Calcium-dependent protein kinases (CPKs) are important Ca2+ sensors playing crucial roles in plant growth and development as well as responses to stresses. However, the molecular mechanisms of many CPKs in ABA signaling and drought tolerance remain largely unknown. Here we combined protein interaction studies, and biochemical and genetic approaches to identify and characterize substrates that were phosphorylated by CPK6 and elucidated the mechanism that underlines the role of CPK6 in ABA signaling and drought tolerance. The expression of CPK6 is induced by ABA and dehydration. Two cpk6 T-DNA insertion mutants are insensitive to ABA during seed germination and root elongation of seedlings; in contrast, overexpression of CPK6 showed the opposite phenotype. Moreover, CPK6-overexpressing lines showed enhanced drought tolerance. CPK6 interacts with and phosphorylates a subset of core ABA signaling-related transcription factors, ABA-responsive element-binding factors (ABFs/AREBs), and enhances their transcriptional activities. The phosphorylation sites in ABF3 and ABI5 were also identified through MS and mutational analyses. Taken together, we present evidence that CPK6 mediates ABA signaling and drought tolerance through phosphorylating ABFs/AREBs. This work thus uncovers a rather conserved mechanism of calcium-dependent Ser/Thr kinases in ABA signaling.

CALCIUM-DEPENDENT PROTEIN KINASE5 Associates with the Truncated NLR Protein TIR-NBS2 to Contribute to exo70B1-Mediated Immunity.

Calcium-dependent protein kinases (CPKs) function as calcium sensors and play important roles in plant immunity. Loss of function of the exocyst complex subunit EXO70B1 leads to autoimmunity caused by activation of TN2, a truncated Toll/interleukin-1 receptor-nucleotide binding sequence protein. Here we show, based on a screen for suppressors of exo70B1, that exo70B1-activated autoimmune responses require CPK5 However, the CPK5 homologs CPK4, CPK6, and CPK11, which were previously reported to function redundantly with CPK5 in effector-triggered immunity, did not contribute to exo70B1-associated phenotypes, indicating that CPK5 plays a unique role in plant immunity. Overexpressing CPK5 results in TN2-dependent autoimmunity and enhanced disease resistance, reminiscent of the exo70B1 phenotypes. Ectopic expression of CPK5 in the exo70B1 mutant led to constitutive CPK5 protein kinase activity, which was not detectable in tn2 mutants. Furthermore, TN2 interacts with the CPK5 N-terminal variable and kinase domains, stabilizing CPK5 kinase activity in vitro. This work uncovers a direct functional link between an atypical immune receptor and a crucial component of early immune signaling: increased immunity in exo70B1 depends on TN2 and CPK5 and, in a positive feedback loop, TN2 keeps CPK5 enzymatically active beyond the initiating stimulus.

Regulation of Multifunctional Calcium/Calmodulin Stimulated Protein Kinases by Molecular Targeting.

Multifunctional calcium/calmodulin-stimulated protein kinases control a broad range of cellular functions in a multitude of cell types. This family of kinases contain several structural similarities and all are regulated by phosphorylation, which either activates, inhibits or modulates their kinase activity. As these protein kinases are widely or ubiquitously expressed, and yet regulate a broad range of different cellular functions, additional levels of regulation exist that control these cell-specific functions. Of particular importance for this specificity of function for multifunctional kinases is the expression of specific binding proteins that mediate molecular targeting. These molecular targeting mechanisms allow pools of kinase in different cells, or parts of a cell, to respond differently to activation and produce different functional outcomes.

Calcium-dependent protein kinase CPK9 negatively functions in stomatal abscisic acid signaling by regulating ion channel activity in Arabidopsis.

KEY MESSAGE: In this manuscript, we demonstrated the negative role of CPK9 in stomatal ABA signaling, and both CPK9 and CPK33 for accurate guard cell function was explored via cpk9/cpk33 double mutants' phenotype. Abscisic acid (ABA) can inhibit stomatal opening and promote stomatal closure by regulating ion channel activity in guard cell membranes. As an important second messenger, calcium (Ca2+) is essentially needed in ABA regulation of stomatal movement. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central Ca2+ signal transduction in plants. Here, we report the functional characterization of CPK9 in Arabidopsis stomatal ABA signaling. CPK9 had high expression in guard cells and the protein was subcellularly located in the cell membrane. A loss-of-function mutant cpk9 showed a much more sensitive phenotype to ABA regulation of stomatal movement and ion channel activity, while CPK9 overexpression lines had opposite phonotypes. These findings demonstrated the negative role of CPK9 in stomatal ABA signaling. As the closest homolog of CPK33, we also proved that stomatal movement of the cpk9/cpk33 double mutants was more sensitive to ABA than either single mutants. These results revealed the role of CPK9 in guard cells, and the need of both CPK9 and CPK33 for accurate guard cell function.

Kinase activity-dependent stability of calcium/calmodulin-dependent protein kinase of Lotus japonicus.

MAIN CONCLUSION: Accumulation of calcium/calmodulin-dependent protein kinase (CCaMK) in root cell nucleus depends on its kinase activity but not on nuclear symbiotic components crucial for nodulation. Plant calcium/calmodulin-dependent protein kinase (CCaMK) is a key regulator of symbioses with rhizobia and arbuscular mycorrhizal fungi as it decodes symbiotic calcium signals induced by microsymbionts. CCaMK is expressed mainly in root cells and localizes to the nucleus, where microsymbiont-triggered calcium oscillations occur. The molecular mechanisms that control CCaMK localization are unknown. Here, we analyzed the expression and subcellular localization of mutated CCaMK in the roots of Lotus japonicus and found a clear relation between CCaMK kinase activity and its stability. Kinase-defective CCaMK variants showed lower protein levels than the variants with kinase activity. The levels of transcripts driven by the CaMV 35S promoter were similar among the variants, indicating that stability of CCaMK is regulated post-translationally. We also demonstrated that CCaMK localized to the root cell nucleus in several symbiotic mutants, including cyclops, an interaction partner and phosphorylation target of CCaMK. Our results suggest that kinase activity of CCaMK is required not only for the activation of downstream symbiotic components but also for its stability in root cells.

Guard Cell Salicylic Acid Signaling Is Integrated into Abscisic Acid Signaling via the Ca2+/CPK-Dependent Pathway.

The phenolic hormone salicylic acid (SA) induces stomatal closure. It has been suggested that SA signaling is integrated with abscisic acid (ABA) signaling in guard cells, but the integration mechanism remains unclear. The Ca2+-independent protein kinase Open Stomata1 (OST1) and Ca2+-dependent protein kinases (CPKs) are key for ABA-induced activation of the slow-type anion channel SLAC1 and stomatal closure. Here, we show that SA-induced stomatal closure and SA activation of slow-type anion channel are impaired in the CPK disruption mutant cpk3-2 cpk6-1 but not in the OST1 disruption mutant ost1-3 We also found that the key phosphorylation sites of SLAC1 in ABA signaling, serine-59 and serine-120, also are important for SA signaling. Chemiluminescence-based detection of superoxide anion revealed that SA did not require CPK3 and CPK6 for the induction of reactive oxygen species production. Taken together, our results suggest that SA activates peroxidase-mediated reactive oxygen species signal that is integrated into Ca2+/CPK-dependent ABA signaling branch but not the OST1-dependent signaling branch in Arabidopsis (Arabidopsis thaliana) guard cells.

Characterization and functional analysis of calcium/calmodulin-dependent protein kinases (CaMKs) in the nematode-trapping fungus Arthrobotrys oligospora.

Ca2+/calmodulin-dependent protein kinases (CaMKs) are unique second-messenger molecules that impact almost all cellular processes in eukaryotes. In this study, five genes encoding different CaMKs were characterized in the nematode-trapping fungus Arthrobotrys oligospora. These CaMKs, which were retrieved from the A. oligospora genome according to their orthologs in fungi such as Aspergillus nidulans and Neurospora crassa, were expressed at a low level in vitro during mycelial growth stages. Five deletion mutants corresponding to these CaMKs led to growth defects in different media and increased sensitivity to several environmental stresses, including H2O2, menadione, SDS, and Congo red; they also reduced the ability to produce conidia and traps, thus causing a deficiency in nematicidal ability as well. In addition, the transcriptional levels of several typical sporulation-related genes, such as MedA, VelB, and VeA, were down-regulated in all ΔCaMK mutants compared with the wild-type (WT) strain. Moreover, these mutants exhibited hypersensitivity to heat shock and ultraviolet-radiation stresses compared with the WT strain. These results suggest that the five CaMKs in A. oligospora are involved in regulating multiple cellular processes, such as growth, environmental stress tolerance, conidiation, trap formation, and virulence.

Death-associated protein kinase 1 phosphorylates Pin1 and inhibits its prolyl isomerase activity and cellular function.

Pin1 is a phospho-specific prolyl isomerase that regulates numerous key signaling molecules and whose deregulation contributes to disease notably cancer. However, since prolyl isomerases are often believed to be constitutively active, little is known whether and how Pin1 catalytic activity is regulated. Here, we identify death-associated protein kinase 1 (DAPK1), a known tumor suppressor, as a kinase responsible for phosphorylation of Pin1 on Ser71 in the catalytic active site. Such phosphorylation fully inactivates Pin1 catalytic activity and inhibits its nuclear location. Moreover, DAPK1 inhibits the ability of Pin1 to induce centrosome amplification and cell transformation. Finally, Pin1 pSer71 levels are positively correlated with DAPK1 levels and negatively with centrosome amplification in human breast cancer. Thus, phosphorylation of Pin1 Ser71 by DAPK1 inhibits its catalytic activity and cellular function, providing strong evidence for an essential role of the Pin1 enzymatic activity for its cellular function.

Revisiting paradigms of Ca2+ signaling protein kinase regulation in plants.

Calcium (Ca2+) serves as a universal second messenger in eukaryotic signal transduction. Understanding the Ca2+ activation kinetics of Ca2+ sensors is critical to understanding the cellular signaling mechanisms involved. In this review, we discuss the regulatory properties of two sensor classes: the Ca2+-dependent protein kinases (CPKs/CDPKs) and the calcineurin B-like (CBL) proteins that control the activity of CBL-interacting protein kinases (CIPKs) and identify emerging topics and some foundational points that are not well established experimentally. Most plant CPKs are activated by physiologically relevant Ca2+ concentrations except for those with degenerate EF hands, and new results suggest that the Ca2+-dependence of kinase activation may be modulated by both protein-protein interactions and CPK autophosphorylation. Early results indicated that activation of plant CPKs by Ca2+ occurred by relief of autoinhibition. However, recent studies of protist CDPKs suggest that intramolecular interactions between CDPK domains contribute allosteric control to CDPK activation. Further studies are required to elucidate the mechanisms regulating plant CPKs. With CBL-CIPKs, the two major activation mechanisms are thought to be (i) binding of Ca2+-bound CBL to the CIPK and (ii) phosphorylation of residues in the CIPK activation loop. However, the relative importance of these two mechanisms in regulating CIPK activity is unclear. Furthermore, information detailing activation by physiologically relevant [Ca2+] is lacking, such that the paradigm of CBLs as Ca2+ sensors still requires critical, experimental validation. Developing models of CPK and CIPK regulation is essential to understand how these kinases mediate Ca2+ signaling and to the design of experiments to test function in vivo.