Eukaryotic Signal Transduction Research Articles

Robust approach for production of the human oncology target Aurora kinase B in complex with its binding partner INCENP

Protein kinases are key players in many eukaryotic signal transduction cascades and are as a result often linked to human disease. In humans, the mitotic protein kinase family of Aurora kinases consist of three members: Aurora A, B and C. All three members are involved in cell division with proposed implications in various human cancers. The human Aurora kinase B has in particular proven challenging to study with structural biology approaches, and this is mainly due to difficulties in producing the large quantities of active enzyme required for such studies. Here, we present a novel and E. coli-based production system that allows for production of milligram quantities of well-folded and active human Aurora B in complex with its binding partner INCENP. The complex is produced as a continuous polypeptide chain and the resulting fusion protein is cleaved with TEV protease to generate a stable and native heterodimer of the Aurora B:INCENP complex. The activity, stability and degree of phosphorylation of the protein complex was quantified by using a coupled ATPase assay, 31P NMR spectroscopy and mass spectrometry. The developed production system enables isotope labeling and we here report the first 1H–15N-HSQC of the human Aurora B:INCENP complex. Our developed production strategy paves the way for future structural and functional studies of Aurora B and can as such assist the development of novel anticancer drugs targeting this important mitotic protein kinase.

Actinomycetes and Fungi Isolated From Maliau Basin, Sabah and The Screening For Novel Secondary Metabolites Against Eukaryotic Signal Transduction and Mycobacterium

Maliau Basin is one of the world's hot spot of biodiversity.

A peroxiredoxin-P38 MAPK scaffold increases MAPK activity by MAP3K-independent mechanisms

Peroxiredoxins (Prdxs) utilize reversibly oxidized cysteine residues to reduce peroxides and promote H2O2 signal transduction, including H2O2-induced activation of P38 MAPK. Prdxs form H2O2-induced disulfide complexes with many proteins, including multiple kinases involved in P38 MAPK signaling. Here, we show that a genetically encoded fusion between a Prdx and P38 MAPK is sufficient to hyperactivate the kinase in yeast and human cells by a mechanism that does not require the H2O2-sensing cysteine of the Prdx. We demonstrate that a P38-Prdx fusion protein compensates for loss of the yeast scaffold protein Mcs4 and MAP3K activity, driving yeast into mitosis. Based on our findings, we propose that the H2O2-induced formation of Prdx-MAPK disulfide complexes provides an alternative scaffold and signaling platform for MAPKK-MAPK signaling. The demonstration that formation of a complex with a Prdx is sufficient to modify the activity of a kinase has broad implications for peroxide-based signal transduction in eukaryotes.

The FHA domain protein ArnA functions as a global DNA damage response repressor in the hyperthermophilic archaeon Saccharolobus islandicus.

Forkhead-associated (FHA) domain proteins specifically recognize phosphorylated threonine via the FHA domain and are involved in signal transduction in various processes especially DNA damage response (DDR) and cell cycle regulation in eukaryotes. Although FHA domain proteins are found in prokaryotes, archaea, and bacteria, their functions are far less clear as compared to the eukaryotic counterparts, and it has not been studied whether archaeal FHA proteins play a role in DDR. Here, we have characterized an FHA protein from the hyperthermophilic Crenarchaeon Saccharolobus islandicus (SisArnA) by genetic, biochemical, and transcriptomic approaches. We find that ΔSisarnA exhibits higher resistance to DNA damage agent 4-nitroquinoline 1-oxide (NQO). The transcription of ups genes, encoding the proteins for pili-mediated cell aggregation and cell survival after DDR, is elevated in ΔSisarnA. The interactions of SisArnA with two predicted partners, SisvWA1 (SisArnB) and SisvWA2 (designated as SisArnE), were enhanced by phosphorylation in vitro. ΔSisarnB displays higher resistance to NQO than the wild type. In addition, the interaction between SisArnA and SisArnB, which is reduced in the NQO-treated cells, is indispensable for DNA binding in vitro. These indicate that SisArnA and SisArnB work together to inhibit the expression of ups genes in vivo. Interestingly, ΔSisarnE is more sensitive to NQO than the wild type, and the interaction between SisArnA and SisArnE is strengthened after NQO treatment, suggesting a positive role of SisArnE in DDR. Finally, transcriptomic analysis reveals that SisArnA represses a number of genes, implying that archaea apply the FHA/phospho-peptide recognition module for extensive transcriptional regulation. IMPORTANCE Cellular adaption to diverse environmental stresses requires a signal sensor and transducer for cell survival. Protein phosphorylation and its recognition by forkhead-associated (FHA) domain proteins are widely used for signal transduction in eukaryotes. Although FHA proteins exist in archaea and bacteria, investigation of their functions, especially those in DNA damage response (DDR), is limited. Therefore, the evolution and functional conservation of FHA proteins in the three domains of life is still a mystery. Here, we find that an FHA protein from the hyperthermophilic Crenarchaeon Saccharolobus islandicus (SisArnA) represses the transcription of pili genes together with its phosphorylated partner SisArnB. SisArnA derepression facilitates DNA exchange and repair in the presence of DNA damage. The fact that more genes including a dozen of those involved in DDR are found to be regulated by SisArnA implies that the FHA/phosphorylation module may serve as an important signal transduction pathway for transcriptional regulation in archaeal DDR.

PsnGPCRdb: The Structure-network Database of G Protein Coupled Receptors

G protein coupled receptors (GPCRs) are critical eukaryotic signal transduction gatekeepers and represent the largest protein superfamily in the human proteome, with more than 800 members. They share seven transmembrane helices organized in an up-down bundle architecture. GPCR-mediated signaling pathways have been linked to numerous human diseases, and GPCRs are the targets of approximately 35% of all drugs currently on the market.Structure network analysis, a graph theory-based approach, represents a cutting-edge tool to deeply understand GPCR function, which strongly relies on communication between the extracellular and intracellular poles of their structure.psnGPCRdb stores the structure networks (i.e., linked nodes, hubs, communities and communication pathways) computed on all updated GPCR structures in the Protein Data Bank, in their isolated states or in complex with extracellular and/or intracellular molecules. The structure communication signatures of a sub-family or family of GPCRs as well as of their small-molecule activators or inhibitors are stored as consensus networks. The database stores also all meaningful structure network-based comparisons (i.e., difference networks) of functionally different states (i.e., inactive or active) of a given receptor sub-type, or of consensus networks representative of a receptor sub-type, type, sub-family or family. Single or consensus GPCR networks hold also information on amino acid conservation. The database allows to graphically analyze 3D structure networks together with interactive data-tables. Ligand-centric networks can be analyzed as well.psnGPCRdb is unique and represents a powerful resource to unravel GPCR function with important implications in cell signaling and drug design.psnGPCRdb is freely available at: http://webpsn.hpc.unimo.it/psngpcr.php.

Transcriptomic Dynamics of Active and Inactive States of Rho GTPase MoRho3 in Magnaporthe oryzae.

The small Rho GTPase acts as a molecular switch in eukaryotic signal transduction, which plays a critical role in polar cell growth and vesicle trafficking. Previous studies demonstrated that constitutively active (CA) mutant strains, of MoRho3-CA were defective in appressorium formation. While dominant-negative (DN) mutant strains MoRho3-DN shows defects in polar growth. However, the molecular dynamics of MoRho3-mediated regulatory networks in the pathogenesis of Magnaporthe oryzae still needs to be uncovered. Here, we perform comparative transcriptomic profiling of MoRho3-CA and MoRho3-DN mutant strains using a high-throughput RNA sequencing approach. We find that genetic manipulation of MoRho3 significantly disrupts the expression of 28 homologs of Saccharomyces cerevisiae Rho3-interacting proteins, including EXO70, BNI1, and BNI2 in the MoRho3 CA, DN mutant strains. Functional enrichment analyses of up-regulated DEGs reveal a significant enrichment of genes associated with ribosome biogenesis in the MoRho3-CA mutant strain. Down-regulated DEGs in the MoRho3-CA mutant strains shows significant enrichment in starch/sucrose metabolism and the ABC transporter pathway. Moreover, analyses of down-regulated DEGs in the in MoRho3-DN reveals an over-representation of genes enriched in metabolic pathways. In addition, we observe a significant suppression in the expression levels of secreted proteins suppressed in both MoRho3-CA and DN mutant strains. Together, our results uncover expression dynamics mediated by two states of the small GTPase MoRho3, demonstrating its crucial roles in regulating the expression of ribosome biogenesis and secreted proteins.

Integrated Strategy for Unbiased Profiling of the Histidine Phosphoproteome.

Histidine phosphorylation (pHis), which plays a key role in signal transduction in bacteria and lower eukaryotes, has been shown to be involved in tumorigenesis. Due to its chemical instability, substoichiometric properties, and lack of specific enrichment reagents, there is a lack of approaches for specific and unbiased enrichment of pHis-proteins/peptides. In this study, an integrated strategy was established and evaluated as an unbiased tool for exploring the histidine phosphoproteome. First, taking advantage of the lower charge states of pHis-peptides versus the non-modified naked peptides at weak acid solution (∼pH 2.7), strong cation exchange (SCX) chromatography was used to differentiate modified and non-modified naked peptides. Furthermore, selective enrichment of the pHis-peptide was performed by applying Cu-IDA beads enrichment. Finally, stable isotope dimethyl labeling was introduced to guarantee high-confidence assignment of pHis-peptides. Using this integrated strategy, 563 different pHis-peptides (H = 1) in 385 proteins were identified from HeLa lysates. Motif analysis revealed that pHis prefers hydrophobic amino acids and has the consensus motif-HxxK, which covered the reports from different approaches. Thus, our method may provide an unbiased and effective tool to reveal histidine phosphoproteome and to study the biological process and function of histidine phosphorylation.

Bioactive Ether Lipids: Primordial Modulators of Cellular Signaling.

The primacy of lipids as essential components of cellular membranes is conserved across taxonomic domains. In addition to this crucial role as a semi-permeable barrier, lipids are also increasingly recognized as important signaling molecules with diverse functional mechanisms ranging from cell surface receptor binding to the intracellular regulation of enzymatic cascades. In this review, we focus on ether lipids, an ancient family of lipids having ether-linked structures that chemically differ from their more prevalent acyl relatives. In particular, we examine ether lipid biosynthesis in the peroxisome of mammalian cells, the roles of selected glycerolipids and glycerophospholipids in signal transduction in both prokaryotes and eukaryotes, and finally, the potential therapeutic contributions of synthetic ether lipids to the treatment of cancer.

How to study intertwined and autoregulated eukaryotic signal transduction pathways.

Mitogen-activated protein kinase (MAPK) pathways are a major means of eukaryotic cells to adapt to environmental changes, in the case of microorganisms, and to nutritional and hormonal signals, in the case of multicellular organisms. Numerous defects in such architecturally conserved pathways have been associated with different human cancers. These signaling cascades usually commence with sensors located in the plasma membrane, which through specific protein kinases activate a conserved tripartite MAPK module. Phosphorylation of their targets, that is, cytosolic proteins and/or transcription factors, then triggers the proper cellular response. In the model yeast Saccharomycescerevisiae and other fungi, the cell wall integrity pathway (CWI) has been extensively studied and its components may serve as targets for antifungal drugs of clinical and agricultural importance. Another well-known MAPK cascade, the high osmolarity glycerol (HOG) pathway, is required to cope with osmotic stress. In the past decade, it has become increasingly evident that such pathways do not act in a linear top-down fashion, but are highly regulated by internal feedback mechanisms as well as by cross-pathway interactions. The work of Jiménez-Gutiérrez et al. in this issue provides an elegant way to identify new players in these complex networks. Comment on: https://doi.org/10.1111/febs.15288.

Kinobead/LC-MS Phosphokinome Profiling Enables Rapid Analyses of Kinase-Dependent Cell Signaling Networks.

Kinase-catalyzed protein phosphorylation is fundamental to eukaryotic signal transduction, regulating most cellular processes. Kinases are frequently dysregulated in cancer, inflammation, and degenerative diseases, and because they can be inhibited with small molecules, they became important drug targets. Accordingly, analytical approaches that determine kinase activation states are critically important to understand kinase-dependent signal transduction and to identify novel drug targets and predictive biomarkers. Multiplexed inhibitor beads (MIBs or kinobeads) efficiently enrich kinases from cell lysates for liquid chromatography-mass spectrometry (LC-MS) analysis. When combined with phosphopeptide enrichment, kinobead/LC-MS can also quantify the phosphorylation state of kinases, which determines their activation state. However, an efficient kinobead/LC-MS kinase phospho-profiling protocol that allows routine analyses of cell lines and tissues has not yet been developed. Here, we present a facile workflow that quantifies the global phosphorylation state of kinases with unprecedented sensitivity. We also found that our kinobead/LC-MS protocol can measure changes in kinase complex composition and show how these changes can indicate kinase activity. We demonstrate the utility of our approach in specifying kinase signaling pathways that control the acute steroidogenic response in Leydig cells; this analysis establishes the first comprehensive framework for the post-translational control of steroid biosynthesis.

Association of Both Inhibitory and Stimulatory Gα Subunits Implies Adenylyl Cyclase 5 Deactivation.

Adenylyl cyclase (AC) generates cyclic AMP required for a variety of cellular functions, and its regulation plays a major role in cellular signal transduction in eukaryotes and prokaryotes. All membrane-bound AC isoforms in eukaryotes can be activated by stimulatory G-proteins, but only AC1, AC5, and AC6 can be both stimulated and inhibited by active Gα subunits, Gαs and Gαi, respectively. In principle, these Gαi-sensitive AC isoforms could form both binary and ternary complexes with Gα subunits due to the noncompetitive association of inhibitory and stimulatory Gα. However, the formation and possible catalytic activity of a putative ternary complex have not yet been experimentally confirmed due to its proposed short-lived nature. Here, the catalytic activity of such a ternary complex consisting of apo AC5, stimulatory Gαolf, and inhibitory Gαi1 is investigated via classical molecular dynamics simulations. Trajectories of inhibited and stimulated binary complexes, AC5:Gαi1 and AC5:Gαolf, respectively, as well as Gα-free AC5 were also obtained to compare the sampled AC5 conformation in the ternary complex to those sampled under different Gα conditions. This comparison suggests that association of both Gα subunits results in an AC5 conformation similar to that sampled by the AC5:Gαi1 complex, indicating that the ternary complex mainly samples an inactive conformation.

Phospholipid homeostasis plays an important role in fungal development, fungicide resistance and virulence in Fusarium graminearum

Phospholipids are major structural components of all cell membranes and participate in energy storage, signal transduction and environmental adaptability in eukaryotes. To date, the enzymes involved in phospholipid biosynthesis have been well characterized in budding yeast. However, their functions in filamentous fungi are largely unclear, especially their contribution to the interaction between phytopathogenic filamentous fungi and plants. In this study, we identified 10 phospholipid biosynthesis-related genes and genetically analyzed their functions in the Fusarium head blight pathogen Fusarium graminearum. The results of this study indicate that phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are critical for fungal vegetative growth. The biosynthesis of PE and PC is largely dependent on FgPsd2, FgCho2 and FgOpi3 in the de novo pathway of phospholipid biosynthesis in F. graminearum. Phospholipid biosynthetic gene mutants showed abnormal conidiation, increased sensitivity to fungicides and the oxidative stress agent H2O2, and defective endocytosis, especially the ΔFgpsd2, ΔFgcho2 and ΔFgopi3 mutants. Importantly, this study shows for the first time that the de novo pathway of phospholipid biosynthesis is required for mycotoxin production and full virulence in plant pathogenic fungi.

An endogenous chemorepellent directs cell movement by inhibiting pseudopods at one side of cells.

Eukaryotic chemoattraction signal transduction pathways, such as those used by Dictyostelium discoideum to move toward cAMP, use a G protein–coupled receptor to activate multiple conserved pathways such as PI3 kinase/Akt/PKB to induce actin polymerization and pseudopod formation at the front of a cell, and PTEN to localize myosin II to the rear of a cell. Relatively little is known about chemorepulsion. We previously found that AprA is a chemorepellent protein secreted by Dictyostelium cells. Here we used 29 cell lines with disruptions of cAMP and/or AprA signal transduction pathway components, and delineated the AprA chemorepulsion pathway. We find that AprA uses a subset of chemoattraction signal transduction pathways including Ras, protein kinase A, target of rapamycin (TOR), phospholipase A, and ERK1, but does not require the PI3 kinase/Akt/PKB and guanylyl cyclase pathways to induce chemorepulsion. Possibly as a result of not using the PI3 kinase/Akt/PKB pathway and guanylyl cyclases, AprA does not induce actin polymerization or increase the pseudopod formation rate, but rather appears to inhibit pseudopod formation at the side of cells closest to the source of AprA.

MAPK/ERK and JNK pathways regulate lipid synthesis and cell growth of Chlamydomonas reinhardtii under osmotic stress, respectively

Microalgae have great potential for the production of biofuels due to the ability of the organism to accumulate large quantities of storage lipids under stress conditions. Mitogen activated protein kinase (MAPK) signaling cascades are widely recognized for their role in stress response signal transduction in eukaryotes. To assess the correlation between MAPK activation and lipid productivity, Chlamydomonas reinhardtii was studied under various concentrations of NaCl. The results demonstrated that C. reinhardtii exhibits elevated levels of extracellular-signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activities after undergoing osmotic stress, as well as an increase in cellular lipid content. To establish a more direct causal link between both kinases and lipid productivity, C. reinhardtii was subjected to biochemically induced regulation of ERK and JNK pathways. Activating the MEK-ERK pathway via C6 ceramide treatment increased ERK activation and lipid production simultaneously, while PD98059 mediated inhibition of the pathway yielded opposite results. Interestingly, suppression of the JNK pathway with SP600125 resulted in a substantial decrease in cell viability under osmotic stress. These results suggest that ERK and JNK MAP kinases have important roles in microalgal lipid accumulation and cell growth under osmotic stress, respectively.

Insights into the GTP-dependent allosteric control of c-di-GMP hydrolysis from the crystal structure of PA0575 protein from Pseudomonasaeruginosa.

Bis-(3'-5')-cyclic diguanylic acid (c-di-GMP) belongs to the class of cyclic dinucleotides, key carriers of cellular information in prokaryotic and eukaryotic signal transduction pathways. In bacteria, the intracellular levels of c-di-GMP and their complex physiological outputs are dynamically regulated by environmental and internal stimuli, which control the antagonistic activities of diguanylate cyclases (DGCs) and c-di-GMP specific phosphodiesterases (PDEs). Allostery is one of the major modulators of the c-di-GMP-dependent response. Both the c-di-GMP molecule and the proteins interacting with this second messenger are characterized by an extraordinary structural plasticity, which has to be taken into account when defining and possibly predicting c-di-GMP-related processes. Here, we report a structure-function relationship study on the catalytic portion of the PA0575 protein from Pseudomonasaeruginosa, bearing both putative DGC and PDE domains. The kinetic and structural studies indicate that the GGDEF-EAL portion is a GTP-dependent PDE. Moreover, the crystal structure confirms the high degree of conformational flexibility of this module. We combined structural analysis and protein engineering studies to propose the possible molecular mechanism guiding the nucleotide-dependent allosteric control of catalysis; we propose that the role exerted by GTP via the GGDEF domain is to allow the two EAL domains to form a dimer, the species competent to enter PDE catalysis.

Genome-wide analyses of the bHLH superfamily in crustaceans: reappraisal of higher-order groupings and evidence for lineage-specific duplications.

The basic helix-loop-helix (bHLH) proteins represent a key group of transcription factors implicated in numerous eukaryotic developmental and signal transduction processes. Characterization of bHLHs from model species such as humans, fruit flies, nematodes and plants have yielded important information on their functions and evolutionary origin. However, relatively little is known about bHLHs in non-model organisms despite the availability of a vast number of high-throughput sequencing datasets, enabling previously intractable genome-wide and cross-species analyses to be now performed. We extensively searched for bHLHs in 126 crustacean species represented across major Crustacea taxa and identified 3777 putative bHLH orthologues. We have also included seven whole-genome datasets representative of major arthropod lineages to obtain a more accurate prediction of the full bHLH gene complement. With focus on important food crop species from Decapoda, we further defined higher-order groupings and have successfully recapitulated previous observations in other animals. Importantly, we also observed evidence for lineage-specific bHLH expansions in two basal crustaceans (branchiopod and copepod), suggesting a mode of evolution through gene duplication as an adaptation to changing environments. In-depth analysis on bHLH-PAS members confirms the phenomenon coined as ‘modular evolution’ (independently evolved domains) typically seen in multidomain proteins. With the amphipod Parhyale hawaiensis as the exception, our analyses have focused on crustacean transcriptome datasets. Hence, there is a clear requirement for future analyses on whole-genome sequences to overcome potential limitations associated with transcriptome mining. Nonetheless, the present work will serve as a key resource for future mechanistic and biochemical studies on bHLHs in economically important crustacean food crop species.

The Ancient Link between G-Protein-Coupled Receptors and C-Terminal Phospholipid Kinase Domains.

ABSTRACTSensing external signals and transducing these into intracellular responses requires a molecular signaling system that is crucial for every living organism. Two important eukaryotic signal transduction pathways that are often interlinked are G-protein signaling and phospholipid signaling. Heterotrimeric G-protein subunits activated by G-protein-coupled receptors (GPCRs) are typical stimulators of phospholipid signaling enzymes such as phosphatidylinositol phosphate kinases (PIPKs) or phospholipase C (PLC). However, a direct connection between the two pathways likely exists in oomycetes and slime molds, as they possess a unique class of GPCRs that have a PIPK as an accessory domain. In principle, these so-called GPCR-PIPKs have the capacity of perceiving an external signal (via the GPCR domain) that, via PIPK, directly activates downstream phospholipid signaling. Here we reveal the sporadic occurrence of GPCR-PIPKs in all eukaryotic supergroups, except for plants. Notably, all species having GPCR-PIPKs are unicellular microorganisms that favor aquatic environments. Phylogenetic analysis revealed that GPCR-PIPKs are likely ancestral to eukaryotes and significantly expanded in the last common ancestor of oomycetes. In addition to GPCR-PIPKs, we identified five hitherto-unknown classes of GPCRs with accessory domains, four of which are universal players in signal transduction. Similarly to GPCR-PIPKs, this enables a direct coupling between extracellular sensing and downstream signaling. Overall, our findings point to an ancestral signaling system in eukaryotes where GPCR-mediated sensing is directly linked to downstream responses.

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.

Combined 15N-Labeling and TandemMOAC Quantifies Phosphorylation of MAP Kinase Substrates Downstream of MKK7 in Arabidopsis.

Reversible protein phosphorylation is a widespread posttranslational modification that plays a key role in eukaryotic signal transduction. Due to the dynamics of protein abundance, low stoichiometry and transient nature of protein phosphorylation, the detection and accurate quantification of substrate phosphorylation by protein kinases remains a challenge in phosphoproteome research. Here, we combine tandem metal-oxide affinity chromatography (tandemMOAC) with stable isotope 15N metabolic labeling for the measurement and accurate quantification of low abundant, transiently phosphorylated peptides by mass spectrometry. Since tandemMOAC is not biased toward the enrichment of acidophilic, basophilic, or proline-directed kinase substrates, the method is applicable to identify targets of all these three types of protein kinases. The MKK7-MPK3/6 module, for example, is involved in the regulation of plant development and plant basal and systemic immune responses, but little is known about downstream cascade components. Using our here described phosphoproteomics approach we identified several MPK substrates downstream of the MKK7-MPK3/6 phosphorylation cascade in Arabidopsis. The identification and validation of dynamin-related protein 2 as a novel phosphorylation substrate of the MKK7-MPK3/6 module establishes a novel link between MPK signaling and clathrin-mediated vesicle trafficking.

Proposed Role for KaiC-Like ATPases as Major Signal Transduction Hubs in Archaea.

ABSTRACTAll organisms must adapt to ever-changing environmental conditions and accordingly have evolved diverse signal transduction systems. In bacteria, the most abundant networks are built around the two-component signal transduction systems that include histidine kinases and receiver domains. In contrast, eukaryotic signal transduction is dominated by serine/threonine/tyrosine protein kinases. Both of these systems are also found in archaea, but they are not as common and diversified as their bacterial and eukaryotic counterparts, suggesting the possibility that archaea have evolved other, still uncharacterized signal transduction networks. Here we propose a role for KaiC family ATPases, known to be key components of the circadian clock in cyanobacteria, in archaeal signal transduction. The KaiC family is notably expanded in most archaeal genomes, and although most of these ATPases remain poorly characterized, members of the KaiC family have been shown to control archaellum assembly and have been found to be a stable component of the gas vesicle system in Halobacteria. Computational analyses described here suggest that KaiC-like ATPases and their homologues with inactivated ATPase domains are involved in many other archaeal signal transduction pathways and comprise major hubs of complex regulatory networks. We predict numerous input and output domains that are linked to KaiC-like proteins, including putative homologues of eukaryotic DEATH domains that could function as adapters in archaeal signaling networks. We further address the relationships of the archaeal family of KaiC homologues to the bona fide KaiC of cyanobacteria and implications for the existence of a KaiC-based circadian clock apparatus in archaea.