The reduced kinome of Ostreococcus tauri: core eukaryotic signalling components in a tractable model species
© Hindle et al.; licensee BioMed Central Ltd. 2014
Received: 24 January 2014
Accepted: 8 July 2014
Published: 2 August 2014
The current knowledge of eukaryote signalling originates from phenotypically diverse organisms. There is a pressing need to identify conserved signalling components among eukaryotes, which will lead to the transfer of knowledge across kingdoms. Two useful properties of a eukaryote model for signalling are (1) reduced signalling complexity, and (2) conservation of signalling components. The alga Ostreococcus tauri is described as the smallest free-living eukaryote. With less than 8,000 genes, it represents a highly constrained genomic palette.
Our survey revealed 133 protein kinases and 34 protein phosphatases (1.7% and 0.4% of the proteome). We conducted phosphoproteomic experiments and constructed domain structures and phylogenies for the catalytic protein-kinases. For each of the major kinases families we review the completeness and divergence of O. tauri representatives in comparison to the well-studied kinomes of the laboratory models Arabidopsis thaliana and Saccharomyces cerevisiae, and of Homo sapiens. Many kinase clades in O. tauri were reduced to a single member, in preference to the loss of family diversity, whereas TKL and ABC1 clades were expanded. We also identified kinases that have been lost in A. thaliana but retained in O. tauri. For three, contrasting eukaryotic pathways – TOR, MAPK, and the circadian clock – we established the subset of conserved components and demonstrate conserved sites of substrate phosphorylation and kinase motifs.
We conclude that O. tauri satisfies our two central requirements. Several of its kinases are more closely related to H. sapiens orthologs than S. cerevisiae is to H. sapiens. The greatly reduced kinome of O. tauri is therefore a suitable model for signalling in free-living eukaryotes.
KeywordsConserved eukaryote signalling Protein kinase phylogeny Ostreococcus tauri Model kinome Phosphorylation TOR signalling MAPK cascade Circadian clock
Protein kinases are a major component of the complex signalling networks that coordinate all fundamental cellular processes, including transcription, cell cycle and metabolism. Protein kinases and phosphatases elicit reversible phosphorylation, which enable the rapid cellular responses that are crucial for survival in a continually changing environment. Protein kinases activate and deactivate proteins by addition of the gamma-phosphate from ATP to serine (S), threonine (T), tyrosine (Y), aspartate (D) or histidine (H) amino acid residues . Cascades of consecutive kinase-mediated phosphorylation events constitute the backbone of signalling pathways . The complexity of the signalling networks scales with size. Part of this complexity is constrained by the number of genes encoding protein kinases, also known as the kinome. The number of encoded protein kinases in free-living eukaryotes ranges from as little as 126 kinases in Saccharomyces cerevisiae to ~1000 in Arabidopsis thaliana. Between these extremes, surveyed organisms include Dictyostelium discoideum with 285 kinases , the fruit fly Drosophila melanogaster with 251, and Homo sapiens with 518 kinases . Minimal kinomes are present in parasites that are not obviously representative of other tractable species. The kinome of the parasitic fungus Encephalitozoon cuniculi has only 32 kinases and lacks sequences that are ubiquitous in the kinomes of free-living eukaryotes, including the STE family, TOR and AMPK. E. cuniculi kinases are also highly divergent within fungi: 9 are reported to have no clear orthologs. The protozoan Giardia lamblia can be grown in pure culture and has a small genome of only 6,500 ORFs with a core of only 80 kinases, of which 14 have no clear orthologs and 5 are Giardia-specific . The remainder of the kinome is composed of a large expansion of 198 Nek kinases, 139 of which are likely to be catalytically inactive. G. lamblia kinase domains were also found to have a mean sequence identity of only 40% with H. sapiens, lower than plant and fungal kinases (49-50%).
It was originally thought that S/T and Y kinases were unique to eukaryotes, and that bacteria and archaea operated a parallel system of H and D phosphorylation. However it is now known that S/T and Y phosphorylation is also important in both bacteria  and archaea . While many eukaryote-like kinases (ELK) in bacteria share only remote sequence similarity with eukaryotic protein kinases (ePK) they share strong structural similarities [11, 12]. The Rio and Bud32 families of kinases are common to both eukaryote and archaea . Conversely, the Histidine kinases (HK) are also found in eukaryotes, where their roles include osmoregulation in several species  and ethylene hormone signalling in A. thaliana.
A well conserved 250 – 300 amino acid catalytic domain, known as the ePK domain , is present in most protein kinases and mediates protein phosphorylation. A small subset of kinases do not possess the ePK domain and are regarded as atypical protein kinases (aPK) . As ePKs are structurally related, a common evolutionary ancestry, distinct from aPKs has been proposed . Members of the protein kinase ePK family  are divided into the following major groups: AGC (named after protein kinases A, G and C), TK (Tyrosine Kinases), TKL (Tyrosine Kinase-Like kinases), CaMK (Calcium/Calmodulin-dependent Kinases), CMGC (containing Cyclin-Dependent Kinases (CDK); Mitogen-Activated Protein Kinases (MAPK); Glycogen Synthase Kinase 3 (GSK3) and Cyclin-Dependent Kinase-Like (CKL)), CK1 (Casein Kinase 1), CK2 (Casein Kinase 2), STE (containing homologs of the yeast Sterile kinases), and AUR (Aurora Kinases). The TK family, particularly transmembrane receptor kinases, account for the majority of receptor kinases in humans and serve as cell-surface receptors for growth factors that trigger cell growth, proliferation and differentiation . Non metazoan-eukaryotes, including the green lineage, do not possess genuine TKs . Instead, Y phosphorylation is substituted by dual-specificity kinases that phosphorylate S/T as well as Y [17, 18].
In this study we survey the kinase components of O. tauri and assess its suitability as a model organism for eukaryotic signalling, based on two criteria: (1) reduced signalling complexity and (2) conservation of signalling components. O. tauri is a promising candidate as it is the smallest free-living eukaryote , with a 12.6 Mb genome, encoding 7,989 proteins with minimal genome duplication . This reduced genome might impose simplified signalling. O. tauri is part of the Chlorophyta clade within the Plantae supergroup , and is taxonomically positioned at the base of the green-plant lineage. Given its size and taxonomic position, it is a promising candidate for generating hypotheses that can be transferred to more complex eukaryotes. O. tauri has a streamlined cell structure comprising a single nucleus, mitochondrion, Golgi body and chloroplast . It possesses several benefits as an experimental model, cells can be readily and rapidly cultured in controlled laboratory conditions, where they undergo simple binary cell-division which can be synchronised by light/dark cycles. It has already been used as a model for the eukaryotic cell-cycle, helping to unify current understanding of cell-cycle regulation across eukaryotes . The lack of a cellulose plant cell wall facilitates transformation [24, 25] as well as organelle enrichment and protein extraction [26, 27]. These genetic and proteomic tools have already been applied to studies of protein turnover , nutrient deprivation  and the plant circadian clock in experimental [25, 28, 29] and mathematical approaches .
We survey the O. tauri kinome and examine conservation of protein sequences, through phylogenies of kinase orthologs in A. thaliana, H. sapiens and S. cerevisiae as the most widely studied models of plant, metazoan and fungal kinomes respectively. We then focus on three pathways, 1) TOR signalling in H. sapiens, 2) MAPK-mediated GSK3 signalling in A. thaliana and 3) the core circadian clock. We evaluate the capacity of O. tauri components to support signalling in current models of these exemplar pathways. Building on our recent proteomic surveys [26, 31, 32], we examine a large set of phosphorylated peptides detected by mass spectrometry and use these to validate phosphorylation-mediated signalling events in O. tauri. In combination with the phylogenetic evidence, we discuss the suitability of O. tauri as a model species to study protein kinase signalling.
Results and discussion
We compared the number of protein kinases for each family in O. tauri with other model organisms, using an existing, high-level classification derived from 22 eukaryotic kinomes . This data-mining approach was augmented by experimental identification of 5,563 phosphorylated O. tauri peptides from 107 liquid-chromatography-coupled mass spectrometry (LC-MS) experiments. These correspond to 3,994 uniquely identified phosphorylations of 2,214 peptide sequences of 1,252 proteins (Additional file 1: Table S1), including several conserved protein kinases, discussed below. In the process of identifying and categorising kinases in O. tauri, we identified a novel gene locus, corrected 9 existing gene models, and patched sequencing gaps in 25 gene loci with sequence information from Ostreococcus lucimarinus data to generate a more complete database for peptide identification. Protein domain diagrams are attached as Additional file 2: Figure S1 while the new and patched gene models and sequences are detailed in Additional file 3: Figure S2. Evidence of phosphorylation motifs conserved between species is presented in Additional file 4: Figure S3.
O. tauriprotein kinase and phosphatase survey
A survey of the 7,989 gene models  currently annotated in the O. tauri genome revealed 133 genes encoding catalytic protein-kinases and 32 protein phosphatases, respectively amounting to 1.7% and 0.4% of the known O. tauri loci (Figure 1B, Additional file 5: Table S2). The O. tauri kinome occupies a similar proportion of the genome to that found in S. cerevisiae (2%, 130 kinases)  and H. sapiens (2%, 426 kinases)  and is proportionally smaller than the A. thaliana kinome (3.1%, 981 kinases) , with which it shares the greatest sequence similarity of components (Additional file 6: Figure S4).
Phosphatases, in contrast, do not scale with the size of the genome. The Human Phosphatase Portal (HuPho)  reports 135 protein phosphatases of which 107 are Protein Tyrosine Phosphatases (PTPs). The remaining 28 S/T phosphatases consist of two families, Metal Dependent Protein Phosphatases (PPMs or PP2Cs) and Phosphoserine Protein Phosphatases (PPPs). A. thaliana contains 131 phosphatases of which 10 are PTPs and the remaining S/T phosphatases contain 38 PPPs and 83 PPMs . S. cerevisiae contains 25 protein phosphatases , which are composed of 6 PTPs, 12 PPPs and 7 PPMs. The O. tauri genome contains 32 protein phosphatases, which are composed of 8 PTPs, 10 PPPs and 14 PPMs. The higher proportions of S/T phosphatases to PTPs in O. tauri resemble the proportions found in higher A. thaliana more than S. cerevisiae and H. sapiens. The dominance of the PPM family within the S/T phosphatases in O. tauri is consistent with A. thaliana and H. sapiens but is in contrast to S. cerevisiae.
A categorisation of kinases into families by sequence similarity and phylogenetic analysis with the A. thaliana, S. cerevisiae, and H. sapiens kinomes confirmed the presence in O. tauri of all major ePK families (TKL, CaMK, CMGC, AGC, STE and CK1) present in the green lineage (Figure 1B). We also observed six small, conserved families of ePK-related protein kinases, which are classified as other-ePKs  and five families of aPKs. No Receptor-Like Kinases (RLKs) were found in O. tauri. The main ePK families account for a large proportion of the kinome in all the eukaryotes. O. tauri contains 13 TKL-like kinases, which is consistent with a large expansion of this family in the green lineage . In contrast the TKL family is absent in S. cerevisiae and many other fungal genomes . For such a small kinome, O. tauri contains a surprising abundance of 20 ABC1-like kinases, which have few functionally-characterised orthologs in other species [42–45]. Recent experimental technologies for targeted gene knock-out in O. tauri will therefore greatly assist in the elucidation of their function . Within ePK subfamilies, not all branches are equally conserved, as is evident in the following phylogenetic analyses (Additional file 7: Figures S5, Additional file 8: Figure S6 and Additional file 5: Table S2).
The TOR pathway: PIKK, CMGC and AGC kinase families
Target of rapamycin (TOR) mediated signalling is vital to the regulation of growth and the key components exist throughout eukaryotes . Here, we describe the phylogenetic relationships within the kinase families that participate in the TOR signalling pathway , aPK PI3K-related kinases (PIKK), and the ePK CMGC and AGC kinases.
PI3K-related kinases (PIKK): TOR, ATR, ATM, TRRAP and DNA-PK
A conserved family of cell-cycle control proteins, phosphatidyl-inositol-3-kinases (PI3Ks) are a class of kinases originally named after their ability to phosphorylate the 3′-hydroxyl group of phosphatidylinositols. The PI3Ks that also act as S/T protein kinases are called PI3K-related kinases (PIKK). Six PIKKs are present in eukaryotic genomes. Several of these couple the DNA damage sensing and repair pathway with the control of cell-cycle checkpoints, thereby maintaining the genetic integrity of the genome .
CMGC cell cycle family: CDK, MAPK, GSK3
MAPKs are S/T-specific protein kinases, closely related to CDKs, their growth and stress-response functions – including osmotic shock, oxidative stress and temperature response in plants have been extensively reviewed [57, 58]. We identified 3 plant-like MAPKs in O. tauri (Ot08g00430, Ot09g04000 and Ot15g00120, Figure 3D, Additional file 7: Figure S5A), which have 8, 7 and 3 groups of paraologous genes respectively in A. thaliana. For MAPK (Ot08g00430) we observed phosphorylation of a conserved Y on the T-X-Y motif of the activation loop (Additional file 6: Figure S4A), which indicates conserved modes of activation. The greatly reduced set of MAPKs in O. tauri is a remarkable feature of a highly reduced kinome.
GSK3 is a highly conserved eukaryote CMGC kinase. The chaperone Heat Shock Protein 90 (HSP90) regulates the autophosphorylation of the activating Y in GSK3 . Pharmacological evidence links both HSP90 and GSK3 with circadian timekeeping in O. tauri. O. tauri, like other algae, has a single copy of GSK3 (Ot04g00510), compared to the ten found in A. thaliana (Figure 3B). The O. tauri GSK3 kinase domain diverges considerably on its branch between H. sapiens and A. thaliana. However, the O. tauri GSK3 sequence is closer to H. sapiens (distance 0.71) than S. cerevisiae (1.24). O. tauri also contains a single ortholog candidate for HSP90 (Ot10g00440) (Figure 3C), while A. thaliana has four HSP90 paralogs . Two closely HSP90-related clades in Figure 3C, acting as outgroups to confirm HSP90 orthology, reveals further A. thaliana specialisation of HSPs that is shared in the O. tauri genome.
AGC Kinases: PDK1, S6K and PKG
PDK1 is the most important member of the AGC family in terms of phylogeny as it represents a highly conserved kinase, which has changed little since the divergence of eukaryotic AGCs . PDK1 in O. tauri is most similar to the two A. thaliana orthologs (1.26). It also groups closer to H. sapiens (1.67) than S. cerevisiae orthologs (1.81) (Figure 4A). PDK1 is thought to be a basal conserved kinase, which predates the divergence of ePKs , and is therefore used to root AGC phylogenies (Figure 4C and D). PDK1 has also been termed the ‘master kinase’ of AGC signal transduction  because of its critical role in cellular survival through the activation of Protein Kinase B (PKB, also known as Akt) and S6K in humans . However, out of these two PDK1 targets only S6K (Figure 4D) is conserved in A. thaliana and O. tauri.
The cAMP-dependent protein kinases (PKAs) and cGMP-dependent protein kinases (PKGs) are part of the same sub-family of kinase domains  and have similar domain components, and quaternary structure . PKG is composed of a single protein with cGMP binding and protein-kinase activity. PKA is a heterodimer composed of separate protein-kinase and cAMP binding subunits. The inactive complex disassociates when cAMP binds to the regulatory subunit, which releases the active protein-kinase component . As with many AGC proteins, a conserved C-terminal tail acts as a phosphorylation site for priming the protein-kinase active-site . There are five AGC kinases with cNMP binding domains in O. tauri. Two of these (Ot02g05760 and Ot13g01150) contain all three domain components and have kinase domains with the strongest similarity to H. sapiens PKAs (Figure 4B and Additional file 2: Figure S1). Ot13g01150, has the closest domain structure to H. sapiens PKA/PKG, and appears at the base of a subclade with two other kinases (90% confidence), branching prior to the divergence of PKA and PKG (Figure 4C).
Ot02g05760 is assigned with low confidence (59%) to the base of the PKG branch. However, it diverges near to the root of H. sapiens PKA-like kinases, which results in the domain being closer to PRKX (1.28) than to PKG (1.30); PRKX is part of the family of PKA catalytic subunits . This supports a PKA like activity for the domain, rather than the more constrained PKG substrate specificity .
The minimal TOR Pathway in O. tauri: An inventory
TOR is highly conserved across eukaryotes and acts as a master regulator for nutrient-responsive growth in yeast, metazoa , and plants . S6K1 and S6K2 are targets of the TOR pathway in A. thaliana, and rapamycin inhibits this pathway, as in other organisms. S6K contains a conserved C-terminal motif that is a target for TOR phosphorylation and PDK1 binding, and this motif is highly conserved in O. tauri. In mammals, complexes of mTOR with RAPTOR (TORC1) and RICTOR (TORC2) mediate distinct signalling pathways. The LST8 protein is a common component of both complexes. Equivalents for both mTOR complexes exist in yeast [72, 73]. O. tauri, like the rest of the green lineage, only contains components of TORC1. The kinase targets of TORC2 (PKB, PKC) are absent from O. tauri, and across the green lineage. In contrast, S6K is a conserved as a target of TORC1 in the green lineage , suggesting that the TORC1-containing mTOR complex could be the prototypical pathway for TOR signalling.
In mammals, the first of these phosphorylation events is by GSK3 to the equivalent residue of S398 on the S6K turn motif . The GSK3 target S/T-X-X-X-S/T motif at this site is conserved in S. cerevisiae, A. thaliana, and O. tauri (Additional file 4: Figure S3B). This residue is constitutively phosphorylated in mammals, and is a dephosphorylation target of PP2C. The presence of GSK3 is proposed to infer resistance to PP2C-mediated inactivation of S6K by countering dephosphorylation . The phosphorylation of S6K by GSK3 at S398 is a pre-requisite for the subsequent phosphorylation of the C-terminal T415 by TOR. The phosphorylation by TOR in turn enables the binding of active PDK1  to the C-terminal motif. The activation of human PDK1 requires autophosphorylation of a S in the activation loop, which is also a 14-3-3 binding motif . Human 14-3-3 binds to the phosphorylated motif in PDK1 ; 14-3-3 also regulates PDK1 in A. thaliana. The activation-loop S is conserved in A. thaliana and O. tauri PDK1 (S210). There are only two 14-3-3 proteins in O. tauri (Ot18g01040 and Ot08g00720), providing a limited number of candidates for PDK1 regulation. Active PDK1 binds to the primed TOR motif at the C-terminus of S6K (T415). This allows PDK1 to phosphorylate a T residue in the S6K activation loop [74, 75], which is also conserved in A. thaliana and O. tauri (S260). Yeast and human TOR phosphorylates TAP42 (or α4 in humans), which affects the formation of a TAP42:PP2A complex . PP2A has been shown to dephosphorylate S6K , but it is unclear what role this potential signalling pathway has on S6K regulation in higher eukaryotes . Both TAP42 (Additional file 4: Figure S3) and PP2A (Ot07g01700) are found within O. tauri and the green lineage, though the TAP42 ortholog in O. tauri was previously unannotated.
We have shown conservation of the AGC kinases in the TORC1 pathway in O. tauri. Key phosphorylation motifs and binding sites are also conserved, for all the components of the model proposed by Shin et al.. No phosphorylation was detected for the three key residues of S6K in our phosphoproteomic surveys. S6K was present, as phosphorylation at S61, S65, and S76 were detected and similar samples observed the unphosphorylated protein . However, the lack of detected phosphorylation in O. tauri cannot be taken as contrary evidence as the quantity of observed phosphorylation in proportion to expected phosphorylation in O. tauri is still relatively low. For example, 28 phosphorylations of human S6K are currently known , and assuming a similar quantity of modification in O. tauri we have observed in the order of 10% of phosphorylations.
The CaMK family: CPK and SnRK1 (ePK)
SnRK are an important subfamily of conserved CaMKs, which are related to SNF1 in yeast. SnRK1 kinases are the founding members and are most closely related to SNF1. It is also the only member of the SnRK family which is present across all eukaryotes and core members are involved in energy regulation in the cell [86, 87], with a primary function in glycogen metabolism . The O. tauri SnRK1 (Ot06g03970) is most closely related to the A. thaliana SnRK1s (Figure 6A; Additional file 7: Figure S5D). In addition to the core SnRKs, A. thaliana contains a functionally diversified set of SnRK subfamilies , which are absent from O. tauri. The human ortholog 5′ AMP-activated Protein Kinase (AMPKa) is equidistant to O. tauri and the S. cerevisiae ortholog SNF1, indicating O. tauri may also be a suitable model for SnRK signalling in humans (Figure 6A).
CDPKs have many roles in biotic and abiotic signalling pathways . Two kinases exist in O. tauri (Ot09g03470 and Ot03g03430) with clear CDPK domain architectures and kinase domains. These have recently been classified as group I algal CDPKs . CDPKs are typically a large family, involved in a variety of roles specific to higher plants, including herbivore defence  and abscisic acid signalling [91–93]. These specialised functions indicate that kingdom-specific adaptations have driven the sequence diversity of CDPKs. A Phosphoenolpyruvate carboxylase-Related Kinases (PEPKR) Ot01g05370 is also present in O. tauri (Additional file 7: Figure S5C). Two other O. tauri kinases align more closely to the S. cerevisiae RAD53 (Ot15g01210 and Ot07g01980) than CDPKs. Ot15g01210 has previously been classified as a group IV algal CDPK . Recent CDPK phylogenies by Hamel et al. have shown that plant CaMKs are likely to have diverged between the emergence of group IV and I, which places Ot15g01210 and Ot07g01980 within two distinct clades for plant CDPKs. The retention of group IV and I clades within the reduced O. tauri kinome suggests an important conserved role for these kinase in calcium signalling.
The MAP2K pathway and STE kinase family
The downstream effects of extracellular signals, which are mediated by kinases such as the CaMKs and AGCs, are the MAPK cascades. These form signalling connections from the cellular environment into the nucleus, in order to affect transcriptional changes . All but the final target of the MAPK cascade are found within the STE family. Here we examine the STE family of kinases with a view to understanding an exemplar MAPK pathway from A. thaliana: the GSK3-mediated regulation of stomatal opening through a MAP2K target [94, 95]. As for the previously described TOR pathway, we first assess the relevant kinase orthologs in O. tauri.
The STE kinases contain the MAP4K, MAP3K and MAP2K components of the MAPK cascade . Eight STE family kinases and an additional six STE-like kinases were identified in O. tauri (Additional file 7: Figure S5E). These include one MAP2K (Ot04g04050), two MAP3K (Ot13g01170 and Ot17g02120, Figure 6B), and two MAP4K (Ot02g05830 and Ot13g02030) kinase candidates. In contrast, A. thaliana has 10, 11 and 7 orthologous genes respectively, again emphasizing the potential of O. tauri as an experimental model for gene manipulation in MAPK signalling studies. Within MAP3Ks Ot13g01170 is the only member of the MEKK clade and Ot17g02120 is a CDC15-like protein. The STEs are closely related to the Tyrosine-Kinase like (TKL) family, and contain the plant-RAF kinase, which also act as MAP3Ks . Ot12g01310 is the only confirmed plant-RAF kinase (Additional file 5: Table S2). It contains a Constitutive Triple Response 1 (CTR1) domain (Additional file 2: Figure S1), confirming it as an ortholog of the CTR1 gene: a potential-MAP3K that in A. thaliana is negatively regulated by the ethylene responsive histidine kinase ETR1 .
O. tauri also contains a single plant-like APG1 kinase (Ot06g01800) with four orthologous proteins in A. thaliana. C-terminal phosphorylation of APG1 was observed in O. tauri. APG1 kinases in yeast and A. thaliana are a target for the negative regulation of autophagy by TOR [98, 99], highlighting another conserved facet of the TOR pathway.
The minimal MAP2K Pathway in O. tauri
The brassinosteroid signalling pathway acts upstream of GSK3 in A. thaliana, to initiate GSK3-mediated inhibition of the MAPK pathway, leading to stomatal regulation . The central components of this pathway are found in O. tauri; however, neither the upstream brassinosteroid signalling pathway nor the downstream stomatal regulation components are present. Similarly, in human and yeast cells MAPK cascades create complex signalling networks in a diverse array of processes [2, 100], many of which are absent in O. tauri. Despite the diversity of processes, these central MAPK components from the CMGC and STE kinase families are among the most conserved protein kinase families in O. tauri (Additional file 7: Figure S5).
Circadian signalling: CK1 and CK2
Circadian rhythms are ≈ 24 h biological cycles, which arose as adaptations to daily changes in the environment. The circadian clock regulates diverse processes across eukaryotes, from the sleep–wake cycle of metazoa to photosynthesis . O. tauri is already in use as a clock model for both in vivo and in silico studies [25, 30, 104]. In particular CK1 and CK2 have been shown to be part of conserved transcriptional/translational feedback loops in eukaryotes that regulate circadian clocks, based on pharmacological and overexpression results [28, 31, 32].
Casein Kinase 1 family
The CK1 family of kinases are named after the highly conserved CK1 protein. CK1 has a variety of cellular functions, including regulation of membrane trafficking, DNA replication, Wnt signalling, RNA metabolism  and cell cycle regulation through tubulin binding [106, 107]. CK1 isoforms have also been shown to affect circadian rhythmicity in metazoa [108, 109], in the fungus Neurospora crassa and in O. tauri[31, 32].
Based on taxonomic studies of eukaryotes [21, 111] we generally expect the Chlorophyta branch of algae and plant proteins to be more closely related, with a smaller distance between sequences from metazoa and yeast. However, our data shows that the distance of the O. tauri CK1 to the base of the branch that contains H. sapiens sequences is less than half the distance of S. cerevisiae to this branch. A. thaliana CK1 sequences also appear to have diverged considerably. The O. tauri CK1 is the closest among these three model organisms to human CK1δ and CK1ϵ. This indicates that O. tauri may be an interesting model organism to study CK1.
Casein Kinase 2 (CK2)
Casein Kinase 2 (CK2) is a highly conserved kinase, found across all eukaryotes. CK2 is centrally important in many signalling pathways and is one of the most ubiquitous kinases in terms of substrate phosphorylation . CK2 is a tetramer composed of a CK2β dimer and two CK2α subunits. O. tauri contains a single catalytic CK2α (Ot12g02620) and regulatory CK2β (Ot02g03010) subunit. The topology of the phylogenies for both subunits is very similar, the O. tauri CK2 appears to be more similar to the A. thaliana, D. melanogaster and H. sapiens than S. cerevisiae sequences. S. cerevisiae CK2 subunits have diverged considerably, similar to CK1, indicating that O. tauri may be interesting alternative model species for CK2.
CK1, CK2 and the circadian clock
A naturally-occurring short circadian clock period phenotype of 20 hours was first observed in Syrian hamsters (Mesocricetus auratus) and attributed to the tau mutation in CK1ϵ . The tau mutation increases PER1 and PER2 phosphorylation, increasing proteasomal degradation, and shortening the circadian period . CK1 control of the clock is exemplified by familial advanced sleep phase syndrome, a condition associated with early sleep time followed by early morning awakening, whereby a mutation to human PER2 or CK1δ advances period [124–126]. Mutations to D. melanogaster CK1δ lengthen period, suggesting differences in the regulation between mammalian and insect clocks . Until recently, CK1 has not been implicated in plant clocks. There are early indications for a functional role for CK1 in the O. tauri clock [31, 32], however the exact targets of CK1 are unknown.
Smaller kinase families in O. tauri
Additional file 5: Table S2 shows that the remaining protein kinases span a wide range of families, which are discussed in Additional file 8: Figure S6: two of the highly-conserved RIO family, a Polo-like kinase (PLK), an Aurora kinase, BUD32, five STN-like kinases, two BUB1-like kinases, Haspin, and two HKs. The O. tauri kinome therefore comprises a suitably diverse set to represent many of the known protein kinase functions in eukaryotes.
We identified 133 gene loci encoding catalytic protein kinases in O. tauri, constituting a small kinome of a similar order to S. cerevisiae (130 genes). As a photosynthetic model for core signalling, it is nearly ten-fold smaller than the A. thaliana kinome . Comparing A. thaliana, S. cerevisiae and H. sapiens sequences, we found O. tauri kinases were frequently more closely related to the H. sapiens sequences than were the S. cerevisiae orthologs (the PIKK kinases are one exception). Thus genome reduction in O. tauri has not led to divergence in its kinome, in contrast to the minimal kinomes of parasitic species. DNA-PK is present in O. tauri and H. sapiens but absent in A. thaliana; PKG in O. tauri is also closer to the metazoan and fungal sequences than the closest A. thaliana sequences; several other components are conserved in exemplar signalling pathways (genes, phosphorylated amino acids and binding motifs), such as S6K activation via the TORC1 pathway. Together with other conserved components that are otherwise absent or poorly conserved in much of the green lineage, such as the cell cycle phosphatase CDC25 , our kinome survey indicates that O. tauri is a reduced but representative laboratory model species for signalling research, which incorporates many eukaryote-wide signalling components.
Identification of OrthoMCL ortholog-groups
We obtained ortholog groups assignment for H. sapiens, S. cerevisiae and A. thaliana from the OrthoMCL version 5 database and used the proteome upload service  to annotate the O. tauri peptide sequence from BEG (Additional file 3: Figure S2), which we supplemented with our corrected gene models.
Identification of kinases in O. tauri
In an approach similar to Vilella et al. we clustered all sequences from H. sapiens, S. cerevisiae, A. thaliana, O. lucimarinus, and O. tauri into related protein families. For O. tauri we used the December 2006 peptides sequences, and for O. lucimarinus we used the JGI November 2011 peptide sequences, both retrieved from BEG (Additional file 3: Figure S2). The TAIR (version 10) representative gene models for A. thaliana and Uniprot reference proteomes for S. cerevisiae and H. sapiens were retrieved in November 2012. We searched all proteins, against all proteins, using the NCBI blastp tool (version 2.2.25; BLOSUM62) with an e-value cut-off of 0.01. We calculated the BLAST Ratio Score (BSR) for each hit found  and we retained best-reciprocal hits and BSR scores greater than 1/3. We created a distance matrix using the BSR scores and applied the Markov Cluster (MCL) algorithm, (version 12–068), with inflation values 1.1 and 1.4. We extracted groups of kinases and phosphatases from the subsequent clusters. We searched for kinase and phosphatase catalytic domains with the hmmsearch algorithm (HMMER 3.0, GA cut-off)  using the models provided in Pfam-A (January 2013) . We also ran a sequence similarity search with an e-value cut-off of 0.07, using the blastp algorithm (BLOSUM62), from O. tauri against the PlantsP database. All these results were manually curated to extract all candidate kinases and phosphatases into a database.
Constructing alignments and phylogenies
Kinase alignments for each family in O. tauri were constructed by whole sequence alignment of protein sequences to whole families of proteins. The KinBase database was used as a source of S. cerevisiae and H. sapiens kinases annotations (Additional file 9) and family. The PlantsP  database provided A. thaliana kinase annotations. We aligned sequences using MAFFT  version 6 within JalView [135, 136]. We used the high quality global alignment algorithm G-INS-i, with BLOSUM62, 2-tree rebuilds, gap open and extension penalties of 1.53 and 0.12 respectively, and a limit of 1,000 iterations. Poorly aligned sequences were manually removed from the alignment. For editing alignments of more than 8 sequences we used guidance version 1.3.1, with the same MAFFT parameters previously described, and 100 guidance bootstraps . We retained columns with a confidence value greater than 0.93, and sequences with a confidence value above 0.6. Columns with gaps were excluded. Inference of phylogenetic trees on the conserved alignment columns was performed using a Maximum Likelihood (ML) approach. Phylogenies were built with RaXML version 7.2.8 . We used a γ model of evolutionary rate heterogeneity combined with an estimation of the proportion of invariant sites. Amino acid replacement scoring was determined using the WAG matrix . Support for branches on the ML tree was evaluated using bootstrap analysis, using the frequency-based criteria (FC) parameter to determine the number of iterations. We used the FigTree version 1.4.0 tool for the visualisation of trees.
When alignments of O. tauri proteins contained gaps, extended inserts which were not found in other species, or poor alignments, we investigated and where appropriate corrected underlying gene models. Where gaps where present in the O. tauri genomic sequence, we used the closest gene from O. lucimarinus to infer the gap sequence, when there was a high degree of conservation in the adjacent region (Additional file 3: Figure S2).
Phosphorylation-site identification by tandem mass spectrometry
Protein extract from O. tauri cells was prepared in a similar manner as described previously , with the digestion performed on 300 μg protein extract. Peptides were cleaned by reverse phase and phosphopeptide enrichment and LC-MS analysis were performed as described previously .
All multi-charged ions (2+, 3+, 4+) were extracted from each LC-MS file and MSMS data was searched using MASCOT Version 2.4 (Matrix Science Ltd, UK) against the O. tauri subset of the NCBI protein database (12/01/2011; 8,726 sequences) using a maximum missed-cut value of 2, variable oxidation (M), N-terminal protein acetylation, phosphorylation (S, T, and Y) and fixed carbamidomethylation (C). Precursor mass tolerance was 7 ppm and MSMS tolerance 0.4 amu. The significance threshold (p) was set below 0.05 (MudPIT scoring). A minimum peptide cut off score of 20 was set, corresponding to <3% global false discovery rate (FDR) using a decoy database search.
Ambiguous sites were confirmed by cross-referencing (by sequence, charge, and quantity of residue modifications) with most probable site predictions from MaxQuant (version 126.96.36.199 in singlet mode, same Mascot settings) .
Availability of supporting data
All sequences and supporting data are included as additional files and are available at http://hdl.handle.net/10283/563.
We are grateful to Bram Verhelst from VIB Department of Plant Systems Biology, Ghent University, for advice concerning O. tauri gene models. GvO is a Royal Society University Research Fellow (UF110173) supported by Royal Society research grant RG120372. This work was supported by BBSRC and EPSRC awards BB/D019621 and BB/J009423.
- Hanks SK, Hunter T: Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 1995, 9: 576-596.PubMedGoogle Scholar
- Keshet Y, Seger R: The MAP Kinase Signaling Cascades: A System of Hundreds of Components Regulates a Diverse Array of Physiological Functions. MAP Kinase Signaling Protocols. Methods in Molecular Biology, vol. 661. Edited by: Seger R. 2010, Totowa: Humana Press, 3-38.Google Scholar
- Breitkreutz A, Choi H, Sharom JR, Boucher L, Neduva V, Larsen B, Lin Z-Y, Breitkreutz B-J, Stark C, Liu G, Ahn J, Dewar-Darch D, Reguly T, Tang X, Almeida R, Qin ZS, Pawson T, Gingras A-C, Nesvizhskii AI, Tyers M: A global protein kinase and phosphatase interaction network in yeast. Science. 2010, 328: 1043-1046.PubMed CentralPubMedGoogle Scholar
- Champion A, Kreis M, Mockaitis K, Picaud A, Henry Y: Arabidopsis kinome: after the casting. Funct Integr Genomics. 2004, 4: 163-187.PubMedGoogle Scholar
- Goldberg JM, Manning G, Liu A, Fey P, Pilcher KE, Xu Y, Smith JL: The Dictyostelium kinome–analysis of the protein kinases from a simple model organism. PLoS Genet. 2006, 2: e38-PubMed CentralPubMedGoogle Scholar
- Manning G, Plowman GD, Hunter T, Sudarsanam S: Evolution of protein kinase signaling from yeast to man. Trends Biochem Sci. 2002, 27: 514-520.PubMedGoogle Scholar
- Miranda-Saavedra D, Stark MJR, Packer JC, Vivares CP, Doerig C, Barton GJ: The complement of protein kinases of the microsporidium Encephalitozoon cuniculi in relation to those of Saccharomyces cerevisiae and Schizosaccharomyces pombe. BMC Genomics. 2007, 8: 309-PubMed CentralPubMedGoogle Scholar
- Manning G, Reiner DS, Lauwaet T, Dacre M, Smith A, Zhai Y, Svard S, Gillin FD: The minimal kinome of Giardia lamblia illuminates early kinase evolution and unique parasite biology. Genome Biol. 2011, 12: R66-PubMed CentralPubMedGoogle Scholar
- Deutscher J, Saier MH: Ser/Thr/Tyr protein phosphorylation in bacteria - for long time neglected, now well established. J Mol Microbiol Biotechnol. 2005, 9: 125-131.PubMedGoogle Scholar
- Kennelly PJ: Protein Ser/Thr/Tyr phosphorylation in the archaea. J Biol Chem. 2014, 289: 9480-9487.PubMed CentralPubMedGoogle Scholar
- Kannan N, Taylor SS, Zhai Y, Venter JC, Manning G: Structural and functional diversity of the microbial kinome. PLoS Biol. 2007, 5: e17-PubMed CentralPubMedGoogle Scholar
- Scheeff ED, Bourne PE: Structural evolution of the protein kinase–like superfamily. PLoS Comput Biol. 2005, 1: e49-PubMed CentralPubMedGoogle Scholar
- Kumar MN, Jane W-N, Verslues PE: Role of the putative osmosensor Arabidopsis histidine kinase1 in dehydration avoidance and low-water-potential response. Plant Physiol. 2013, 161: 942-953.PubMed CentralPubMedGoogle Scholar
- Hall BP, Shakeel SN, Amir M, Ul Haq N, Qu X, Schaller GE: Histidine kinase activity of the ethylene receptor ETR1 facilitates the ethylene response in Arabidopsis. Plant Physiol. 2012, 159: 682-695.PubMed CentralPubMedGoogle Scholar
- Hanks SK, Quinn AM, Hunter T: The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988, 241: 42-52.PubMedGoogle Scholar
- Leonard CJ, Aravind L, Koonin EV: Novel families of putative protein kinases in bacteria and archaea: evolution of the “eukaryotic” protein kinase superfamily. Genome Res. 1998, 8: 1038-1047.PubMedGoogle Scholar
- Brinkworth RI, Munn AL, Kobe B: Protein kinases associated with the yeast phosphoproteome. BMC Bioinformatics. 2006, 7: 47-PubMed CentralPubMedGoogle Scholar
- Ghelis T: Signal processing by protein tyrosine phosphorylation in plants. Plant Signal Behav. 2011, 6: 942-951.PubMed CentralPubMedGoogle Scholar
- Courties C, Vaquer A, Troussellier M, Lautier J, Chrétiennot-Dinet MJ, Neveux J, Machado C, Claustre H: Smallest eukaryotic organism. Nature. 1994, 370: 255-255.Google Scholar
- Palenik B, Grimwood J, Aerts A, Rouzé P, Salamov A, Putnam N, Dupont C, Jorgensen R, Derelle E, Rombauts S, Zhou K, Otillar R, Merchant SS, Podell S, Gaasterland T, Napoli C, Gendler K, Manuell A, Tai V, Vallon O, Piganeau G, Jancek S, Heijde M, Jabbari K, Bowler C, Lohr M, Robbens S, Werner G, Dubchak I, Pazour GJ, et al: The tiny eukaryote Ostreococcus provides genomic insights into the paradox of plankton speciation. Proc Natl Acad Sci U S A. 2007, 104: 7705-7710.PubMed CentralPubMedGoogle Scholar
- Keeling PJ, Burger G, Durnford DG, Lang BF, Lee RW, Pearlman RE, Roger AJ, Gray MW: The tree of eukaryotes. Trends Ecol Evol. 2005, 20: 670-676.PubMedGoogle Scholar
- Henderson GP, Gan L, Jensen GJ: 3-D ultrastructure of O. tauri: electron cryotomography of an entire eukaryotic cell. PLoS One. 2007, 2: e749-PubMed CentralPubMedGoogle Scholar
- Farinas B, Mary C, De Manes C-L O, Bhaud Y, Peaucellier G, Moreau H: Natural synchronisation for the study of cell division in the green unicellular alga Ostreococcus tauri. Plant Mol Biol. 2006, 60: 277-292.PubMedGoogle Scholar
- van Ooijen G, Knox K, Kis K, Bouget F-Y, Millar AJ: Genomic transformation of the picoeukaryote Ostreococcus tauri. J Vis Exp. 2012, e4074-65
- Corellou F, Schwartz C, Motta J-P, Djouani-Tahri EB, Sanchez F, Bouget F-Y: Clocks in the green lineage: comparative functional analysis of the circadian architecture of the picoeukaryote ostreococcus. Plant Cell. 2009, 21: 3436-3449.PubMed CentralPubMedGoogle Scholar
- Le Bihan T, Martin SF, Chirnside ES, van Ooijen G, Barrios-Llerena ME, O’Neill JS, Shliaha PV, Kerr LE, Millar AJ: Shotgun proteomic analysis of the unicellular alga Ostreococcus tauri. J Proteomics. 2011, 74: 2060-2070.PubMedGoogle Scholar
- Martin SF, Munagapati VS, Salvo-Chirnside E, Kerr LE, Le Bihan T: Proteome turnover in the green alga Ostreococcus tauri by time course 15 N metabolic labeling mass spectrometry. J Proteome Res. 2012, 11: 476-486.PubMedGoogle Scholar
- O’Neill JS, van Ooijen G, Dixon LE, Troein C, Corellou F, Bouget F-Y, Reddy AB, Millar AJ: Circadian rhythms persist without transcription in a eukaryote. Nature. 2011, 469: 554-558.PubMed CentralPubMedGoogle Scholar
- van Ooijen G, Dixon LE, Troein C, Millar AJ: Proteasome function is required for biological timing throughout the twenty-four hour cycle. Curr Biol. 2011, 21: 869-875.PubMed CentralPubMedGoogle Scholar
- Troein C, Corellou F, Dixon LE, van Ooijen G, O’Neill JS, Bouget F-Y, Millar AJ: Multiple light inputs to a simple clock circuit allow complex biological rhythms. Plant J. 2011, 66: 375-385.PubMed CentralPubMedGoogle Scholar
- van Ooijen G, Hindle M, Martin SF, Barrios-Llerena M, Sanchez F, Bouget F-Y, O’Neill JS, Bihan TL, Millar AJ: Functional analysis of casein kinase 1 in a minimal circadian system. PLoS One. 2013, 8: e70021-PubMed CentralPubMedGoogle Scholar
- van Ooijen G, Martin SF, Barrios-Llerena ME, Hindle M, Le Bihan T, O’Neill JS, Millar AJ: Functional analysis of the rodent CK1tau mutation in the circadian clock of a marine unicellular alga. BMC Cell Biol. 2013, 14: 46-PubMed CentralPubMedGoogle Scholar
- Chen F, Mackey AJ, Stoeckert CJ, Roos DS: OrthoMCL-DB: querying a comprehensive multi-species collection of ortholog groups. Nucl Acids Res. 2006, 34 (suppl 1): D363-D368.PubMed CentralPubMedGoogle Scholar
- Shiu S-H, Bleecker AB: Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. PNAS. 2001, 98: 10763-10768.PubMed CentralPubMedGoogle Scholar
- Miranda-Saavedra D, Barton GJ: Classification and functional annotation of eukaryotic protein kinases. Proteins Struct Funct Bioinformatics. 2007, 68: 893-914.Google Scholar
- Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S: The protein kinase complement of the human genome. Science. 2002, 298: 1912-1934.PubMedGoogle Scholar
- Tchieu JH, Fana F, Fink JL, Harper J, Nair TM, Niedner RH, Smith DW, Steube K, Tam TM, Veretnik S, Wang D, Gribskov M: The plantsP and plantsT functional genomics databases. Nucleic Acids Res. 2003, 31: 342-344.PubMed CentralPubMedGoogle Scholar
- Liberti S, Sacco F, Calderone A, Perfetto L, Iannuccelli M, Panni S, Santonico E, Palma A, Nardozza AP, Castagnoli L, Cesareni G: HuPho: the human phosphatase portal. FEBS J. 2013, 280: 379-387.PubMedGoogle Scholar
- Lehti-Shiu MD, Zou C, Hanada K, Shiu S-H: Evolutionary history and stress regulation of plant receptor-like kinase/pelle genes. Plant Physiol. 2009, 150: 12-26.PubMed CentralPubMedGoogle Scholar
- Lehti-Shiu MD, Shiu S-H: Diversity, classification and function of the plant protein kinase superfamily. Phil Trans R Soc B. 2012, 367: 2619-2639.PubMed CentralPubMedGoogle Scholar
- Kosti I, Mandel-Gutfreund Y, Glaser F, Horwitz BA: Comparative analysis of fungal protein kinases and associated domains. BMC Genomics. 2010, 11: 133-PubMed CentralPubMedGoogle Scholar
- Christie JM, Yang H, Richter GL, Sullivan S, Thomson CE, Lin J, Titapiwatanakun B, Ennis M, Kaiserli E, Lee OR, Adamec J, Peer WA, Murphy AS: Phot1 inhibition of ABCB19 primes lateral auxin fluxes in the shoot apex required for phototropism. PLoS Biol. 2011, 9: e1001076-PubMed CentralPubMedGoogle Scholar
- Gao Q, Yang Z, Zhou Y, Yin Z, Qiu J, Liang G, Xu C: Characterization of an Abc1 kinase family gene OsABC1-2 conferring enhanced tolerance to dark-induced stress in rice. Gene. 2012, 498: 155-163.PubMedGoogle Scholar
- Yang S, Zeng X, Li T, Liu M, Zhang S, Gao S, Wang Y, Peng C, Li L, Yang C: AtACDO1, an ABC1-like kinase gene, is involved in chlorophyll degradation and the response to photooxidative stress in Arabidopsis. J Exp Bot. 2012, 63: 3959-3973.PubMedGoogle Scholar
- Yang S: AtSIA1, an ABC1-like kinase, regulates salt response in Arabidopsis. Biologia. 2012, 67: 1107-1111.Google Scholar
- Lozano J-C, Schatt P, Botebol H, Vergé V, Lesuisse E, Blain S, Carré IA, Bouget F-Y: Efficient gene targeting and foreign DNA removal by homologous recombination in the picoeukaryote Ostreococcus. Plant J. 2014, 76: 6-Google Scholar
- van Dam TJP, Zwartkruis FJT, Bos JL, Snel B: Evolution of the TOR pathway. J Mol Evol. 2011, 73: 209-220.PubMed CentralPubMedGoogle Scholar
- Shin S, Wolgamott L, Yu Y, Blenis J, Yoon S-O: Glycogen synthase kinase (GSK)-3 promotes p70 ribosomal protein S6 kinase (p70S6K) activity and cell proliferation. PNAS. 2011, 108: E1204-E1213.PubMed CentralPubMedGoogle Scholar
- Warmerdam DO, Kanaar R: Dealing with DNA damage: relationships between checkpoint and repair pathways. Mutat Res. 2010, 704: 2-11.PubMedGoogle Scholar
- Templeton GW, Moorhead GBG: The phosphoinositide-3-OH-kinase-related kinases of Arabidopsis thaliana. EMBO Rep. 2005, 6: 723-728.PubMed CentralPubMedGoogle Scholar
- Lloyd JPB, Davies B: SMG1 is an ancient nonsense-mediated mRNA decay effector. Plant J. 2013, 76: 800-810.PubMedGoogle Scholar
- Lieber MR, Ma Y, Pannicke U, Schwarz K: Mechanism and regulation of human non-homologous DNA end-joining. Nat Rev Mol Cell Biol. 2003, 4: 712-720.PubMedGoogle Scholar
- Liu S, Opiyo SO, Manthey K, Glanzer JG, Ashley AK, Amerin C, Troksa K, Shrivastav M, Nickoloff JA, Oakley GG: Distinct roles for DNA-PK, ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress. Nucl Acids Res. 2012, 40: 10780-10794.PubMed CentralPubMedGoogle Scholar
- Marwedel T, Ishibashi T, Lorbiecke R, Jacob S, Sakaguchi K, Sauter M: Plant-specific regulation of replication protein A2 (OsRPA2) from rice during the cell cycle and in response to ultraviolet light exposure. Planta. 2003, 217: 457-465.PubMedGoogle Scholar
- Ferguson BJ, Mansur DS, Peters NE, Ren H, Smith GL: DNA-PK is a DNA sensor for IRF-3-dependent innate immunity. ELife. 2012, 1: e00047-PubMed CentralPubMedGoogle Scholar
- Robbens S, Khadaroo B, Camasses A, Derelle E, Ferraz C, Inzé D, van de Peer Y, Moreau H: Genome-wide analysis of core cell cycle genes in the unicellular green alga Ostreococcus tauri. Mol Biol Evol. 2005, 22: 589-597.PubMedGoogle Scholar
- CristinaRodriguez M, Petersen M, Mundy J: Mitogen-activated protein kinase signaling in plants. Annu Rev Plant Biol. 2010, 61: 621-649.Google Scholar
- Seger R, Krebs EG: The MAPK signaling cascade. FASEB J. 1995, 9: 726-735.PubMedGoogle Scholar
- Lochhead PA, Kinstrie R, Sibbet G, Rawjee T, Morrice N, Cleghon V: A chaperone-dependent GSK3beta transitional intermediate mediates activation-loop autophosphorylation. Mol Cell. 2006, 24: 627-633.PubMedGoogle Scholar
- Saidi Y, Hearn TJ, Coates JC: Function and evolution of “green” GSK3/Shaggy-like kinases. Trends Plant Sci. 2012, 17: 39-46.PubMedGoogle Scholar
- Krishna P, Gloor G: The Hsp90 family of proteins in Arabidopsis thaliana. Cell Stress Chaperones. 2001, 6: 238-246.PubMed CentralPubMedGoogle Scholar
- Dittrich ACN, Devarenne TP: Perspectives in PDK1 evolution. Plant Signal Behav. 2012, 7: 642-649.PubMed CentralPubMedGoogle Scholar
- Rademacher EH, Offringa R: Evolutionary adaptations of plant AGC kinases: from light signaling to cell polarity regulation. Front Plant Sci. 2012, 3: 250-PubMed CentralPubMedGoogle Scholar
- Mora A, Komander D, van Aalten DMF, Alessi DR: PDK1, the master regulator of AGC kinase signal transduction. Semin Cell Dev Biol. 2004, 15: 161-170.PubMedGoogle Scholar
- Biondi RM, Kieloch A, Currie RA, Deak M, Alessi DR: The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB. EMBO J. 2001, 20: 4380-4390.PubMed CentralPubMedGoogle Scholar
- Osborne BW, Wu J, McFarland CJ, Nickl CK, Sankaran B, Casteel DE, Woods VL, Kornev AP, Taylor SS, Dostmann WR: Crystal structure of cGMP-dependent protein kinase reveals novel site of interchain communication. Structure. 2011, 19: 1317-1327.PubMed CentralPubMedGoogle Scholar
- Taylor SS, Ilouz R, Zhang P, Kornev AP: Assembly of allosteric macromolecular switches: lessons from PKA. Nat Rev Mol Cell Biol. 2012, 13: 646-658.PubMed CentralPubMedGoogle Scholar
- Zimmermann B, Chiorini JA, Ma Y, Kotin RM, Herberg FW: PrKX is a novel catalytic subunit of the cAMP-dependent protein kinase regulated by the regulatory subunit type I. J Biol Chem. 1999, 274: 5370-5378.PubMedGoogle Scholar
- Hornbeck PV, Chabra I, Kornhauser JM, Skrzypek E, Zhang B: PhosphoSite: a bioinformatics resource dedicated to physiological protein phosphorylation. Proteomics. 2004, 4: 1551-1561.PubMedGoogle Scholar
- Wullschleger S, Loewith R, Hall MN: TOR signaling in growth and metabolism. Cell. 2006, 124: 471-484.PubMedGoogle Scholar
- Xiong Y, Sheen J: Rapamycin and glucose-target of rapamycin (TOR) protein signaling in plants. J Biol Chem. 2012, 287: 2836-2842.PubMed CentralPubMedGoogle Scholar
- Loewith R: 9 - TORC1 Signaling in Budding Yeast. The Enzymes. Volume 27. Edited by: Michael Hall N. 2010, Fuyuhiko Tamanoi: Academic Press, 147-175.Google Scholar
- Cybulski N, Hall MN: TOR complex 2: a signaling pathway of its own. Trends Biochem Sci. 2009, 34: 620-627.PubMedGoogle Scholar
- Alessi DR, Kozlowski MT, Weng QP, Morrice N, Avruch J: 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro. Curr Biol. 1998, 8: 69-81.PubMedGoogle Scholar
- Pullen N, Dennis PB, Andjelkovic M, Dufner A, Kozma SC, Hemmings BA, Thomas G: Phosphorylation and activation of p70s6k by PDK1. Science. 1998, 279: 707-710.PubMedGoogle Scholar
- Burnett PE, Barrow RK, Cohen NA, Snyder SH, Sabatini DM: RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc Natl Acad Sci U S A. 1998, 95: 1432-1437.PubMed CentralPubMedGoogle Scholar
- Moser BA, Dennis PB, Pullen N, Pearson RB, Williamson NA, Wettenhall RE, Kozma SC, Thomas G: Dual requirement for a newly identified phosphorylation site in p70s6k. Mol Cell Biol. 1997, 17: 5648-5655.PubMed CentralPubMedGoogle Scholar
- Casamayor A, Morrice NA, Alessi DR: Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo. Biochem J. 1999, 342 (Pt 2): 287-292.PubMed CentralPubMedGoogle Scholar
- Sato S, Fujita N, Tsuruo T: Regulation of kinase activity of 3-phosphoinositide-dependent protein kinase-1 by binding to 14-3-3. J Biol Chem. 2002, 277: 39360-39367.PubMedGoogle Scholar
- Otterhag L, Gustavsson N, Alsterfjord M, Pical C, Lehrach H, Gobom J, Sommarin M: Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase. Biochimie. 2006, 88: 11-21.PubMedGoogle Scholar
- Jiang Y, Broach JR: Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast. EMBO J. 1999, 18: 2782-2792.PubMed CentralPubMedGoogle Scholar
- Peterson RT, Desai BN, Hardwick JS, Schreiber SL: Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12–rapamycinassociated protein. PNAS. 1999, 96: 4438-4442.PubMed CentralPubMedGoogle Scholar
- Cygnar KD, Gao X, Pan D, Neufeld TP: The phosphatase subunit tap42 functions independently of target of rapamycin to regulate cell division and survival in Drosophila. Genetics. 2005, 170: 733-740.PubMed CentralPubMedGoogle Scholar
- Hrabak EM, Chan CWM, Gribskov M, Harper JF, Choi JH, Halford N, Kudla J, Luan S, Nimmo HG, Sussman MR, Thomas M, Walker-Simmons K, Zhu J-K, Harmon AC: The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol. 2003, 132: 666-680.PubMed CentralPubMedGoogle Scholar
- Klimecka M, Muszyńska G: Structure and functions of plant calcium-dependent protein kinases. Acta Biochim Pol. 2007, 54: 219-233.PubMedGoogle Scholar
- Ghillebert R, Swinnen E, Wen J, Vandesteene L, Ramon M, Norga K, Rolland F, Winderickx J: The AMPK/SNF1/SnRK1 fuel gauge and energy regulator: structure, function and regulation. FEBS J. 2011, 278: 3978-3990.PubMedGoogle Scholar
- Usaite R, Jewett MC, Oliveira AP, Yates JR, Olsson L, Nielsen J: Reconstruction of the yeast Snf1 kinase regulatory network reveals its role as a global energy regulator. Mol Syst Biol. 2009, 5: 319-PubMed CentralPubMedGoogle Scholar
- Polge C, Thomas M: SNF1/AMPK/SnRK1 kinases, global regulators at the heart of energy control?. Trends Plant Sci. 2007, 12: 20-28.PubMedGoogle Scholar
- Hamel L-P, Sheen J, Séguin A: Ancient signals: comparative genomics of green plant CDPKs. Trends Plant Sci. 2013, 19: 79-89.PubMed CentralPubMedGoogle Scholar
- Kanchiswamy CN, Takahashi H, Quadro S, Maffei ME, Bossi S, Bertea C, Zebelo SA, Muroi A, Ishihama N, Yoshioka H, Boland W, Takabayashi J, Endo Y, Sawasaki T, Arimura G: Regulation of Arabidopsis defense responses against Spodoptera littoralis by CPK-mediated calcium signaling. BMC Plant Biology. 2010, 10: 97-PubMed CentralPubMedGoogle Scholar
- Mori IC, Murata Y, Yang Y, Munemasa S, Wang Y-F, Andreoli S, Tiriac H, Alonso JM, Harper JF, Ecker JR, Kwak JM, Schroeder JI: CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca2 + − permeable channels and stomatal closure. PLoS Biol. 2006, 4: e327-PubMed CentralPubMedGoogle Scholar
- Munemasa S, Hossain MA, Nakamura Y, Mori IC, Murata Y: The Arabidopsis calcium-dependent protein kinase, CPK6, functions as a positive regulator of methyl jasmonate signaling in guard cells. Plant Physiol. 2011, 155: 553-561.PubMed CentralPubMedGoogle Scholar
- Zhu S-Y, Yu X-C, Wang X-J, Zhao R, Li Y, Fan R-C, Shang Y, Du S-Y, Wang X-F, Wu F-Q, Xu Y-H, Zhang X-Y, Zhang D-P: Two calcium-dependent protein kinases, CPK4 and CPK11, regulate abscisic acid signal transduction in Arabidopsis. Plant Cell. 2007, 19: 3019-3036.PubMed CentralPubMedGoogle Scholar
- Khan M, Rozhon W, Bigeard J, Pflieger D, Husar S, Pitzschke A, Teige M, Jonak C, Hirt H, Poppenberger B: Brassinosteroid-regulated GSK3/shaggy-like kinases phosphorylate mitogen-activated protein (MAP) kinase kinases, which control stomata development in Arabidopsis thaliana. J Biol Chem. 2013, 288: 7519-7527.PubMed CentralPubMedGoogle Scholar
- Kim T-W, Michniewicz M, Bergmann DC, Wang Z-Y: Brassinosteroid regulates stomatal development by GSK3-mediated inhibition of a MAPK pathway. Nature. 2012, 482: 419-422.PubMed CentralPubMedGoogle Scholar
- Champion A, Picaud A, Henry Y: Reassessing the MAP3K and MAP4K relationships. Trends Plant Sci. 2004, 9: 123-129.PubMedGoogle Scholar
- Huang Y, Li H, Hutchison CE, Laskey J, Kieber JJ: Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis. Plant J. 2003, 33: 221-233.PubMedGoogle Scholar
- Liu Y, Bassham DC: TOR is a negative regulator of autophagy in Arabidopsis thaliana. PLoS One. 2010, 5: e11883-PubMed CentralPubMedGoogle Scholar
- Díaz-Troya S, Pérez-Pérez ME, Florencio FJ, Crespo JL: The role of TOR in autophagy regulation from yeast to plants and mammals. Autophagy. 2008, 4: 851-865.PubMedGoogle Scholar
- Rudolf F, Pelet S, Peter M: Regulation of MAPK Signaling in Yeast. Stress-Activated Protein Kinases, 20. Edited by: Posas F, Nebreda AR. 2008, Berlin: Springer, 187-204.Google Scholar
- Zheng CF, Guan KL: Activation of MEK family kinases requires phosphorylation of two conserved Ser/Thr residues. EMBO J. 1994, 13: 1123-1131.PubMed CentralPubMedGoogle Scholar
- Kim T-W, Guan S, Sun Y, Deng Z, Tang W, Shang J-X, Sun Y, Burlingame AL, Wang Z-Y: Brassinosteroid signal transduction from cell surface receptor kinases to nuclear transcription factors. Nat Cell Biol. 2009, 11: 1254-1260.PubMed CentralPubMedGoogle Scholar
- Pokhilko A, Fernández AP, Edwards KD, Southern MM, Halliday KJ, Millar AJ: The clock gene circuit in Arabidopsis includes a repressilator with additional feedback loops. Mol Syst Biol. 2012, 8: 574-PubMed CentralPubMedGoogle Scholar
- Cheong JK, Virshup DM: Casein kinase 1: complexity in the family. Int J Biochem Cell Biol. 2011, 43: 465-469.PubMedGoogle Scholar
- Behrend L, Stöter M, Kurth M, Rutter G, Heukeshoven J, Deppert W, Knippschild U: Interaction of casein kinase 1 delta (CK1delta) with post-Golgi structures, microtubules and the spindle apparatus. Eur J Cell Biol. 2000, 79: 240-251.PubMedGoogle Scholar
- Ben-Nissan G, Cui W, Kim D-J, Yang Y, Yoo B-C, Lee J-Y: Arabidopsis casein kinase 1-like 6 contains a microtubule-binding domain and affects the organization of cortical microtubules. Plant Physiol. 2008, 148: 1897-1907.PubMed CentralPubMedGoogle Scholar
- Hirota T, Lee JW, Lewis WG, Zhang EE, Breton G, Liu X, Garcia M, Peters EC, Etchegaray J-P, Traver D, Schultz PG, Kay SA: High-throughput chemical screen identifies a novel potent modulator of cellular circadian rhythms and reveals CKIα as a clock regulatory kinase. PLoS Biol. 2010, 8: e1000559-PubMed CentralPubMedGoogle Scholar
- Lee J-Y: Versatile casein kinase 1. Plant Signal Behav. 2009, 4: 652-654.PubMed CentralPubMedGoogle Scholar
- Syed S, Saez L, Young MW: Kinetics of doubletime kinase-dependent degradation of the Drosophila period protein. J Biol Chem. 2011, jbc.M111.243618Google Scholar
- Querfurth C, Diernfellner ACR, Gin E, Malzahn E, Höfer T, Brunner M: Circadian conformational change of the Neurospora clock protein FREQUENCY triggered by clustered hyperphosphorylation of a basic domain. Mol Cell. 2011, 43: 713-722.PubMedGoogle Scholar
- Adl SM, Simpson AGB, Lane CE, Lukeš J, Bass D, Bowser SS, Brown MW, Burki F, Dunthorn M, Hampl V, Heiss A, Hoppenrath M, Lara E, Le Gall L, Lynn DH, McManus H, Mitchell EAD, Mozley-Stanridge SE, Parfrey LW, Pawlowski J, Rueckert S, Shadwick RS, Shadwick L, Schoch CL, Smirnov A, Spiegel FW: The revised classification of eukaryotes. J Eukaryot Microbiol. 2012, 59: 429-493.PubMed CentralPubMedGoogle Scholar
- Meggio F, Pinna LA: One-thousand-and-one substrates of protein kinase CK2?. FASEB J. 2003, 17: 349-368.PubMedGoogle Scholar
- van Ooijen G, Millar AJ: Non-transcriptional oscillators in circadian timekeeping. Trends Biochem Sci. 2012, 37: 484-492.PubMedGoogle Scholar
- Lu SX, Liu H, Knowles SM, Li J, Ma L, Tobin EM, Lin C: A role for protein kinase CK2 alpha subunits in the Arabidopsis circadian clock. Plant Physiol. 2011Google Scholar
- Sugano S, Andronis C, Green RM, Wang ZY, Tobin EM: Protein kinase CK2 interacts with and phosphorylates the Arabidopsis circadian clock-associated 1 protein. Proc Natl Acad Sci U S A. 1998, 95: 11020-11025.PubMed CentralPubMedGoogle Scholar
- Sugano S, Andronis C, Ong MS, Green RM, Tobin EM: The protein kinase CK2 is involved in regulation of circadian rhythms in Arabidopsis. PNAS. 1999, 96: 12362-12366.PubMed CentralPubMedGoogle Scholar
- Mehra A, Shi M, Baker CL, Colot HV, Loros JJ, Dunlap JC: A role for casein kinase 2 in the mechanism underlying circadian temperature compensation. Cell. 2009, 137: 749-760.PubMed CentralPubMedGoogle Scholar
- Portolés S, Más P: The functional interplay between protein kinase CK2 and CCA1 transcriptional activity is essential for clock temperature compensation in Arabidopsis. PLoS Genet. 2010, 6: e1001201-PubMed CentralPubMedGoogle Scholar
- Lin J-M, Kilman VL, Keegan K, Paddock B, Emery-Le M, Rosbash M, Allada R: A role for casein kinase 2α in the Drosophila circadian clock. Nature. 2002, 420: 816-820.PubMedGoogle Scholar
- Akten B, Jauch E, Genova GK, Kim EY, Edery I, Raabe T, Jackson FR: A role for CK2 in the Drosophila circadian oscillator. Nat Neurosci. 2003, 6: 251-257.PubMedGoogle Scholar
- Maier B, Wendt S, Vanselow JT, Wallach T, Reischl S, Oehmke S, Schlosser A, Kramer A: A large-scale functional RNAi screen reveals a role for CK2 in the mammalian circadian clock. Genes Dev. 2009, 23: 708-718.PubMed CentralPubMedGoogle Scholar
- Lowrey PL, Shimomura K, Antoch MP, Yamazaki S, Zemenides PD, Ralph MR, Menaker M, Takahashi JS: Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau. Science. 2000, 288: 483-491.PubMed CentralPubMedGoogle Scholar
- Gallego M, Eide EJ, Woolf MF, Virshup DM, Forger DB: An opposite role for tau in circadian rhythms revealed by mathematical modeling. Proc Natl Acad Sci U S A. 2006, 103: 10618-10623.PubMed CentralPubMedGoogle Scholar
- Jones CR, Campbell SS, Zone SE, Cooper F, DeSano A, Murphy PJ, Jones B, Czajkowski L, Ptácek LJ: Familial advanced sleep-phase syndrome: A short-period circadian rhythm variant in humans. Nat Med. 1999, 5: 1062-1065.PubMedGoogle Scholar
- Toh KL, Jones CR, He Y, Eide EJ, Hinz WA, Virshup DM, Ptácek LJ, Fu YH: An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome. Science. 2001, 291: 1040-1043.PubMedGoogle Scholar
- Xu Y, Padiath QS, Shapiro RE, Jones CR, Wu SC, Saigoh N, Saigoh K, Ptácek LJ, Fu Y-H: Functional consequences of a CKIdelta mutation causing familial advanced sleep phase syndrome. Nature. 2005, 434: 640-644.PubMedGoogle Scholar
- Khadaroo B, Robbens S, Ferraz C, Derelle E, Eychenié S, Cooke R, Peaucellier G, Delseny M, Demaille J, van de Peer Y, Picard A, Moreau H: The first green lineage cdc25 dual-specificity phosphatase. Cell Cycle. 2004, 3: 513-518.PubMedGoogle Scholar
- Fischer S, Brunk BP, Chen F, Gao X, Harb OS, Iodice JB, Shanmugam D, Roos DS, Stoeckert CJ: Using OrthoMCL to Assign Proteins to OrthoMCL-DB Groups or to Cluster Proteomes Into New Ortholog Groups. Current Protocols in Bioinformatics. Edited by: Bateman A, Pearson WR, Stein LD, Stormo GD, Yates JR. 2002, Hoboken: John Wiley & Sons, IncGoogle Scholar
- Vilella AJ, Severin J, Ureta-Vidal A, Heng L, Durbin R, Birney E: EnsemblCompara GeneTrees: complete, duplication-aware phylogenetic trees in vertebrates. Genome Res. 2009, 19: 327-335.PubMed CentralPubMedGoogle Scholar
- Rasko DA, Myers GS, Ravel J: Visualization of comparative genomic analyses by BLAST score ratio. BMC Bioinformatics. 2005, 6: 2-PubMed CentralPubMedGoogle Scholar
- Eddy SR: Accelerated Profile HMM Searches. PLoS Comput Biol. 2011, 7: e1002195-PubMed CentralPubMedGoogle Scholar
- Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunasekaran P, Ceric G, Forslund K, Holm L, Sonnhammer ELL, Eddy SR, Bateman A: The Pfam protein families database. Nucl Acids Res. 2010, 38 (Suppl 1): D211-D222.PubMed CentralPubMedGoogle Scholar
- Hunter T, Plowman GD: The protein kinases of budding yeast: six score and more. Trends Biochem Sci. 1997, 22: 18-22.PubMedGoogle Scholar
- Katoh K, Toh H: Parallelization of the MAFFT multiple sequence alignment program. Bioinformatics. 2010, 26: 1899-1900.PubMed CentralPubMedGoogle Scholar
- Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ: Jalview Version 2–a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009, 25: 1189-1191.PubMed CentralPubMedGoogle Scholar
- Troshin PV, Procter JB, Barton GJ: Java bioinformatics analysis web services for multiple sequence alignment–JABAWS:MSA. Bioinformatics. 2011, 27: 2001-2002.PubMed CentralPubMedGoogle Scholar
- Penn O, Privman E, Landan G, Graur D, Pupko T: An alignment confidence score capturing robustness to guide tree uncertainty. Mol Biol Evol. 2010, 27: 1759-1767.PubMed CentralPubMedGoogle Scholar
- Stamatakis A, Hoover P, Rougemont J: A rapid bootstrap algorithm for the RAxML Web servers. Syst Biol. 2008, 57: 758-771.PubMedGoogle Scholar
- Whelan S, Goldman N: A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach. Mol Biol Evol. 2001, 18: 691-699.PubMedGoogle Scholar
- Cox J, Mann M: MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 2008, 26: 1367-1372.PubMedGoogle Scholar
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