Marta Riera

Distinctive features of plant protein kinase CK2

In plants, protein kinase CK2 is involved in different processes that control many aspects of metabolism and development. In mammals and yeast the enzyme is a heterotetramer composed of two types of subunits. During years the subunit composition of the maize protein kinase CK2 enzyme has been a source of controversy. We have recently characterized the maize holoenzyme subunits. Our results show that multiple catalytic and regulatory subunits are expressed in maize and are able to specifically interact with other α and β subunits suggesting a high level of heterogeneity in the typical heterotetrameric structure. Here, we summarize data available on plant CK2 enzymes, in order to clarify the distinctive features and functions of plant protein kinase CK2. (Mol Cell Biochem 227: 119-127, 2001)

A MYB/ZML Complex Regulates Wound-Induced Lignin Genes in Maize

A methyl jasmonate-dependent MYB-ZML regulatory mechanism links wounding stress to the derepression of lignin genes in maize. Lignin is an essential polymer in vascular plants that plays key structural roles in vessels and fibers. Lignification is induced by external inputs such as wounding, but the molecular mechanisms that link this stress to lignification remain largely unknown. In this work, we provide evidence that three maize (Zea mays) lignin repressors, MYB11, MYB31, and MYB42, participate in wound-induced lignification by interacting with ZML2, a protein belonging to the TIFY family. We determined that the three R2R3-MYB factors and ZML2 bind in vivo to AC-rich and GAT(A/C) cis-elements, respectively, present in a set of lignin genes. In particular, we show that MYB11 and ZML2 bind simultaneously to the AC-rich and GAT(A/C) cis-elements present in the promoter of the caffeic acid O-methyl transferase (comt) gene. We show that, like the R2R3-MYB factors, ZML2 also acts as a transcriptional repressor. We found that upon wounding and methyl jasmonate treatments, MYB11 and ZML2 proteins are degraded and comt transcription is induced. Based on these results, we propose a molecular regulatory mechanism involving a MYB/ZML complex in which wound-induced lignification can be achieved by the derepression of a set of lignin genes.

Association of protein kinase CK2 with eukaryotic translation initiation factor eIF-2 and with grp94/endoplasmin

Protein kinase CK2 forms complexes with some protein substrates what may be relevant for the physiological control of this protein kinase. In previous studies in rat liver cytosol we had detected that the trimeric form of eukaryotic translation initiation factor 2 (eIF-2) co-eluted with protein kinase CK2. We have now observed that the ratio between eIF-2 and cytosolic CK2 contents in testis, liver and brain is quite similar, being eIF-2 levels about 5-fold higher than those of CK2. Furthermore eIF-2 was present in liver samples immunoprecipitated with anti-CK2α/α′ antibodies, confirming the existence of complexes containing both proteins. Nonetheless, these complexes would represent only a fraction of total cytosolic CK2 and eIF-2.

Functional analysis of the isoforms of an ABI3-like factor of Pisum sativum generated by alternative splicing.

At least seven isoforms (PsABI3-1 to PsABI3-7) of a putative, pea ABI3-like factor, originated by alternative splicing, have been identified after cDNA cloning. A similar variability had previously only been described for monocot genes. The full-length isoform, PsABI3-1, contains the typical N-terminal acidic domains and C-terminal basic subdomains, B1 to B3. Reverse transcriptase-PCR analysis revealed that the gene is expressed just in seeds, starting at middle embryogenesis; no gene products are observed in embryo axes after 18 h post-imbibition although they are more persistent in cotyledons. The activity of the isoforms was studied by yeast one-hybrid assays. When yeast was transformed with the isoforms fused to the DNA binding domain of Gal4p, only the polypeptides PsABI3-2 and PsABI3-7 failed to complement the activity of Gal4p. Acidic domains A1 and A2 exhibit transactivating activity, but the former requires a small C-terminal extension to be active. Yeast two-hybrid analysis showed that PsABI3 is able to heterodimerize with Arabidopsis thaliana ABI5, thus proving that PsABI3 is functionally active. The minimum requirement for the interaction PsABI3–AtABI5 is the presence of the subdomain B1 with an extension, 81 amino acids long, at their C-terminal side. Finally, a transient onion transformation assay showed that both the active PsABI3-1 and the inactive PsABI3-2 isoforms are localized to nuclei. Considering that the major isoforms remain approximately constant in developing seeds although their relative proportion varied, the possible role of splicing in the regulatory network of ABA signalling is discussed.

Maize AKINβγ dimerizes through the KIS/CBM domain and assembles into SnRK1 complexes

MINT‐7039978: SnRK1 (uniprotkb:Q8H1L5) and AKIN (uniprotkb:B4FX20) physically interact (MI:0915) by bimolecular fluorescence complementation (MI:0809)

Emerging roles of protein kinase CK2 in abscisic acid signaling

The phytohormone abscisic acid (ABA) regulates many aspects of plant growth and development as well as responses to multiple stresses. Post-translational modifications such as phosphorylation or ubiquitination have pivotal roles in the regulation of ABA signaling. In addition to the positive regulator sucrose non-fermenting-1 related protein kinase 2 (SnRK2), the relevance of the role of other protein kinases, such as CK2, has been recently highlighted. We have recently established that CK2 phosphorylates the maize ortholog of open stomata 1 OST1, ZmOST1, suggesting a role of CK2 phosphorylation in the control of ZmOST1 protein degradation (Vilela et al., 2015). CK2 is a pleiotropic enzyme involved in multiple developmental and stress-responsive pathways. This review summarizes recent advances that taken together suggest a prominent role of protein kinase CK2 in ABA signaling and related processes.

Role of Plant-Specific N-Terminal Domain of Maize CK2β1 Subunit in CK2β Functions and Holoenzyme Regulation

Protein kinase CK2 is a highly pleiotropic Ser/Thr kinase ubiquituous in eukaryotic organisms. CK2 is organized as a heterotetrameric enzyme composed of two types of subunits: the catalytic (CK2α) and the regulatory (CK2β). The CK2β subunits enhance the stability, activity and specificity of the holoenzyme, but they can also perform functions independently of the CK2 tetramer. CK2β regulatory subunits in plants differ from their animal or yeast counterparts, since they present an additional specific N-terminal extension of about 90 aminoacids that shares no homology with any previously characterized functional domain. Sequence analysis of the N-terminal domain of land plant CK2β subunit sequences reveals its arrangement through short, conserved motifs, some of them including CK2 autophosphorylation sites. By using maize CK2β1 and a deleted version (ΔNCK2β1) lacking the N-terminal domain, we have demonstrated that CK2β1 is autophosphorylated within the N-terminal domain. Moreover, the holoenzyme composed with CK2α1/ΔNCK2β1 is able to phosphorylate different substrates more efficiently than CK2α1/CK2β1 or CK2α alone. Transient overexpression of CK2β1 and ΔNCK2β1 fused to GFP in different plant systems show that the presence of N-terminal domain enhances aggregation in nuclear speckles and stabilizes the protein against proteasome degradation. Finally, bimolecular fluorescence complementation (BiFC) assays show the nuclear and cytoplasmic location of the plant CK2 holoenzyme, in contrast to the individual CK2α/β subunits mainly observed in the nucleus. All together, our results support the hypothesis that the plant-specific N-terminal domain of CK2β subunits is involved in the down-regulation of the CK2 holoenzyme activity and in the stabilization of CK2β1 protein. In summary, the whole amount of data shown in this work suggests that this domain was acquired by plants for regulatory purposes.

Specific characteristics of CK2β regulatory subunits in plants

In all eukaryotes, the typical CK2 holoenzyme is an heterotetramer composed of two catalytic (CK2α and CK2α′) and two regulatory (CK2β) subunits. One of the distinctive traits of plant CK2 is that they present a greater number of genes encoding for CK2α/β subunits than animals or yeasts, for instance, in Arabidopsis and maize both CK2α/β subunits belong to multigenic families composed by up to four genes. Here, we conducted a genome-wide survey examining 34 different plant genomes in order to investigate if the multigenic property of CK2β genes is a common feature through the entire plant kingdom. Also, at the level of structure, the plant CK2β regulatory subunits present distinctive features as (i) they lack about 20 aminoacids in the C-terminal domain, (ii) they present a specific N-terminal extension of about 90 aminoacids that shares no homology with any previously characterized functional domain, and (iii) the acidic loop region is poorly conserved at the aminoacid level. Since there is no data about CK2β or holoenzyme structure in plants, in this study, we use human CK2β as a template to predict a structure for Zea mays CK2β1 by homology modeling and we discuss about possible structural changes in the acidic loop region that could affect the enzyme regulation.

Novel CK2α and CK2β subunits in maize reveal functional diversification in subcellular localization and interaction capacity

In plants, CK2α/β subunits are encoded by multigenic families. They assemble as heterotetrameric holoenzymes or remain as individual subunits and are usually located in distinct cell compartments. Here we revise the number of maize CK2α/β genes, bringing them up to a total of eight (four CK2α catalytic and four CK2β regulatory subunits). We characterize CK2β4, which presents nuclear localization and interacts with CK2α1, CK2α3, CK2β1, and CK2β3. We also describe two CK2α isoforms (CK2α2 and CK2α4) containing N-terminal extensions that correspond to putative cTPs (chloroplast transit peptides). These cTPs are functional and responsible for the subcellular localization of CK2α2 and CK2α4 in chloroplasts. Phylogenetic analysis of the CK2α gene family, further supported by the gene structure and architecture of conserved protein domains, reveals the evolutionary expansion and diversification of this family. The subcellular localization of all four CK2α isoforms was found to be altered when were co-expressed with CK2β, thereby pointing to the latter as regulators of CK2α localization.

Maize RNA-Binding Protein ZmTGH: A New Partner for CK2β1 Regulatory Subunit

In plants, as in humans, splicing regulation controls plant growth and development. Plant CK2 is involved in multiple signaling pathways. The maize CK2β1 regulatory subunit is preferentially accumulated in nuclear speckles, and little is known about the role of CK2 in these nuclear structures that contain splicing regulators among other proteins. Here we perform a yeast two-hybrid screening using CK2β1 as a bait, and between other putative partners, we found an RNA-binding protein that presents high homology with Arabidopsis TGH, an evolutionary conserved protein required for proper plant development. In addition, transient co-transformation of both proteins in maize embryo cells confirms that maize CK2β1 regulatory subunit and ZmTGH colocalize in nuclear speckles. The interaction and colocalization of both proteins suggest a possible role of CK2 modulating proteins involved in splicing processes in plants.