The Arabidopsis cyclin-dependent kinase-activating kinase CDKF;1 is a major regulator of cell proliferation and cell expansion but is dispensable for CDKA activation

Cyclin-dependent kinases (CDKs) play an essential role in cell cycle regulation during the embryonic and post-embryonic development of various organisms. Full activation of CDKs requires not only binding to cyclins but also phosphorylation of the T-loop domain. This phosphorylation is catalysed by CDK-activating kinases (CAKs). Plants have two distinct types of CAKs, namely CDKD and CDKF; in Arabidopsis, CDKF;1 exhibits the highest CDK kinase activity in vitro . We have previously shown that CDKF;1 also functions in the activation of CDKD;2 and CDKD;3 by T-loop phosphorylation. Here, we isolated the knockout mutants of CDKF;1 and showed that they had severe defects in cell division, cell elongation and endoreduplication. No defect was observed during embryogenesis, suggesting that CDKF;1 function is primarily required for post-embryonic development. In the cdkf;1 mutants, T-loop phosphorylation of CDKA;1, an orthologue of yeast Cdc2/Cdc28p, was comparable to that in wild-type plants, and its kinase activity did not decrease. In contrast, the protein level and kinase activity of CDKD;2 were significantly reduced in the mutants. Substitution of threonine-168 with a non-phosphorylatable alanine residue made CDKD;2 unstable in Arabidopsis tissues. These results indicate that CDKF;1 is dispensable for CDKA;1 activation but is essential for maintaining a steady-state level of CDKD;2, thereby suggesting the quantitative regulation of a vertebrate-type CAK in a plant-specific manner.     

Auxin is required for the assembly of A-type cyclin-dependent kinase complexes in tobacco cell suspension culture

Although activation of A-type cyclin-dependent kinase (CDKA) is required for plant cell division, little is known about how CDKA is activated before commitment to cell division. Here, we show that auxin is required for the formation of active CDKA-associated complexes, promoting assembly of the complex in tobacco suspension culture Bright Yellow-2 (BY-2) cells. Protein gel blot analysis revealed that CDKA levels increased greatly after stationary-phase BY-2 cells were subcultured into fresh medium to re-enter the cell cycle. However, these increasing levels subsided when cells were subcultured into auxin-deprived medium, and a subtle increase was observed after subculturing into sucrose-deprived medium. Additionally, p13(suc1)-associated kinase activity did not increase significantly after subculturing into either auxin- or sucrose-deprived medium, but increased strongly after subculturing into medium containing both auxin and sucrose. Using gel filtration, we found that p13(suc1)-associated kinase activity against tobacco retinoblastoma-related protein was maximal in fractions corresponding to the molecular mass of the cyclin/CDKA complex. Interestingly, this peak distribution of high molecular-mass fractions of CDKA disappeared after cells were subcultured into auxin-deprived medium. These findings suggest an important role for auxin in the assembly of active CDKA-associated complexes.     

Phosphorylation of mixed lineage kinase MLK3 by cyclin-dependent kinases CDK1 and CDK2 controls ovarian cancer cell division

Mixed lineage kinase 3 (MLK3) is a serine/threonine mitogen-activated protein kinase kinase kinase that promotes the activation of multiple mitogen-activated protein kinase pathways and is required for invasion and proliferation of ovarian cancer cells. Inhibition of MLK activity causes G2/M arrest in HeLa cells; however, the regulation of MLK3 during ovarian cancer cell cycle progression is not known. Here, we found that MLK3 is phosphorylated in mitosis and that inhibition of cyclin-dependent kinase 1 (CDK1) prevented MLK3 phosphorylation. In addition, we observed that c-Jun N-terminal kinase, a downstream target of MLK3 and a direct target of MKK4 (SEK1), was activated in G2 phase when CDK2 activity is increased and then inactivated at the beginning of mitosis concurrent with the increase in CDK1 and MLK3 phosphorylation. Using in vitro kinase assays and phosphomutants, we determined that CDK1 phosphorylates MLK3 on Ser548 and decreases MLK3 activity during mitosis, whereas CDK2 phosphorylates MLK3 on Ser770 and increases MLK3 activity during G1/S and G2 phases. We also found that MLK3 inhibition causes a reduction in cell proliferation and a cell cycle arrest in ovarian cancer cells, suggesting that MLK3 is required for ovarian cancer cell cycle progression. Taken together, our results suggest that phosphorylation of MLK3 by CDK1 and CDK2 is important for the regulation of MLK3 and c-Jun N-terminal kinase activities during G1/S, G2, and M phases in ovarian cancer cell division.     

Phosphorylation of the homer-binding domain of group I metabotropic glutamate receptors by cyclin-dependent kinase 5

Phosphorylation of neurotransmitter receptors can modify their activity and regulate neuronal excitability. Cyclin-dependent kinase 5 (cdk5) is a proline-directed serine/threonine kinase involved not only in neuronal development, but also in synaptic function and plasticity. Here we demonstrate that group I metabotropic glutamate receptors (mGluRs), which modulate post-synaptic signaling by coupling to intracellular signal transduction pathways, are phosphorylated by cdk5. In vitro kinase assays reveal that cdk5 phosphorylates mGluR5 within the domain of the receptor that interacts with the scaffolding protein homer. Using a novel phosphospecific mGluR antibody, we show that the homer-binding domain of both mGluR1 and mGluR5 are phosphorylated in vivo , and that inhibition of cdk5 with siRNA decreases the amount of phosphorylated receptor. Furthermore, kinetic binding analysis, by surface plasmon resonance, indicates that phosphorylation of mGluR5 enhances its association with homer. Homer protein complexes in the post-synaptic density, and their disruption by an activity-dependent short homer 1a isoform, have been shown to regulate the trafficking and signaling of the mGluRs and impact many neuroadaptive processes. Phosphorylation of the mGluR homer-binding domain, in contrast to homer 1a induction, provides a novel mechanism for potentially regulating a subset of homer interactions.     

Expression of the plant cyclin-dependent kinase inhibitor ICK1 affects cell division, plant growth and morphology

The plant CDK inhibitor ICK1 was identified previously from Arabidopis thaliana with its inhibitory activity characterized in vitro. ICK1 displayed several structural and functional features that are distinct from known animal CDK inhibitors. Despite the initial characterization, there is no information on the functions of any plant CDK inhibitor in plants. To gain insight into ICK1 functions in vivo and the role of cell division during plant growth and development, transgenic plants were generated expressing ICK1 driven by the cauliflower mosaic virus 35S promoter. In comparison to control plants, growth was significantly inhibited in transgenic 35S-ICK1 plants, with some plants weighing <10% of wild-type plants at the 3 week stage. Most organs of 35S-ICK1 plants were smaller. There were also modifications in plant morphology such as shape and serration of leaves and petals. The changes were so drastic that 35S-ICK1 plants with strong phenotype no longer resembled wild-type plants morphologically. Analyses showed that increased ICK1 expression resulted in reduced CDK activity and reduced the number of cells in these plants. Cells in 35S-ICK1 plants were larger than corresponding cells in control plants. These results demonstrate that ICK1 acts as a CDK inhibitor in the plant, and the inhibition of cell division by ICK1 expression has profound effects on plant growth and development. They also suggest that alterations of plant organ shape can be achieved by restriction of cell division.     

The ACR4 receptor-like kinase is required for surface formation of epidermis-related tissues in Arabidopsis thaliana

In higher plants, an outer layer of meristematic cells, the protoderm, forms early in embryogenesis and this layer gives rise to the epidermis in differentiating tissues. We proposed previously that an Arabidopsis thaliana homolog of crinkly4 (ACR4), a gene for a receptor-like protein kinase, would be involved in differentiation and/or maintenance of epidermis-related tissues. In the present study, we isolated loss-of-function acr4 mutants by a reverse genetic approach. Our extensive analyses using the transmission electron microscopy and the toluidine blue test -- a method that has recently been developed for the rapid visualization of defects in the leaf cuticle -- showed that the acr4 mutations significantly affected the differentiation of leaf epidermal cells, suggesting similar roles for ACR4 and CR4 in the differentiation of leaf epidermis. Our acr4 mutants also had various abnormalities related to epidermal differentiation, which included disorganized cell layers in the integument and endothelium of ovules. In addition, the green fluorescent protein fused to ACR4 was localized preferentially on the lateral and basal plasma membranes in the epidermis of the leaf primordia, suggesting a role for ACR4 in epidermal differentiation at cell surfaces that make contact with adjacent cells. Furthermore, the loss-of-function mutations in the ACR4 and ABNORMAL LEAF SHAPE1 (ALE1) genes, which encode a putative subtilisin-like serine protease, synergistically affected the function of the epidermis such that most leaves fused. Thus, ACR4 seems to play an essential role in the differentiation of proper epidermal cells in both vegetative and reproductive tissues.     

Phosphorylation of CAP-G is required for its chromosomal DNA localization during mitosis

Condensin is a 5 subunit complex that plays an important role in the structure of chromosomes during mitosis. It is known that phosphorylation of condensin subunits by cdc2/cyclin B at the beginning of mitosis is important for condensin activity, but the sites of these phosphorylation events have not been identified nor has their role in regulating condensin function. Here we identify two threonine residues in the CAP-G subunit of condensin, threonines 308 and 332, that are targets of cdc2/cyclin B phosphorylation. Mutation of these threonines to alanines results in defects in CAP-G localization with chromosomes during mitosis. These results are the first to identify phosphorylation sites within the condensin complex that regulate condensin localization with chromosomal DNA.     

Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis

We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (Plk1). MKlp2 is a target for Plk1, and phosphorylated MKlp2 binds to the polo box domain of Plk1. Plk1 also binds directly to microtubules and targets to the central spindle via its polo box domain, and this interaction controls the activity of Plk1 toward MKlp2. An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by Plk1 causes a cytokinesis defect when introduced into cells. We propose that phosphorylation of MKlp2 by Plk1 is necessary for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.     

A specific activation of the mitogen-activated protein kinase kinase 1 (MEK1) is required for Golgi fragmentation during mitosis

Incubation of permeabilized cells with mitotic extracts results in extensive fragmentation of the pericentriolarly organized stacks of cisternae. The fragmented Golgi membranes are subsequently dispersed from the pericentriolar region. We have shown previously that this process requires the cytosolic protein mitogen-activated protein kinase kinase 1 (MEK1). Extracellular signal-regulated kinase (ERK) 1 and ERK2, the known downstream targets of MEK1, are not required for this fragmentation (Acharya et al. 1998). We now provide evidence that MEK1 is specifically phosphorylated during mitosis. The mitotically phosphorylated MEK1, upon partial proteolysis with trypsin, generates a different peptide population compared with interphase MEK1. MEK1 cleaved with the lethal factor of the anthrax toxin can still be activated by its upstream mitotic kinases, and this form is fully active in the Golgi fragmentation process. We believe that the mitotic phosphorylation induces a change in the conformation of MEK1 and that this form of MEK1 recognizes Golgi membranes as a target compartment. Immunoelectron microscopy analysis reveals that treatment of permeabilized normal rat kidney (NRK) cells with mitotic extracts, treated with or without lethal factor, converts stacks of pericentriolar Golgi membranes into smaller fragments composed predominantly of tubuloreticular elements. These fragments are similar in distribution, morphology, and size to the fragments observed in the prometaphase/metaphase stage of the cell cycle in vivo.     

A quantitative model for cyclin-dependent kinase control of the cell cycle: revisited

The eukaryotic cell division cycle encompasses an ordered series of events. Chromosomal DNA is replicated during S phase of the cell cycle before being distributed to daughter cells in mitosis. Both S phase and mitosis in turn consist of an intricately ordered sequence of molecular events. How cell cycle ordering is achieved, to promote healthy cell proliferation and avert insults on genomic integrity, has been a theme of Paul Nurse's research. To explain a key aspect of cell cycle ordering, sequential S phase and mitosis, Stern & Nurse proposed 'A quantitative model for cdc2 control of S phase and mitosis in fission yeast'. In this model, S phase and mitosis are ordered by their dependence on increasing levels of cyclin-dependent kinase (Cdk) activity. Alternative mechanisms for ordering have been proposed that rely on checkpoint controls or on sequential waves of cyclins with distinct substrate specificities. Here, we review these ideas in the light of experimental evidence that has meanwhile accumulated. Quantitative Cdk control emerges as the basis for cell cycle ordering, fine-tuned by cyclin specificity and checkpoints. We propose a molecular explanation for quantitative Cdk control, based on thresholds imposed by Cdk-counteracting phosphatases, and discuss its implications.     

Phosphorylation of HtrA2 by cyclin-dependent kinase-5 is important for mitochondrial function

The role of the serine protease HtrA2 in neuroprotection was initially identified by the demonstration of neurodegeneration in mice lacking HtrA2 expression or function, and the interesting finding that mutations adjacent to two putative phosphorylation sites (S142 and S400) have been found in Parkinson's disease patients. However, the mechanism of this neuroprotection and the signalling pathways associated with it remain mostly unknown. Here we report that cyclin-dependent kinase-5 (Cdk5), a kinase implicated in the pathogenesis of several neurodegenerative diseases, is responsible for phosphorylating HtrA2 at S400. HtrA2 and Cdk5 interact in human and mouse cell lines and brain, and Cdk5 phosphorylates S400 on HtrA2 in a p38-dependent manner. Phosphorylation of HtrA2 at S400 is involved in maintaining mitochondrial membrane potential under stress conditions and is important for mitochondrial function, conferring cells protection against cellular stress.     

Phosphorylation of histone H2A is associated with centromere function and maintenance in meiosis

Histone phosphorylation is dynamically regulated during cell division, for example phosphorylation of histone H3 (H3)-Ser10, H3-Thr11 and H3-Ser28. Here we analyzed maize (Zea mays L) for Thr133-phosphorylated histone H2A, which is important for spindle checkpoint control and localization of the centromere cohesion protector Shugoshin in mammals and yeast. Immunostaining results indicate that phosphorylated H2A-Thr133 signals bridged those of the centromeric H3 histone variant CENH3 by using a plant displaying yellow fluorescent protein-CENH3 signals and H2A-Thr133 is phosphorylated in different cell types. During mitosis, H2A-Thr133 phosphorylation becomes strong in metaphase and is specific to centromere regions but drops during later anaphase and telophase. Immunostaining for several maize dicentric chromosomes revealed that the inactive centromeres have lost phosphorylation of H2A-Thr133. During meiosis in maize meiocytes, H2A phosphorylation becomes strong in the early pachytene stage and increases to a maximum at metaphase I. In the maize meiotic mutant afd1 (absence of first division), sister chromatids show equational separation at metaphase I, but there are no changes in H2A-Thr-133 phosphorylation during meiosis compared with the wild type. In sgo1 mutants, sister chromatids segregate randomly during meiosis II, and phosphorylation of H2A-Thr-133 is observed on the centromere regions during meiosis II. The availability of such mutants in maize that lack sister cohesion and Shugoshin indicate that the signals for phosphorylation are not dependent on cohesion but on centromere activity.     

Expression of telomerase activity is closely correlated with the capacity for cell division in tobacco plants

Telomerase is a specialized RNA-directed DNA polymerase that adds telomeric repeats to the ends of linear eukaryotic chromosomes. This activity is developmentally regulated in mammals. Here, we investigated the expression of telomerase activities in various cell types of tobacco plants using the telomere repeat amplification protocol (TRAP) assay. The greatest telomerase activity was detected in BY-2 suspension culture cells, while a relatively high level of activity was also found in roots. Because these two cell types contain a high proportion of actively dividing cells, our results indicate a close correlation between telomerase activity and the capacity for division in tobacco cells. Consistent with this observation was the very low level of telomerase activity in stems, leaves, and flowers, all tissues that had negligible activity of cell division. The specific expression of telomerase in actively dividing plant cells suggests that the pattern of telomerase regulation is largely conserved between higher plants and mammals.     

Silencing of subfamily I of protein phosphatase 2A catalytic subunits results in activation of plant defense responses and localized cell death

The central importance of protein phosphorylation in plant defense responses has been demonstrated by the isolation of several disease-resistance genes that encode protein kinases. In addition, there are many reports of changes in protein phosphorylation accompanying plant responses to pathogens. In contrast, little is known about the role of protein dephosphorylation in regulating plant defenses. We report that expression of the LePP2Ac1 gene, which encodes a catalytic subunit of the heterotrimeric protein phosphatase 2A (PP2Ac), is rapidly induced in resistant tomato leaves upon inoculation with an avirulent strain of Pseudomonas syringae pv. tomato. By analysis of PP2Ac gene sequences from several plant species, we found that PP2Ac genes cluster into two subfamilies, with LePP2Ac1 belonging to subfamily I. Virus-induced gene silencing (VIGS) in Nicotiana benthamiana was used to suppress expression of genes from subfamily I and not from subfamily II. The PP2Ac-silenced plants had greatly decreased PP2A activity, constitutively expressed pathogenesis-related (PR) genes, and developed localized cell death in stems and leaves. In addition, the plants were more resistant to a virulent strain of P. syringae pv. tabaci and showed an accelerated hypersensitive response (HR) to effector proteins from both P. syringae and the fungal pathogen, Cladosporium fulvum. Thus, catalytic subunits of PP2Ac subfamily I act as negative regulators of plant defense responses likely by de-sensitizing protein phosphorylation cascades.     

Phosphorylation at serine 318 is not required for inhibition of T cell activation by ALX

The activation of T cells and the initiation of an immune response is tightly controlled by both positive and negative regulators. Two adaptors which function as negative regulators of T cell activation are ALX and LAX. ALX constitutively associates with LAX in T cells, and T cells from mice deficient in ALX and LAX display similar hyper-responsiveness upon T cell receptor (TCR)/CD28 stimulation, including increased production of interleukin-2. During T cell activation, ALX is inducibly phosphorylated, however the site of ALX phosphorylation had not been previously identified and the role of phosphorylation in the inhibitory function of ALX was not known. Here, using mass spectrometry, we demonstrate that ALX is phosphorylated on a serine at position 318. Substitution of alanine for serine at this position (ALX S318A) leads to an abrogation of the mobility shift in ALX induced upon TCR/CD28 stimulation. However, ALX S318A retained the ability to bind to and stimulate tyrosine phosphorylation of LAX. In addition, overexpression of ALX S318A inhibited RE/AP activation upon TCR/CD28 stimulation to a similar extent as wild-type ALX. Therefore, although ALX is inducibly phosphorylated upon TCR/CD28 stimulation, this phosphorylation is not required for ALX to inhibit T cell activation.