Cryptogein-induced cell cycle arrest at G2 phase is associated with inhibition of cyclin-dependent kinases, suppression of expression of cell cycle-related genes and protein degradation in synchronized tobacco BY-2 cells

Induction of defense responses by pathogens or elicitors is often accompanied by growth inhibition in planta, but its molecular mechanisms are poorly understood. In this report, we characterized the molecular events that occur during cryptogein-induced cell cycle arrest at G(2) phase in synchronously cultured tobacco Bright Yellow-2 (BY-2) cells. Concomitant with the proteinaceous elicitor-induced G(2) arrest, we observed inhibition of the histone H1 kinase activity of cyclin-dependent kinases (CDKs), which correlated with a decrease in mRNA and protein levels of CDKB1. In contrast, the amount of CDKA was almost unaffected by cryptogein even at M phase. Cryptogein rapidly inhibited the expression not only of positive, e.g. A- and B-type cyclins and NtCAK, but also of negative cell cycle regulators such as WEE1, suggesting that cryptogein affects multiple targets to inactivate CDKA to induce G(2) arrest by mechanisms distinct from known checkpoint regulation. Moreover, we show that CDKB1 and cyclin proteins are also rapidly degraded by cryptogein and that the proteasome-dependent protein degradation has a crucial role in the control of cryptogein-induced hypersensitive cell death.     

Natural Synchronisation for the Study of Cell Division in the Green Unicellular Alga Ostreococcus tauri

Ostreococcus tauri (Prasinophyceae) is a marine unicellular green alga which diverged early in the green lineage. The interest of O. tauri as a potential model to study plant cell division is based on its key phylogenetic position, its simple binary division, a very simple cellular organisation and now the availability of the full genome sequence. In addition O. tauri has a minimal yet complete set of cell cycle control genes. Here we show that division can be naturally synchronised by light/dark cycles and that organelles divide before the nucleus. This natural synchronisation, although being only partial, enables the study of the expression of CDKs throughout the cell cycle. The expression patterns of OtCDKA and OtCDKB were determined both at the mRNA and protein levels. The single OtCDKA gene is constantly expressed throughout the cell cycle, whereas OtCDKB is highly regulated and expressed only in S/G2/M phases. More surprisingly, OtCDKA is not phosphorylated at the tyrosine residue, in contrast to OtCDKB which is strongly phosphorylated during cell division. OtCDKA kinase activity appears before the S phase, indicating a possible role of this protein in the G1/S transition. OtCDKB kinase activity occurs later than OtCDKA, and its tyrosine phosphorylation is correlated to G2/M, suggesting a possible control of the mitotic activity. To our knowledge this is the first organism in the green lineage which showed CDKB tyrosine phosphorylation during cell cycle progression.     

Phosphorylation of retinoblastoma-related protein by the cyclin D/cyclin-dependent kinase complex is activated at the G1/S-phase transition in tobacco

In mammals, D-type cyclin-associated kinases mainly regulate the G1/S transition by phosphorylating the retinoblastoma (Rb) protein. We previously demonstrated that in tobacco, cyclin D (Nicta; CycD3;3) is complexed with the PSTAIRE-containing cyclin-dependent kinase (CDKA) from tobacco. Here, we show that Nicta; CycD3;3-associated kinases phosphorylate both the tobacco Rb-related protein (NtRb1) and histone H1. Although NtRb1 kinase activity was detected only during the middle G1- to early S-phase, histone H1 kinase activity was observed as two peaks in G1- to S-phase and G2/M- to M-phase. Importantly, we show that the proportion of cells in the G1-phase was reduced in transgenic Bright Yellow-2 cells overexpressing Nicta; CycD3;3-GFP. Mutational analyses revealed that phosphorylation of Thr-191 in Nicta; CycD3;3 possibly is required for both full kinase activity and localization predominantly to the nucleus. These data suggest that Nicta; CycD3;3 acts as a rate-limiting regulator in the G1/S transition by forming active complexes with CDKA or its related kinases to phosphorylate Rb-related protein and potentially plays a novel role during G2/M and mitosis.     

Genetic framework of cyclin-dependent kinase function in Arabidopsis

Cyclin-dependent kinases (CDKs) are at the heart of eukaryotic cell-cycle control. The yeast Cdc2/CDC28 PSTAIRE kinase and its orthologs such as the mammalian Cdk1 have been found to be indispensable for cell-cycle progression in all eukaryotes investigated so far. CDKA;1 is the only PSTAIRE kinase in the flowering plant Arabidopsis and can rescue Cdc2/CDC28 mutants. Here, we show that cdka;1 null mutants are viable but display specific cell-cycle and developmental defects, e.g., in S phase entry and stem cell maintenance. We unravel that the crucial function of CDKA;1 is the control of the plant Retinoblastoma homolog RBR1 and that codepletion of RBR1 and CDKA;1 rescued most defects of cdka;1 mutants. Our work further revealed a basic cell-cycle control system relying on two plant-specific B1-type CDKs, and the triple cdk mutants displayed an early germline arrest. Taken together, our data indicate divergent functional differentiation of Cdc2-type kinases during eukaryote evolution.     

Cell cycle function of a rice B2-type cyclin interacting with a B-type cyclin-dependent kinase

Cyclin-dependent kinases (CDKs) are involved in the control of cell cycle progression. Plant A-type CDKs are functional homologs of yeast Cdc2/Cdc28 and are expressed throughout the cell cycle. In contrast, B-type CDK (CDKB) is a family of mitotic CDKs expressed during the S/M phase, and its precise function remains unknown. Here, we identified two B2-type cyclins, CycB2;1 and CycB2;2, as a specific partner of rice CDKB2;1. The CDKB2;1-CycB2 complexes produced in insect cells showed a significant level of kinase activity in vitro, suggesting that CycB2 binds to and activates CDKB2. We then expressed green fluorescent protein (GFP)-fused CDKB2;1 and CycB2;2 in tobacco BY2 cells to investigate their subcellular localization during mitosis. Surprisingly, the fluorescence signal of CDKB2;1-GFP was tightly associated with chromosome alignment as well as with spindle structure during the metaphase. During the telophase, the signal was localized to the spindle midzone and the separating sister chromosomes, and then to the phragmoplast. On the other hand, the CycB2;2-GFP fluorescence signal was detected in nuclei during the interphase and prophase, moved to the metaphase chromosomes, and then disappeared completely after the cells passed through the metaphase. Co-localization of CDKB2;1-GFP and CycB2;2-GFP on chromosomes aligned at the center of the metaphase cells suggests that the CDKB2-CycB2 complex may function in retaining chromosomes at the metaphase plate. Overexpression of CycB2;2 in rice plants resulted in acceleration of root growth without any increase in cell size, indicating that CycB2;2 promoted cell division probably through association with CDKB2 in the root meristem.     

Cyclin dependent kinases and their role in regulation of plant cell cycle

Plants have capability to optimize its architecture by using CDK pathways. It involves diverse types of cyclin dependent kinase enzymes (CDKs). CDKs are classified in to eight classes (CDKA to CDKG and CKL) based on the recognized cyclin-binding domains. These enzymes require specific cyclin proteins to get activated. They form complex with cyclin subunits and phosphorylate key target proteins. Phosphorylation of these target proteins is essential to drive cell cycle further from one phase to another phase. During cell division, the activity of cyclin dependent kinase is controlled by CDK interactor/inhibitor of CDKs (ICK) and Kip-related proteins (KRPs). They bind with specific CDK/cyclin complex and help in controlling CDKs activity. Since cell cycle can be progressed further only by synthesis and destruction of cyclins, they are quickly degraded using ubiquitination-proteasome pathway. Ubiquitylation reaction is followed by DNA duplication and cell division process. These two processes are regulated by two complexes known as Skp1/cullin/F-box (SCF)-related complex and the anaphase-promoting complex/cyclosome (APC/C). SCF allows cell to enter from G1 to S phase and APC/C allows cell to enter from G2 to M phase. When all these above processes of cell division are going on, genes of cyclin dependent kinases gets activated one by one simultaneously and help in regulation of CDK pathways. How cell cycle is regulated by CDKs is discussed.     

Regulation of tomato fruit pericarp development by an interplay between CDKB and CDKA1 cell cycle genes

Growth of tomato fruits is determined by cell division and cell expansion, which are tightly controlled by factors that drive the core cell cycle. The cyclin-dependent kinases (CDKs) and their interacting partners, the cyclins, play a key role in the progression of the cell cycle. In this study the role of CDKA1, CDKB1, and CDKB2 in fruit development was characterized by fruit-specific overexpression and down-regulation. CDKA1 is expressed in the pericarp throughout development, but is strongly up-regulated in the outer pericarp cell layers at the end of the growth period, when CDKB gene expression has ceased. Overexpression of the CDKB genes at later stages of development and the down-regulation of CDKA1 result in a very similar fruit phenotype, showing a reduction in the number of cell layers in the pericarp and alterations in the desiccation of the fruits. Expression studies revealed that CDKA1 is down-regulated by the expression of CDKB1/2 in CDKB1 and CDKB2 overexpression mutants, suggesting opposite roles for these types of CDK proteins in tomato pericarp development.     

Arabidopsis KRPs have distinct inhibitory activity toward cyclin D2-associated kinases, including plant-specific B-type cyclin-dependent kinase

Arabidopsis contains seven Kip-related protein (KRP) genes encoding CDK (cyclin-dependent kinase) inhibitors (CKIs), which shares a restricted similarity with mammalian p27Kip1. Here, we analyze the characteristics of the KRPs. Although KRP1-KRP7 interact with active cyclin D2 (CYCD2)/CDKA and CYCD2/CDKB complexes to a similar extent, they inhibit kinase activity to a different extent. Our results suggest that inhibitory activity is related to the binding ability between KRP proteins and cyclin/CDK complexes, but secondary and tertiary structure may be also involved. These data provide the first evidence that KRPs inhibit kinase activity associated with plant-specific CDKB.     

Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6.

Cyclin D-dependent kinases act as mitogen-responsive, rate-limiting controllers of G1 phase progression in mammalian cells. Two novel members of the mouse INK4 gene family, p19 and p18, that specifically inhibit the kinase activities of CDK4 and CDK6, but do not affect those of cyclin E-CDK2, cyclin A-CDK2, or cyclin B-CDC2, were isolated. Like the previously described human INK4 polypeptides, p16INK4a/MTS1 and p15INK4b/MTS2, mouse p19 and p18 are primarily composed of tandemly repeated ankyrin motifs, each ca. 32 amino acids in length, p19 and p18 bind directly to CDK4 and CDK6, whether untethered or in complexes with D cyclins, and can inhibit the activity of cyclin D-bound cyclin-dependent kinases (CDKs). Although neither protein interacts with D cyclins or displaces them from preassembled cyclin D-CDK complexes in vitro, both form complexes with CDKs at the expense of cyclins in vivo, suggesting that they may also interfere with cyclin-CDK assembly. In proliferating macrophages, p19 mRNA and protein are periodically expressed with a nadir in G1 phase and maximal synthesis during S phase, consistent with the possibility that INK4 proteins limit the activities of CDKs once cells exit G1 phase. However, introduction of a vector encoding p19 into mouse NIH 3T3 cells leads to constitutive p19 synthesis, inhibits cyclin D1-CDK4 activity in vivo, and induces G1 phase arrest.     

G(1)/S CDK is inhibited to restrain mitotic onset when DNA replication is blocked in fission yeast

Cyclin-dependent kinase (CDK) Tyr15 phosphorylation plays a major role in regulating G(2)/M CDKs, but the role of this phosphorylation in regulating G(1)/S CDKs is less clear. We have studied the regulation and function of Cdc2-Tyr15 phosphorylation in the fission yeast Schizosaccharomyces pombe G(1)/S CDK Cig2/Cdc2. This complex is subject to high level Cdc2-Tyr15 phosphorylation inhibiting its kinase activity in hydroxyurea-treated cells blocked in S-phase. We show that this Tyr15 phosphorylation is required to maintain efficient mitotic checkpoint arrest, because Cig2 accumulates during the block and this accumulation can advance mitotic onset. This mitotic induction operates, at least in part, through activation of the normal G(2)/M CDK complex Cdc13/Cdc2. Thus, Tyr15 phosphorylation of G(1)/S CDK complexes is important in the checkpoint control blocking mitotic onset when DNA replication is inhibited.     

A cell-cycle-regulated kinase activity phosphorylates plant retinoblastoma protein and contains, in Arabidopsis, a CDKA/cyclin D complex

The activity of cyclin-dependent kinases (CDK) is crucial for cell-cycle transitions. Here, we report the identification of a CDK activity that phosphorylates the retinoblastoma-related (RBR) protein. A CDK/cyclin complex that binds to and phosphorylates RBR may be isolated from various plant sources, e.g. wheat, maize, Arabidopsis thaliana and tobacco, and from cells growing under various conditions. The presence of an RBR-associated CDK activity correlates with the proliferative activity, suggesting that phosphorylation of RBR is a major event in actively proliferating tissues. In A. thaliana, this activity comprises a PSTAIRE CDKA and at least cyclin D2. Furthermore, this CDK activity is cell-cycle-regulated, as revealed by studies with highly synchronized tobacco BY-2 cells where it is maximal in late G1 and early S phase cells and progressively decreases until G2 phase. Aphidicolin-arrested but not roscovitine-arrested cells contain a PSTAIRE-type CDK that binds to and phosphorylates RBR. Thus, association with a D-type cyclin is a likely mechanism leading to CDK activation late in G1. Our studies constitute the first report measuring the activity of CDK/cyclin complexes formed in vivo on RBR, an activity that fluctuates in a cell-cycle-dependent manner. This work provides the basis for further studies on the impact of phosphorylation of RBR on its function during the cell cycle and development.     

A PSTAIRE CDK-like protein localizes in nuclei and cytoplasm of Physarum polycephalum and functions in the mitosis

CDKs play key roles in controlling cell cycle progression in all eukaryotes. In plants, multiple CDKs are present,among which the best characterized CDKs are PSTAIRE CDKs. In this study, we carried out Western blot,immunoelectron microscopy and antibody treatment with an anti-PSTAIRE monoclonal antibody to explore the subcellular localization and functions of PSTAIRE CDKs in Physarum polycephalum. The results of Western blot and immunoelectron microscopy showed that in P. polycephalum, a PSTAIRE CDK-like protein was 34 kD in molecular weight and located in both nuclei and cytoplasm. In nuclei, the protein was mainly associated with chromosomes and nucleoli. The expression of the PSTAIRE CDK-like protein in both the plasmodia and nuclei showed little fluctuation through the whole cell cycle. When treated with an anti-PSTAIRE monoclonal antibody at early S phase, the cells were arrested in S phase, and the mitotic onset of P. polycephalum was blocked for about 1 h when treated at early G2 phase.Our data indicated that the PSTAIRE CDK-like protein has a direct bearing on the mitosis.