Misexpression of the cyclin-dependent kinase inhibitor ICK1/KRP1 in single-celled Arabidopsis trichomes reduces endoreduplication and cell size and induces cell death

A positive correlation between cell size and DNA content has been recognized in many plant cell types. Conversely, misexpression of a dominant-negative cyclin-dependent kinase (CDK) or CDK inhibitor proteins (ICK/KRPs) in Arabidopsis and tobacco leaves has revealed that cell growth can be uncoupled from cell cycle progression and DNA content. However, cell growth also appears to be controlled in a non-cell-autonomous manner by organ size, making it difficult in a ubiquitous expression assay to judge the cell-autonomous function of putative cell growth regulators. Here, we investigated the function of the CDK inhibitor ICK1/KRP1 on cell growth and differentiation independent of any compensatory influence of an organ context using Arabidopsis trichomes as a model system. By analyzing cell size with respect to DNA content, we dissected cell growth in a DNA-dependent and a DNA-independent process. We further found that ICK1/KRP1 misexpression interfered with differentiation and induced cell death, linking cell cycle progression, differentiation, and cell death in plants. The function of ICK1/KRP1 in planta was found to be dependent on a C-terminal domain and regulated negatively by an N-terminal domain. Finally, we identified CDKA;1 and a D-type cyclin as possible targets of ICK1/KRP1 expression in vivo.     

The Arabidopsis Cdc2a-interacting protein ICK2 is structurally related to ICK1 and is a potent inhibitor of cyclin-dependent kinase activity in vitro

Cyclin-dependent kinases (CDKs) are important regulators of the eukaryotic cell division cycle. To study protein-protein interactions involving plant CDKs, the Arabidopsis thaliana Cdc2aAt was used as bait in the yeast two-hybrid system. Here we report on the isolation of ICK2, and show that it interacts with Cdc2aAt, but not with a second CDK from Arabidopsis, Cdc2bAt. ICK2 contains a carboxy-terminal domain related to that of ICK1, a previously described CDK inhibitor from Arabidopsis, and to the CDK-binding domain of the mammalian inhibitor p27Kip1. Outside of this domain, ICK2 is distinct from ICK1, p27Kip1, and other proteins. At nanogram levels (8 nM), purified recombinant ICK2 inhibits p13Suc1-associated histone H1 kinase activity from Arabidopsis tissue extracts, demonstrating that it is a potent inhibitor of plant CDK activity in vitro. ICK2 mRNA was present in all tissues analysed by Northern hybridization, and its distribution was distinct from that of ICK1. These results demonstrate that plants possess a family of differentially regulated CDK inhibitors that contain a conserved carboxy terminal but with distinct amino terminal regions.     

Analysis of the subcellular localization, function, and proteolytic control of the Arabidopsis cyclin-dependent kinase inhibitor ICK1/KRP1

Recent studies have shown that cyclin-dependent kinase (CDK) inhibitors can have a tremendous impact on cell cycle progression in plants. In animals, CDK inhibitors are tightly regulated, especially by posttranslational mechanisms of which control of nuclear access and regulation of protein turnover are particularly important. Here we address the posttranslational regulation of INHIBITOR/INTERACTOR OF CDK 1 (ICK1)/KIP RELATED PROTEIN 1 (KRP1), an Arabidopsis (Arabidopsis thaliana) CDK inhibitor. We show that ICK1/KRP1 exerts its function in the nucleus and its presence in the nucleus is controlled by multiple nuclear localization signals as well as by nuclear export. In addition, we show that ICK1/KRP1 localizes to different subnuclear domains, i.e. in the nucleoplasm and to the chromocenters, hinting at specific actions within the nuclear compartment. Localization to the chromocenters is mediated by an N-terminal domain, in addition we find that this domain may be involved in cyclin binding. Further we demonstrate that ICK1/KRP1 is an unstable protein and degraded by the 26S proteasome in the nucleus. This degradation is mediated by at least two domains indicating the presence of at least two different pathways impinging on ICK1/KRP1 protein stability.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> cyclin-dependent kinase inhibitors are nuclear-localized and show different localization patterns within the nucleoplasm

The Arabidopsis genome contains seven cyclin-dependent kinase (CDK) inhibitors (ICK for inhibitor/interactor with cyclin-dependent kinase) which share a small conserved C-terminal domain responsible for the CDK-inhibition activity by these proteins. Different ICK/KRPs have been shown to have unique expression patterns within tissues, organs and during the cell cycle. Previous studies have shown that overexpressing one of the ICK/KRPs inhibits CDK activity, cell division, and profoundly affects plant growth and development. In this study, we investigated the subcellular localization of the seven Arabidopsis ICK proteins and domains responsible for this localization. Using transgenic expression in Arabidopsis plants and transient expression in tobacco leaf cells, all ICK/KRPs fused to green fluorescent protein (GFP) were localized to the nucleus, suggesting that the nucleus is the cellular compartment for the plant CDK inhibitors to function. While ICK2/KRP2, ICK4/KRP6, and ICK5/KRP7 were localized to the nucleoplasm in a homogeneous manner, ICK1/KRP1, ICK3/KRP5, ICK6/KRP3, and ICK7/KRP4 showed a punctate pattern of localization. A small motif conserved amongst the latter group of ICK/KRPs is required to confer this subcellular pattern as deletion of this motif from ICK7/KRP4 resulted in a shift from a punctate to a homogeneous pattern of localization. While a single nuclear localization signal (NLS) is responsible for the nuclear localization of ICK2/KRP2, multiple mechanisms for nuclear localization are suggested to exist for the other six ICK/KRPs since deletion mutants lacking predicted NLS motifs and the conserved C-terminal domain are still localized in the nucleus.     

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.     

ICK1, a cyclin-dependent protein kinase inhibitor from Arabidopsis thaliana interacts with both Cdc2a and CycD3, and its expression is induced by abscisic acid

Cyclin-dependent kinase (CDK) inhibitor genes encode low molecular weight proteins which have important functions in cell cycle regulation, development and perhaps also in tumorigenesis. The first plant CDK inhibitor gene ICK1 was recently identified from Arabidopsis thaliana . Although the C-terminal domain of ICK1 contained an important consensus sequence with the mammalian CDK inhibitor p27^Kip1, the remainder of the deduced ICK1 sequence showed little similarity to any known CDK inhibitors. In vitro assays showed that recombinant ICK1 exhibited unique kinase inhibitory properties. In the present study we characterized ICK1 in terms of its gene structure, its interaction with both A. thaliana Cdc2a and CycD3, and its induction by the plant growth regulator, abscisic acid (ABA). ICK1 was expressed at a relatively low level in the tissues surveyed. However, ICK1 was induced by ABA, and along with ICK1 induction there was a decrease in Cdc2-like histone H1 kinase activity. These results suggest a molecular mechanism by which plant cell division might be inhibited by ABA. ICK1 clones were also identified from independent yeast two-hybrid screens using the CycD3 construct. The implication that ICK1 protein could interact with both Cdc2a and CycD3 was confirmed by in vitro binding assays. Furthermore, deletion analysis indicated that different regions of ICK1 are required for the interactions with Cdc2a and CycD3. These results provide a mechanistic basis for understanding the role of CDK inhibitors in cell cycle regulation in plant cells.     

Activation of a nuclear Cdc2-related kinase within a mitogen-activated protein kinase-like TDY motif by autophosphorylation and cyclin-dependent protein kinase-activating kinase

Male germ cell-associated kinase (MAK) and intestinal cell kinase (ICK) are nuclear Cdc2-related kinases with nearly identical N-terminal catalytic domains and more divergent C-terminal noncatalytic domains. The catalytic domain is also related to mitogen-activated protein kinases (MAPKs) and contains a corresponding TDY motif. Nuclear localization of ICK requires subdomain XI and interactions of the conserved Arg-272, but not kinase activity or, surprisingly, any of the noncatalytic domain. Further, nuclear localization of ICK is required for its activation. ICK is activated by dual phosphorylation of the TDY motif. Phosphorylation of Tyr-159 in the TDY motif requires ICK autokinase activity but confers only basal kinase activity. Full activation requires additional phosphorylation of Thr-157 in the TDY motif. Coexpression of ICK with constitutively active MEK1 or MEK5 fails to increase ICK phosphorylation or activity, suggesting that MEKs are not involved. ICK and MAK are related to Ime2p in budding yeast, and cyclin-dependent protein kinase-activating kinase Cak1p has been placed genetically upstream of Ime2p. Recombinant Cak1p phosphorylates Thr-157 in the TDY motif of recombinant ICK and activates its activity in vitro. Coexpression of ICK with wild-type CAK1 but not kinase-inactive CAK1 in cells also increases ICK phosphorylation and activity. Our studies establish ICK as the prototype for a new group of MAPK-like kinases requiring dual phosphorylation at TDY motifs.     

Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis

CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis thaliana. The C-terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two-thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf-1. Deletion of the N-terminal domain did not elevate the kinase activity of CTR1, indicating that, at least in vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation, ctr1-8, was found to result from an amino acid substitution within a new conserved motif within the N-terminal domain. Ctr1-8 has no detectable effect on the kinase activity of CTR1 in vitro, but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino-terminal domain in transgenic Arabidopsis. These results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway.     

Molecular Control of Nuclear and Subnuclear Targeting of the Plant CDK Inhibitor ICK1 and ICK1-Mediated Nuclear Transport of CDKA

ICK1 is the first member of a family of plant cyclin-dependent kinase (CDK) inhibitors. It has been shown that ICK1 is localized in the nuclei of transgenic Arabidopsis plants. Since cellular localization is important for the functions of cell cycle regulators, a comprehensive analysis was undertaken to identify specific sequences regulating the cellular localization of ICK1. Deletion and site-specific mutants fused to the green fluorescent protein (GFP) were used in transgenic Arabidopsis plants and transfected tobacco cells. Surprisingly, three separate sequences in the N-terminal, central and C-terminal regions of ICK1 could independently confer nuclear localization of the GFP fusion proteins. The central nuclear localization signal NLS^ICK1 could transport the much larger GUS (β-glucuronidase)-GFP fusion protein into nuclei, while the other two sequences were unable to. These results suggest that NLS^ICK1 is a strong NLS that actively transports the fusion protein into nuclei, while the other two sequences are either a weaker NLS or confer the nuclear localization of GFP indirectly. It was further observed that the N-terminal sequence specifies a punctate pattern of subnuclear localization, while the C-terminal sequence suppresses it. Furthermore, co-expression of ICK1 and Arabidopsis CDKA, tagged with different GFP variants, showed that ICK1 could mediate the transport of CDKA into nuclei while a mutant ICK1^1–162 that does not interact with CDKA lost this ability. These results illustrate how the nuclear localization of ICK1 is regulated and also suggest a possible role of ICK1 in regulating the cellular distribution of CDKA.     

Human cyclin-dependent kinase 2-associated protein 1 (CDK2AP1) is dimeric in its disulfide-reduced state, with natively disordered N-terminal region

CDK2AP1 (cyclin-dependent kinase 2-associated protein 1), corresponding to the gene doc-1 (deleted in oral cancer 1), is a tumor suppressor protein. The doc-1 gene is absent or down-regulated in hamster oral cancer cells and in many other cancer cell types. The ubiquitously expressed CDK2AP1 protein is the only known specific inhibitor of CDK2, making it an important component of cell cycle regulation during G(1)-to-S phase transition. Here, we report the solution structure of CDK2AP1 by combined methods of solution state NMR and amide hydrogen/deuterium exchange measurements with mass spectrometry. The homodimeric structure of CDK2AP1 includes an intrinsically disordered 60-residue N-terminal region and a four-helix bundle dimeric structure with reduced Cys-105 in the C-terminal region. The Cys-105 residues are, however, poised for disulfide bond formation. CDK2AP1 is phosphorylated at a conserved Ser-46 site in the N-terminal "intrinsically disordered" region by IκB kinase ε.     

Plant CDK inhibitors: studies of interactions with cell cycle regulators in the yeast two-hybrid system and functional comparisons in transgenic Arabidopsis plants

. The cyclin-dependent kinase (CDK) inhibitors ICK1 and ICK2 have been shown to inhibit plant CDK activity in vitro, and the expression of ICK1 was able to inhibit cell division in the plant and modify plant growth and morphology. In order to characterize other ICK1-related inhibitor genes and understand possible differences among plant CDK inhibitors, the interactions of plant CDK inhibitors with cell cycle regulators were analysed in the yeast two-hybrid system and their functions were compared in transgenic Arabidopsis plants. Yeast two-hybrid results indicate that there are likely two groups of plant CDK inhibitors. The A-group inhibitors ICK1, ICK2, ICK6 and ICK7 interact with Cdc2a and three D-type cyclins (D1, D2 and D3), while the B-group inhibitors ICK4, ICK5 and ICKCr interact with D-type cyclins but not with Arabidopsis Cdc2a. ICK1 (A-group), and ICK4 and ICKCr (B-group) were expressed separately in transgenic Arabidopsis plants. Overexpression of the three inhibitor genes resulted in plants of a smaller size with serrated leaves and modified flowers. These plants also had reduced nuclear DNA content (polyploidy), suggesting that expression of these inhibitors affected endoreduplication. Further, there were apparent differences in the strength of effect among the inhibitors. These results provide the first evidence on the CDK inhibitory function for ICK4 and ICKCr. They also suggest that these CDK inhibitors play important roles in cell division and plant growth.     

Control of petal and pollen development by the plant cyclin-dependent kinase inhibitor ICK1 in transgenic <Emphasis Type="Italic">Brassica</Emphasis> plants

The cyclin-dependent protein kinases (CDKs) have a central role in cell cycle regulation and can be inhibited by the binding of small protein CDK inhibitors. The first plant CDK inhibitor gene ICK1 was previously identified in Arabidopsis thaliana. In comparison to known animal CDK inhibitors, ICK1 protein exhibits unique structural and functional properties. The expression of ICK1 directed by the constitutive CaMV 35S promoter was shown to inhibit cell division and plant growth. The aim of this study was to determine the effects of ICK1 overexpression on particular organs and cells. ICK1 was expressed in specific tissues or cells of Brassica napus L. plants using two tissue-specific promoters, Arabidopsis AP3 and Brassica Bgp1. Transgenic AP3-ICK1 plants were morphologically normal except for some modified flowers either without petals or with petals of reduced size. Surprisingly, petals of novel shapes such as tubular petals were also observed, indicating a profound effect of cell division inhibition on morphogenesis. The cell size in the smaller modified petals was similar to that in control petals, suggesting that the reduction of petal size is mainly due to the reduction of cell numbers and that the inhibition of cell division does not necessarily lead to an increase in cell size. Transgenic Bgp1-ICK1 plants were normal morphologically; however, dramatic decreases in seed production were observed in some plants. In those plants, the ability of pollen to germinate and pollen nuclear number were affected. These results are discussed in relation to the cell cycle and plant development.     

The effect of ICK1, a plant cyclin-dependent kinase inhibitor,on mitosis in living plant cells

The inhibitory activity of Arabidopsis thaliana ICK1, a plant cyclin-dependent kinase inhibitor, has previously been characterised by its effect on plant cyclin-dependent kinase activity in vitro and its effect on growth in transgenic plants. Herein, we examine cyclin-dependent kinase-driven cell-cycle events, probed by testing the sensitivity of living cells to introduced ICK1 protein. The microinjection of ICK1 into individual Tradescantia virginiana stamen hair cells during late prophase and prometaphase resulted in a clear protein-specific increase in the metaphase transit time (time from nuclear envelope breakdown to the onset of anaphase) in a manner dependent on load and injection time. The results indicate a continuing role for cyclin-dependent kinases in mitotic progression and provide in vivo evidence at the cellular level that ICK1 can restrict growth in the plant by inhibiting cell division.     

A short motif in Arabidopsis CDK inhibitor ICK1 decreases the protein level,probably through a ubiquitin‐independent mechanism

The ICK/KRP family of cyclin‐dependent kinase (CDK) inhibitors modulates the activity of plant CDKs through protein binding. Previous work has shown that changing the levels of ICK/KRP proteins by overexpression or downregulation affects cell proliferation and plant growth, and also that the ubiquitin proteasome system is involved in degradation of ICK/KRPs. We show in this study that the region encompassing amino acids 21 to 40 is critical for ICK1 levels in both Arabidopsis and yeast. To determine how degradation of ICK1 is controlled, we analyzed the accumulation of hemagglutinin (HA) epitope‐tagged ICK1 proteins in yeast mutants defective for two ubiquitin E3 ligases. The highest level of HA‐ICK1 protein was observed when both the N‐terminal 1–40 sequence was removed and the SCF (SKP1–Cullin1–F‐box complex) function disrupted, suggesting the involvement of both SCF‐dependent and SCF‐independent mechanisms in the degradation of ICK1 in yeast. A short motif consisting of residues 21–30 is sufficient to render green fluorescent protein (GFP) unstable in plants and had a similar effect in plants regardless of whether it was fused to the N‐terminus or C‐terminus of GFP. Furthermore, results from a yeast ubiquitin receptor mutant rpn10Δ indicate that protein ubiquitination is not critical in the degradation of GFP‐ICK1^1–40 in yeast. These results thus identify a protein‐destabilizing sequence motif that does not contain a typical ubiquitination residue, suggesting that it probably functions through an SCF‐independent mechanism.     

Inhibitor of apoptosis (IAP)-like protein lacks a baculovirus IAP repeat (BIR) domain and attenuates cell death in plant and animal systems

A novel Arabidopsis thaliana inhibitor of apoptosis was identified by sequence homology to other known inhibitor of apoptosis (IAP) proteins. Arabidopsis IAP-like protein (AtILP) contained a C-terminal RING finger domain but lacked a baculovirus IAP repeat (BIR) domain, which is essential for anti-apoptotic activity in other IAP family members. The expression of AtILP in HeLa cells conferred resistance against tumor necrosis factor (TNF)-α/ActD-induced apoptosis through the inactivation of caspase activity. In contrast to the C-terminal RING domain of AtILP, which did not inhibit the activity of caspase-3, the N-terminal region, despite displaying no homology to known BIR domains, potently inhibited the activity of caspase-3 in vitro and blocked TNF-α/ActD-induced apoptosis. The anti-apoptotic activity of the AtILP N-terminal domain observed in plants was reproduced in an animal system. Transgenic Arabidopsis lines overexpressing AtILP exhibited anti-apoptotic activity when challenged with the fungal toxin fumonisin B1, an agent that induces apoptosis-like cell death in plants. In AtIPL transgenic plants, suppression of cell death was accompanied by inhibition of caspase activation and DNA fragmentation. Overexpression of AtILP also attenuated effector protein-induced cell death and increased the growth of an avirulent bacterial pathogen. The current results demonstrated the existence of a novel plant IAP-like protein that prevents caspase activation in Arabidopsis and showed that a plant anti-apoptosis gene functions similarly in plant and animal systems.     

Mutational analysis of the ethylene receptor ETR1. Role of the histidine kinase domain in dominant ethylene insensitivity

The ethylene receptor family of Arabidopsis consists of five members, one of these being ETR1. The N-terminal half of ETR1 contains a hydrophobic domain responsible for ethylene binding and membrane localization. The C-terminal half of the polypeptide contains domains with homology to histidine (His) kinases and response regulators, signaling motifs originally identified in bacteria. The role of the His kinase domain in ethylene signaling was examined in planta. For this purpose, site-directed mutations were introduced into the full-length wild-type ETR1 gene and into etr1-1, a mutant allele that confers dominant ethylene insensitivity on plants. The mutant forms of the receptor were expressed in Arabidopsis and the transgenic plants characterized for their ethylene responses. A mutation that eliminated His kinase activity did not affect the ability of etr1-1 to confer ethylene insensitivity. A truncated version of etr1-1 that lacks the His kinase domain also conferred ethylene insensitivity. Possible mechanisms by which a truncated version of etr1-1 could exert dominance are discussed.     

Induced N- and C-terminal cleavage of p53: a core fragment of p53, generated by interaction with damaged DNA, promotes cleavage of the N-terminus of full-length p53, whereas ssDNA induces C-terminal cleavage of p53.

p53 is able to recognize and bind sites of DNA damage and, in some way, damage to cellular DNA activates a p53 response leading to G1 arrest or apoptosis. We have previously shown that 'damaged DNA' induces N-terminal cleavage of p53 to generate p40(DeltaN) and p35 (core) protein products. We now show that the p35 product has protease activity and is able to cleave between residues 23 and 24 of full-length p53 to generate a novel product, p50(DeltaN23). This activity was inhibited by bestatin, an aminopeptidase inhibitor. Residues 23 and 24 lie within the mdm-2 binding domain of p53 and the possibility that p50(DeltaN23) may be resistant to feedback regulation by mdm-2 is discussed. Unexpectedly, interaction with ssDNA induced two further cleavage products of p53, generated by C-terminal cleavage and designated p50(DeltaC) and p40(DeltaC). In vivo generation of a C-terminal cleavage product of endogenous p53 similar in size to p50(DeltaC) correlated with up-regulation of p21 expression in ML-1 cells exposed to either adriamycin or cisplatin. The possible significance of the various p53 cleavage products in relation to the cellular response to DNA damage is discussed.