Maize D4;1 and D5 cyclin proteins in germinating maize. Associated kinase activity and regulation by phytohormones

We have previously reported the expression of four different maize D cyclins during seed germination and showed that cytokinins and auxins stimulate the expression of every cyclin in a differential way. In this paper we characterize the behavior at the protein level of two of these cyclins, CycD5 and CycD4;1. Antibodies were raised against CycD5;2 (which very likely also recognizes D5;1) and CycD4;1 and Western blot studies demonstrated that neither BA nor indol-3 acetic acid (IAA) stimulate cyclin accumulation during germination, compared with control levels. However, phytohormones, particularly IAA, modify the kinase activity associated to D cyclins preferentially at early hours of germination. The associated kinase moiety to D cyclins appears to be of a Cdk-A type because this protein immunoprecipitates with D cyclins and because kinase activity is strongly inhibited by both olomoucine and also by a peptide corresponding to the carboxy end of a maize kip related protein (KRP) protein. There is thus no correlation between mRNA and protein expression for these maize D cyclins during seed germination, although phytohormones may stimulate a signaling cascade that stimulates activation of protein kinase activity in cyclin–Cdk complexes.     

Expression of maize D-type cyclins: comparison, regulation by phytohormones during seed germination and description of a new D cyclin

While cloning maize D-type cyclins previously reported in databases (described as of D1, D2 and D4 types), a fourth D cyclin was cloned that showed high homology (75%) with the D1 cyclin. Because this D1 cyclin has been recently described as a D5-type cyclin (D5;1), the new cyclin was named D5;2. All maize cyclins have been compared among themselves and among D cyclins from other plant species. All maize D cyclins possess the retinoblastoma protein–binding motif and cyclin boxes but no PEST sequences or destruction box sequences are required for protein degradation. D5 and D2 cyclins also have canonical cyclin-dependent kinase (Cdk)–phosphorylation sites. Every cyclin showed a different expression pattern during seed germination, standing out cyclin D5;2, which seems to be expressed only during the early stages (equivalent to postmitotic interphase), and cyclin D4;1, which progressively accumulates from an almost undetectable level in dry seed embryo axes. Phytohormones like cytokinins and auxins, which accelerate the germination process, change the expression pattern of all cyclins, with cytokinins promoting an increase in expression during the early hours of germination (by 6 h), whereas auxins promote a constant increase in the levels of three out of the four D cyclins (except D5;1). Cyclin D5;1 is the least expressed of all cyclins in all tissues measured (embryo axes, seedlings and plantlets), and all cyclins are expressed in both meristematic and non-meristematic tissues. We discuss their relevance for the germination process and plantlet establishment.     

Phosphatidylinositol 3-kinase activity regulates alpha -thrombin-stimulated G1 progression by its effect on cyclin D1 expression and cyclin-dependent kinase 4 activity

In this study, we present evidence that PI 3-kinase is required for alpha-thrombin-stimulated DNA synthesis in Chinese hamster embryonic fibroblasts (IIC9 cells). Previous results from our laboratory demonstrate that the mitogen-activated protein kinase (extracellular signal-regulated kinase (ERK)) pathway controls transit through G(1) phase of the cell cycle by regulating the induction of cyclin D1 mRNA levels and cyclin dependent kinase 4 (CDK4)-cyclin D1 activity. In IIC9 cells, PI 3-kinase activation also is an important controller of the expression of cyclin D1 protein and CDK4-cyclin D1 activity. Pretreatment of IIC9 cells with the selective PI 3-kinase inhibitor, LY294002 blocks the alpha-thrombin-stimulated increase in cyclin D1 protein and CDK4 activity. However, LY294002 does not affect alpha-thrombin-induced cyclin D1 steady state message levels, indicating that PI 3-kinase acts independent of the ERK pathway. Interestingly, expression of a dominant-negative Ras significantly decreased both alpha-thrombin-stimulated ERK and PI 3-kinase activities. These data clearly demonstrate that the alpha-thrombin-induced Ras activation coordinately regulates ERK and PI 3-kinase activities, both of which are required for expression of cyclin D1 protein and progression through G(1).     

Characterization of the fission yeast mcs2 cyclin and its associated protein kinase activity.

We have previously described the isolation of mcs2-75, a mutation obtained as an allele-specific suppressor of a dominant allele of cdc2. mcs2 was cloned and determined to be an essential gene, the product of which shares homology with the cyclin family of proteins. In contrast to the behavior of some, but not all cyclins, the mcs2 protein is constant in its abundance and localization throughout the cell cycle. A kinase activity that co-precipitates with mcs2 can be detected when myelin basic protein (MBP) is provided as an exogenous substrate. This kinase activity is constant throughout the cell cycle. mcs2 does not appear to associate with the cdc2 protein kinase or an antigenically related kinase. Finally, a protein kinase termed csk1 (cyclin suppressing kinase) was isolated as a high copy suppressor of an mcs2 mutation. csk1 is not essential, however, the level of kinase activity that co-precipitates with mcs2 is reduced approximately 3-fold in strains harboring a csk1 null allele. Therefore, csk1 may encode a protein kinase physically associated with mcs2 or alternatively may function as an upstream activator of the mcs2-associated kinase.     

Nuclear translocation of LIM kinase mediates Rho-Rho kinase regulation of cyclin D1 expression

We previously reported that the Rho-Rho kinase pathway controls cyclin D1 expression by preventing its early G1 phase induction in response to Rac and/or Cdc42, thus increasing its dependence on ERK signaling and actin stress fiber formation. We now show that the Rho kinase effector LIM kinase is responsible for this effect. Surprisingly, inhibition of Rac-dependent cyclin D1 expression by LIM kinase is independent of both cofilin phosphorylation and actin polymerization. Instead, specific mutation of its nuclear localization and export sequences showed that LIM kinase acts in the nucleus to suppress Rac/Cdc42-dependent cyclin D1 expression. Our results therefore describe an unexpected role for LIM kinase that requires nuclear translocation. The effect of nuclear LIM kinase on cyclin D1 expression ultimately regulates the duration of G1 phase and the degree to which G1 phase progression depends on actin stress fiber formation and imposition of cellular tension.     

Xenopus cyclin D2: Cloning and expression in oocytes and during early development

Summary— We have isolated and characterized a cDNA which contains the entire coding sequence of Xenopus laevis cyclin D2 protein. Cyclin D2 mRNA is identified as a member of the class of maternal RNAs. It is rare and stable during embryonic development at least until tadepole. In addition, a second cDNA coding for a truneated version of cyclin D2 was also isolated. Mieroinjection of cyclin D2 into oocytes undergoing meiotic maturation and parthenogenetic activation reveals that the protein is stable for several hours, independently of the ubiquitin-mediated degradation of cyclin B2 that takes place periodically during this process. Microinjected cyclin D2 localizes both in the cytoplasm and in the nucleus of oocyte. In somatic cells, it is well established that cyclin D2 is almost exclusively nuclear and very labile. The unusual behaviour of cyclin D2 upon injection into oocytes may provide indications about a possible role for this protein during meiosis and early development.     

Differential expression of cyclin G2, cyclin-dependent kinase inhibitor 2C and peripheral myelin protein 22 genes during adipogenesis

Increase of fat cells (FCs) in adipose tissue is attributed to proliferation of preadipocytes or immature adipocytes in the early stage, as well as adipogenic differentiation in the later stage of adipose development. Although both events are involved in the FC increase, they are contrary to each other, because the former requires cell cycle activity, whereas the latter requires cell cycle withdrawal. Therefore, appropriate regulation of cell cycle inhibition is critical to adipogenesis. In order to explore the important cell cycle inhibitors and study their expression in adipogenesis, we adopted a strategy combining the Gene Expression Omnibus (GEO) database available on the NCBI website and the results of quantitative real-time PCR (qPCR) data in porcine adipose tissue. Three cell cycle inhibitors – cyclin G2 (CCNG2), cyclin-dependent kinase inhibitor 2C (CDKN2C) and peripheral myelin protein (PMP22) – were selected for study because they are relatively highly expressed in adipose tissue compared with muscle, heart, lung, liver and kidney in humans and mice based on two GEO DataSets (GDS596 and GDS3142). In the latter analysis, they were found to be more highly expressed in differentiating/ed preadipocytes than in undifferentiated preadipocytes in human and mice as shown respectively by GDS2366 and GDS2743. In addition, GDS2659 also suggested increasing expression of the three cell cycle inhibitors during differentiation of 3T3-L1 cells. Further study with qPCR in Landrace pigs did not confirm the high expression of these genes in adipose tissue compared with other tissues in market-age pigs, but confirmed higher expression of these genes in FCs than in the stromal vascular fraction, as well as increasing expression of these genes during in vitro adipogenic differentiation and in vivo development of adipose tissue. Moreover, the relatively high expression of CCNG2 in adipose tissue of market-age pigs and increasing expression during development of adipose tissue was also confirmed at the protein level by western blot analysis. Based on the analysis of the GEO DataSets and results of qPCR and Western blotting we conclude that all three cell cycle inhibitors may inhibit adipocyte proliferation, but promote adipocyte differentiation and hold a differentiated state by inducing and maintaining cell cycle inhibition. Therefore, their expression in adipose tissue is positively correlated with age and mature FC number. By regulating the expression of these genes, we may be able to control FC number, and, thus, reduce excessive fat tissue in animals and humans.     

Combined effect of cyclin D3 expression and abrogation of cyclin D1 prevent mouse skin tumor development

We have previously demonstrated that ras-mediated skin tumorigenesis depends on signaling pathways that act preferentially through cyclin D1 and D2. Interestingly, the expression of cyclin D3 inhibits skin tumor development, an observation that conflicts with the oncogenic role of D-type cyclins in the mouse epidermis. Here, we show that simultaneous up and downregulation of particular members of the D-type cyclin family is a valuable approach to reduce skin tumorigenesis. We developed the K5D3/cyclin D1^−/− compound mouse, which overexpresses cyclin D3 but lacks expression of cyclin D1 in the skin. Similar to K5D3 transgenic mice, keratinocytes from K5D3/cyclin D1^−/− compound mice show a significant reduction of cyclin D2 levels. Therefore, this model allows us to determine the effect of cyclin D3 expression when combined with reduced or absent expression of the remaining two members of the D-type cyclin family in mouse epidermis. Our data show that induced expression of cyclin D3 compensates for the reduced level of cyclin D1 and D2, resulting in normal keratinocyte proliferation. However, simultaneous ablation of cyclin D1 and downregulation of cyclin D2 via cyclin D3 expression resulted in a robust reduction in ras-mediated skin tumorigenesis. We conclude that modulation of the levels of particular members of the D-type cyclin family could be useful to inhibit tumor development and, in particular, ras-mediated tumorigenesis.Key words: cell cycle, D-type cyclins, skin, carcinogenesis, epidermis     

Combined effect of cyclin D3 expression and abrogation of cyclin D1 prevent mouse skin tumor development

We have previously demonstrated that ras-mediated skin tumorigenesis depends on signaling pathways that act preferentially through cyclin D1 and D2. Interestingly, the expression of cyclin D3 inhibits skin tumor development, an observation that conflicts with the oncogenic role of D-type cyclins in the mouse epidermis. Here, we show that simultaneous up and downregulation of particular members of the D-type cyclin family is a valuable approach to reduce skin tumorigenesis. We developed the K5D3/cyclin D1^-/- compound mouse, which overexpresses cyclin D3 but lacks expression of cyclin D1 in the skin. Similar to K5D3 transgenic mice, keratinocytes from K5D3/cyclin D1^-/- compound mice show a significant reduction of cyclin D2 levels. Therefore, this model allows us to determine the effect of cyclin D3 expression when combined with reduced or absent expression of the remaining two members of the D-type cyclin family in mouse epidermis. Our data show that induced expression of cyclin D3 compensates for the reduced level of cyclin D1 and D2, resulting in normal keratinocyte proliferation. However, simultaneous ablation of cyclin D1 and downregulation of cyclin D2 via cyclin D3 expression resulted in a robust reduction in ras-mediated skin tumorigenesis. We conclude that modulation of the levels of particular members of the D-type cyclin family could be useful to inhibit tumor development and, in particular, ras-mediated tumorigenesis.     

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.     

Increased cyclin E expression may obviate the role of cyclin D1 during brain development in cyclin D1 knockout mice

Cyclins D and E play critical roles during the G1 phase of mammalian cell division. Cyclin D1 expression is high and expected to play an important role during mouse brain development. However, in the present study, we found no difference in CNS morphology between cyclin D1 knockout (KO) and control wild-type mice at the ages of 1, 4 and 12 months. Analysis of protein expression in embryonic brains revealed that cyclin E is obviously increased in cyclin D1 KO mice at 13.5 days post coitum. At the same age a high level of cyclin D1 expression is detected in the embryonic brain of wild-type mice. The data indicate that enhanced cyclin E protein expression in cyclin D1 KO mice may obviate the role of cyclin D1 and contribute to the normal brain development of cyclin D1 KO mice.     

Hsc70 regulates accumulation of cyclin D1 and cyclin D1-dependent protein kinase

The cyclin D-dependent kinase is a critical mediator of mitogen-dependent G1 phase progression in mammalian cells. Given the high incidence of cyclin D1 overexpression in human neoplasias, the nature and complexity of cyclin D complexes in vivo have been subjects of intense interest. Besides its catalytic partner, the nature and complexity of cyclin D complexes in vivo remain ambiguous. To address this issue, we purified native cyclin D1 complexes from proliferating mouse fibroblasts by affinity chromatography and began to identify and functionally characterize the associated proteins. In this report, we describe the identification of Hsc70 and its functional importance for cyclin D1 and cyclin D1-dependent kinase maturation. We demonstrate that Hsc70 associates with newly synthesized cyclin D1 and is a component of a mature, catalytically active cyclin D1/CDK4 holoenzyme complex. Our data suggest that Hsc70 promotes stabilization of newly synthesized cyclin D1, thereby increasing its availability for assembly with CDK4. In addition, our data demonstrate that Hsc70 remains bound to cyclin D1 following its assembly with CDK4 and Cip/Kip proteins, where it ensures the formation of a catalytically active complex.     

Identification of G1 kinase activity for cdk6, a novel cyclin D partner.

A family of vertebrate cdc2-related kinases has been identified, and these kinases are candidates for roles in cell cycle regulation. Here, we show that the human PLSTIRE gene product is a novel cyclin-dependent kinase, cdk6. The cdk6 kinase is associated with cyclins D1, D2, and D3 in lysates of human cells and is activated by coexpression with D-type cyclins in Sf9 insect cells. Furthermore, we demonstrate that endogenous cdk6 from human cell extracts is an active kinase which can phosphorylate pRB, the product of the retinoblastoma tumor suppressor gene. The activation of cdk6 kinase occurs during mid-G1 in phytohemagglutinin-stimulated T cells, well prior to the activation of cdk2 kinase. This timing suggests that cdk6, and by analogy its homolog cdk4, links growth factor stimulation with the onset of cell cycle progression.     

The SPARC-related factor SMOC-2 promotes growth factor-induced cyclin D1 expression and DNA synthesis via integrin-linked kinase

Secreted modular calcium-binding protein-2 (SMOC-2) is a recently-identified SPARC-related protein of unknown function. In mRNA profiling experiments we, found that SMOC-2 expression was elevated in quiescent (G0) mouse fibroblasts and repressed after mitogenic stimulation with serum. The G0-specific expression of SMOC-2 was similar to that of platelet-derived growth factor-beta receptor (PDGFbetaR), a major mitogenic receptor. Therefore, we tested a possible role for SMOC-2 in growth factor-induced cell cycle progression. SMOC-2 overexpression augmented DNA synthesis induced by serum and fibroblast mitogens (including PDGF-BB and basic fibroblast growth factor). Conversely, SMOC-2 ablation by using small interfering RNA attenuated DNA synthesis in response to PDGF-BB and other growth factors. Mitogen-induced expression of cyclin D1 was attenuated in SMOC-2-ablated cells, and cyclin D1-overexpressing cells were resistant to inhibition of mitogenesis after SMOC-2 ablation. Therefore, cyclin D1 is limiting for G1 progression in SMOC-2-deficient cells. SMOC-2 ablation did not inhibit PDGF-induced PDGFbetaR autophosphorylation or PDGF-BB-dependent activation of mitogen-activated protein kinase and Akt kinases, suggesting that SMOC-2 is dispensable for growth factor receptor activation. However, integrin-linked kinase (ILK) activity was reduced in SMOC-2-ablated cells. Ectopic expression of hyperactive ILK corrected the defective mitogenic response of SMOC-2-deficient cells. Therefore, SMOC-2 contributes to cell cycle progression by maintaining ILK activity during G1. These results identify a novel role for SMOC-2 in cell cycle control.     

Polyamine contents, ethylene synthesis, and BrACO2 expression during turnip germination

Contents of total free [PA(S)] and conjugated polyamines [PA(SH), PA(PH)] were higher in turnip (Brassica rapa L. cv. Rapa) seeds during imbibition (0–36 h) and radicle protrusion (36–48 h) than during the further growth (10 d). Ethylene production was activated with the protrusion, reaching a maximum at the second day of germination and dropping afterwards. The application of ethrel accelerated radicle emergence but the direct intervention of ethylene in the breaking of the seed coat was not clear from the use of ethylene-biosynthesis inhibitors (CoCl₂ and AVG). Finally, in this work the gene BrACO2 was characterized. Although its expression was not detected in seeds through zygotic embryogenesis, it increased concomitantly with the germination process.     

Focal adhesion kinase mediates endothelin-induced cyclin D3 expression in rat cultured astrocytes

Focal adhesion kinase (FAK), a non-receptor type tyrosine kinase, is involved in the G1/S phase cell cycle transition of astrocytes induced by endothelin-1 (ET-1). In this study, the roles of FAK in the expression of cyclin D1 or D3, which are pivotal in G1/S phase transition, were examined in cultured astrocytes. Accompanied with increases in bromodeoxyuridine (BrdU) incorporation, ET-1 (100 nm) increased the numbers of cyclin D1- and D3-positive astrocytes. PD98059 (a MEK inhibitor) and PP-2 (a Src kinase inhibitor) inhibited ET-induced cyclin D1 expression and BrdU incorporation without affecting cyclin D3 expression. In contrast, cytochalasin D, lovastatin (a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor) and Y-27632 (a rho-kinase inhibitor) prevented both cyclin D3 expression and BrdU incorporation. FAK phosphorylation by ET-1 was inhibited by cytochalasin D, lovastatin and Y-27632, but not by PD98059 or PP-2. Transfection with wild-type FAK increased expression of cyclin D3 in astrocytes, while that of cyclin D1 was not affected. Dominant-negative FAK mutants prevented an ET-induced increase in cyclin D3 expression, but not D1. These results suggest that activation of FAK causes cyclin D3 expression in cultured astrocytes, which would underlie the FAK-mediated astrocytic G1/S phase transition by ET-1.     

Protein kinase activity in meristematic root tips of Pisum sativum during germination

Membranes isolated from pea root meristems contain a protein kinase activity and a number of unidentified endogenous substrates. The enzyme activity increases in presence of MgCl₂ and MnCl₂ but not in presence of CaCl₂; the maximum of phosphorylation activity has been observed after 45 s of incubation. This protein kinase activity is partially solubilized by Triton X-100, although no extensive purification has been attempted. The activity falls during the first hours of germination and is detected only in membranes obtained from meristematic tissues. In the same membrane system, protein phosphatase activity could be present; if this data is confirmed then an interesting mechanism of phospho-dephosphorylation of proteins could exist in pea root meristematic membranes. In this case, such a particular membrane system could become very useful for investigating the physiological role of phosphorylations of protein membrane components during cell proliferation and germination.