Levels of p34cdc2-like protein in dividing,differentiating and dedifferentiating cells of carrot

P34^cdc2 is a key cell-cycle protein in fission yeast that is necessary for progress in the cell cycle from the G1 to the S phase and from G2 through mitosis. Homologues of p34^cdc2 have been found in all eukaryotes that have been investigated. Levels of p34^cdc2-like protein were studied by quantitative Western blotting in developing cotyledons of Daucus carota L. (carrot) seedlings, in expiants from the same seedlings transferred to tissueculture media with and without 2,4-dichlorophenoxyacetic acid (2,4-D), and in nutrient-starved suspension cultures derived from carrot callus. During the cessation of cell division, which accompanies development of the cotyledon to maturity, there was a 16-fold decline in the level of the p34^cdc2-like protein. Auxin-stimulated dedifferentiation in excised tissue from mature cotyledons was accompanied by restoration of the level of p34^cdc2-like protein, and the responding cells formed a callus. These data support our earlier proposition, based upon evidence from wheat leaf, that changes in the level of p34^cdc2-like protein act in the switch between cycling and differentiation. Persisting high levels of p34^cdc2-like protein in suspension cultures, when division was stopped by nutrient limitation, indicated that decline of the protein was not an inevitable consequence of the cessation of division. Decline of p34^cdc2 in differentiation may therefore be a regulated process that determines exit from the cell cycle and the converse increase in p34^cdc2 may be a regulated process controlling dedifferentiation and resumption of cell division.Abbreviations BrdUrd 5-bromodeoxyuridine - 2,4-D 2,4-dichlorophenoxyacetic acid - kDa kilodalton - MS Murashige and Skoog (1962) J.R.G. gratefully acknowledges the support of a National Research Fellowship from the Australian Government during the time this work was done.     

The identification of a Caenorhabditis elegans homolog of p34cdc2 kinase

We have identified a Caenorhabditis elegans homolog of p34^cdc2 kinase. The C. elegans homolog, ncc-1, is -60% identical to p34^cdc2 of Homo sapiens. When expressed from a constitutive yeast promoter, ncc-1 is capable of complementing a conditional lethal mutation in the CDC28 gene of Saccharomyces cerevisiae, indicating that this C. elegans homolog can properly regulate the cell cycle.     

p 34cdc2 kinase homologue in the preprophase band

Summary Immunofluorescence microscopy with a monoclonal antibody raised against the PSTAIR sequence, which corresponds to a peptide conserved in the p 34^cdc2 protein kinase throughout the phylogenetic scale including higher plants, was used to study the intracellular localization of p 34^cdc2 during the cell cycle in onion root tip cells. Although p 34^cdc2 was evenly distributed in the cytoplasm throughout the cell cycle, a more intense staining was observed in the cortical region, where the preprophase band of microtubules (MTs) was located. Double staining with the PSTAIR and plant tubulin antibodies showed that the width of p 34^cdc2 band was narrower than that of MT band. These data raise the interesting question regarding the possible role of p 34^cdc2 protein kinase in determining the division site in plant cells.     

Genetic analysis of human p34CDC2 function in fission yeast

The p34^cdc2 protein kinase plays a key role in the control of the mitotic cell cycle of fission yeast, being required for both entry into S-phase and for entry into mitosis in the mitotic cell cycle, as well as for the initiation of the second meiotic nuclear division. In recent years, structural and functional homologues of p34^cdc2, as well as several of the proteins that interact with and regulate p34^cdc2 function in fission yeast, have been identified in a wide range of higher eukaryotic cell types, suggesting that the control mechanisms uncovered in this simple eukaryote are likely to be well conserved across evolution. Here we describe the construction and characterisation of a fission yeast strain in which the endogenous p34^cdc2 protein is entirely absent and is replaced by its human functional homologue p34^CDC2, We have used this strain to analyse aspects of the function of the human p34^CDC2 protein genetically. We show that the function of the human p34^CDC2 protein in fission yeast cells is dependent upon the action of the protein tyrosine phosphatase p80^cdc25 that it responds to altered levels of both the mitotic inhibitor p107²³³¹ and the p34^cdc2-binding protein p13^suc1, and is lethal in combination with the mutant B-type cyclin p56^cdc13-117. In addition, we demonstrate that the human p34^CDC2 protein is proficient for fission yeast meiosis, and examine the behaviour of two mutant p34^CDC2 proteins in fission yeast.     

Regulation of maturation-promoting factor by protein kinase C in Chaetopterus oocytes

Summary

We present the results of a variety of studies showing that activation of protein kinase C (PKC) in oocytes of Chaetopterus pergamentaceus results in germinal vesicle breakdown (GVBD). Phorbol esters and diacylglycerol can initiate a morphologically normal GVBD accompanied by a spectrum of associated biochemical processes, including increased protein phosphorylation, a shift in protein synthesis and activation of a protein kinase, maturation promoting factor (MPF). MPF activation is essential for GVBD in response to phorbol esters. In addition, inhibitors of PKC can block naturally-induced GVBD. We also present evidence that PKC can phosphorylate p34^cde2, the catalytic subunit of MPF and that phosphorylation by PKC increases the histone H1 kinase activity of immunoprecipitated MPF. Immunoblot studies show that Chaetopterus oocyte p34^cdc2 is not tyrosine phosphorylated prior to the initiation of GVBD, indicating that activation of MPF at GVBD in this species does not require p80^cdc25, the activator of MPF at mitosis. These results suggest that PKC is an essential regulator of GVBD which can directly phosphorylate and regulate p34^cdc2. Since PKC is the intracellular receptor for and is directly activated by tumor-promoters, tumor promotion might involve acceleration of the cell cycle through modification of the enzymatic activity of MPF by PKC.     

Expression of a dominant negative allele of cdc2 prevents activation of the endogenous p34cdc2 kinase

Summary The cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a 34 kDa phosphoprotein with serine/threonine protein kinase activity that acts as the key component in regulation of the eukaryotic cell cycle. We used a repressible promoter fused to the cdc2 cDNA to isolate conditionally dominant negative mutants of cdc2. One of these mutants, DL5, is described in this paper. Overexpression of the mutant protein in a wild-type cdc2 background is lethal and confers cell cycle arrest with a typical cdc phenotype. Sequencing of the mutant cdc2 gene revealed a single amino acid substitution in a region highly conserved in cdc2-like proteins. The mutant protein exhibits no protein kinase activity, but is able to bind a component(s) required for an active protein kinase complex and thereby prevents binding of this component(s) to the co-existing wild-type cdc2 protein. We also demonstrate that S. pombe p34^cdc2 contains no phosphoserine.     

Isolation and characterization of cDNA clones encoding cdc2 homologues from Oryza sativa: a functional homologue and cognate variants.

Summary Using probes obtained by PCR amplification, we have isolated two cognate rice cDNAs (cdc2Os-1 andcdc2Os-2) encoding structural homologues of thecdc2 ⁺/CDC28(cdc2) protein kinase from a cDNA library prepared from cultured rice cells. Comparison of the deduced amino acid sequences of cdc2Os-1 and cdc2Os-2 showed that they are 83 % identical. They are 62 % identical toCDC28 ofSaccharomyces cerevisiae and much more similar to the yeast and mammalian p34^cdc2 kinases than to riceR2, acdc2-related kinase isolated previously by screening the same rice cDNA library with a different oligonucleotide probe. Southern blot analysis indicated that the three rice clones (cdc2Os-1,cdc2Os-2 andR2) are derived from distinct genes and are each found in a single copy per rice haploid genome. RNA blot analysis revealed that these genes are expressed in proliferating rice cells and in young rice seedlings.cdc2Os-1 could complement a temperature-sensitive yeast mutant ofcdc28. However, despite the similarity in structure, bothcdc2Os-2 andR2 were unable to complement the same mutant. Thus, the present results demonstrate the presence of structurally related, but functionally distinct cognates of thecdc2 cell cycle kinase in rice.The nucleotide sequence data in this paper have been deposited in the EMBL database under accession number X60374 (cdc2Os-1) and X60375 (cdc2Os-2)     

Novel alleles ofcdc13 andcdc2 isolated as suppressors of mitotic catastrophe inSchizosaccharomyces pombe

Cell cycle control in the fission yeastSchizosaccharomyces pombe involves interplay amongst a number of regulatory molecules, including thecdc2, cdc13, cdc25, weel, andmik1 gene products. Cdc2, Cdc13, and Cdc25 act as positive regulators of cell cycle progression at the G2/M boundary, while Wee1 and Mik1 play a negative regulatory role. Here, we have screened for suppressors of the lethal premature entry into mitosis, termed mitotic catastrophe, which results from simultaneous loss of function of both Wee1 and Mik1. Through such a screen, we hoped to identify additional components of the cell cycle regulatory network, and/or G2/M-specific substrates of Cdc2. Although we did not identify such molecules, we isolated a number of alleles of bothcdc2 andcdc13, including a novel wee allele ofcdc2, cdc2-5w. Here, we characterizecdc2-5w and two alleles ofcdc13, which have implications for the understanding of details of the interactions amongst Cdc2, Cdc13, and Wee1.     

Cold-sensitive mutants of p34cdc2 that suppress a mitotic catastrophe phenotype in fission yeast.

Summary The p34^cdc2 protein kinase plays a central role in the regulation of the eukaryotic cell cycle, being required both in late G1 for the commitment to S-phase and in late G2 for the initiation of mitosis. p34^cdc2 also determines the precise timing of entry into mitosis in fission yeast, where a number of gene produts that regulate p34^cdc2 activity have been identified and characterised. To investigate further the mitotic role of p34^cdc2 in this organism we have isolated new cold-sensitive p34^cdc2 mutants. These are defective only in their G2 function and are extragenic suppressors of the lethal premature entry into mitosis brought about by mutating the mitotic inhibitor p107^wee1 and overproducing the mitotic activator p80^cdc25. One of the mutant proteins p34^cdc2-E8 is only functional in the absence of p107^wee1, and all the mutant strains have reduced histone H1 kinase activity in vitro. Each mutant allele has been cloned and sequenced, and the lesions responsible for the cold-sensitive phenotypes identified. All the mutations were found to map to regions that are conserved between the fission yeast p34^cdc2 and functional homologues from higher eukaryotes.     

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with ³H and auxin was found to promote conversion of zeatin-type cytokinins to ³H-labelled adenine derivatives. When the very rapid metabolism of exogenous [³H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.Abbreviations Ade adenine - Ados adenosine - BA 6-benzylaminopurine - C control - Con A concanavallin A - CP cellulose phosphate - IPT isopentenyl transferase - NAA 1-naphthylacetic acid - NP normal phase - NPPU N-(3-nitrophenyl)-N-phenylurea - RIA radioimmunoassay - RP reversed phase We wish to thank Dr. J. Zwar for supplying phenylurea derivitives.     

Mutational analysis of the fission yeast p34cdc2 protein kinase gene

Summary The p34^cdc2 protein serine-threonine kinase plays an essential role in the life cycle of fission yeast, being required for both the G1-S and G2-M transitions during mitotic growth, and also for the second meiotic nuclear division. Functional homologues of p34^cdc2 (each ca. 60 % identical to the fission yeast prototype) have been isolated from organisms as diverse as humans, insects and plants, and there is now considerable evidence supporting the view that fundamental aspects of the cell cycle controls uncovered in fission yeast will prove to be conserved in all eukaryotes. By comparing the amino acid sequences of fission yeast p34^cdc2 with its higher eukaryotic counterparts it is possible to identify conserved residues that are likely to be centrally important for p34^cdc2 function. Here the effects are described of mutating a number of these conserved residues. Twenty-three new mutant alleles have been constructed and tested. We show that replacing cysteine 67 with trypthophan renders the resulting mutant protein p80^cdc25-independent (while neither leucine, isoleucine nor valine has this effect) and that several of the amino acids within the highly conserved PSTAIRE region are not absolutely required for p34^cdc2 function. Five acidic amino acids have also been mutated within p34^cdc2, which are invariant across the eukaryotic protein kinase family. Acid-to-base mutations at three of these residues resulted in a dominant-negative, cell cycle arrest phenotype while similar mutations at the other two simply abolished p34^cdc2 protein function. The results are discussed with reference to the predicted tertiary structure of the p34^cdc2 enzyme.     

The effect of auxin concentration on cytokinin stability and metabolism

The stability of [³H]zeatin riboside supplied to freshly excised tobacco pith explants was found to be inversely related to -naphthaleneacetic acid concentration in the incubation medium. At higher concentrations of -naphthaleneacetic acid greater breakdown of [³H]zeatin riboside was indicated by higher levels of degradative metabolites (adenine, adenosine and adenosine nucleotides) formed. This auxin effect on cytokinin metabolism appears to be mediated, at least in part, through cytokinin oxidase. The results of in-vitro assays carried out with partially purified enzyme from corn kernels substantiale this conclusion. These findings are discussed in relation to recent observations of auxin and cytokinin levels in crown-gall tumours with altered morphology.Abbreviations FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - IP isopentenyladenine - NAA naphthaleneacetic acid - ZR zeatin riboside     

Parameters that specify the timing of cytokinesis.

One model for the timing of cytokinesis is based on findings that p34(cdc2) can phosphorylate myosin regulatory light chain (LC20) on inhibitory sites (serines 1 and 2) in vitro (Satterwhite, L.L., M.H. Lohka, K.L. Wilson, T.Y. Scherson, L.J. Cisek, J.L. Corden, and T.D. Pollard. 1992. J. Cell Biol. 118:595-605), and this inhibition is proposed to delay cytokinesis until p34(cdc2) activity falls at anaphase. We have characterized previously several kinase activities associated with the isolated cortical cytoskeleton of dividing sea urchin embryos (Walker, G.R., C.B. Shuster, and D.R. Burgess. 1997. J. Cell Sci. 110:1373-1386). Among these kinases and substrates is p34(cdc2) and LC20. In comparison with whole cell activity, cortical H1 kinase activity is delayed, with maximum levels in cortices prepared from late anaphase/telophase embryos. To determine whether cortical-associated p34(cdc2) influences cortical myosin II activity during cytokinesis, we labeled eggs in vivo with [(32)P]orthophosphate, prepared cortices, and mapped LC20 phosphorylation through the first cell division. We found no evidence of serine 1,2 phosphorylation at any time during mitosis on LC20 from cortically associated myosin. Instead, we observed a sharp rise in serine 19 phosphorylation during anaphase and telophase, consistent with an activating phosphorylation by myosin light chain kinase. However, serine 1,2 phosphorylation was detected on light chains from detergent-soluble myosin II. Furthermore, cells arrested in mitosis by microinjection of nondegradable cyclin B could be induced to form cleavage furrows if the spindle poles were physically placed in close proximity to the cortex. These results suggest that factors independent of myosin II inactivation, such as the delivery of the cleavage stimulus to the cortex, determine the timing of cytokinesis.     

Functional analysis of theDrosophila CDC2 Dm gene in fission yeast

Thecdc2 ⁺ gene product (p34^cdc2) is a protein kinase that regulates entry into mitosis in all eukaryotic cells. The role that p34^cdc2 plays in the cell cycle has been extensively investigated in a number of organisms, including the fission yeastSchizosaccharomyces pombe. To study the degree of functional conservation among evolutionarily distant p34^cdc2 proteins, we have constructed aS. pombe strain in which the yeastcdc2 ⁺ gene has been replaced by itsDrosophila homologue CDC2Dm (theCDC2Dm strain). ThisCDC2Dm S. pombe strain is viable, capable of mating and producing four viable meiotic products, indicating that the fly p34^CDC2Dm recognizes all the essentialS. pombe cdc2 ⁺ substrates, and that it is recognized by cyclin partners and other elements required for its activity. The p34^CDC2Dm protein yields a lethal phenotype in combination with the mutant B-type cyclin p56^cdc13-117, suggesting that thisS. pombe cyclin might interact less efficiently with theDrosophila protein than with its native p34^cdc2 counterpart. ThisCDC2Dm strain also responds to nutritional starvation and to incomplete DNA synthesis, indicating that proteins involved in these signal transduction pathways, interact properly with p34^CDC2Dm (and/or that p34^cdc2-independent pathways are used). TheCDC2Dm gene produces a ‘wee’ phenotype, and it is largely insensitive to the action of theS. pombe weel ⁺ mitotic inhibitor, suggesting thatDrosophila weel ⁺ homologue might not be functionally conserved. ThisCDC2Dm strain is hypersensitive to UV irradiation, to the same degree asweel-deficient mutants. A strain which co-expresses theDrosophila and yeastcdc2⁺ genes shows a dominantwee phenotype, but displays a wild-type sensitivity to UV irradiation, suggesting that p34^cdc2 triggers mitosis and influences the UV sensitivity by independent mechanisms. Communicated by B. J. Kilbey     

Exogenous auxin and cytokinin dependent activation of CDKs and cell division in leaf protoplast-derived cells of alfalfa

Alfalfa leaf protoplast cultures were used to study the role ofexogenously supplied auxin and cytokinin on the level and activity ofCdc2-related protein kinases and progression through the first celldivision cycle after re-activation of cell division. Among the threealfalfa Cdc2-related kinases studied, the Cdc2MsA/B kinase (PSTAIRE)showed only significant activity during the first four days ofprotoplast culture while the Cdc2MsD (PPTALRE) and Cdc2MsF kinases(PPTTLRE) exhibited only low or undetectable activity, respectively,during this period. Although the Cdc2MsA/B protein could be detectedin leaves and freshly isolated protoplasts in variable amounts, thekinase was never active in these cells. The kinase protein disappearedfrom protoplast-derived cells at the beginning (8h) of culture but itssynthesis re-commenced dependent on the presence of exogenous auxin butnot cytokinin. The cytokinin response of alfalfa protoplast-derivedcells varied significantly in different experiments although cytokininwas always required for completion of the first cell division cycle.Frequently both auxin and cytokinin was required for DNA replication asnot more than 5% of cells could incorporate BrdU into their DNAduring three days and significant Cdc2MsA/B activity could not bedetected in the absence of exogenous cytokinin. In other protoplastpopulations, the Cdc2MsA/B kinase was activated by auxin alone andallowed the protoplast-derived cells to enther the S-phase at a similarrate observed in parallel cultures with both auxin and cytokinin. Evenin these cultures, however, ca. 95% of the protoplast-derivedcells were arrested before mitosis without exogenous cytokinin supplywhich could be correlated with decreasing Cdc2MsA/B activity. Theseobservations suggest, that although cytokinin is required for bothG0-G1/S and G2/M cell cycle transitions, in certain cultures theG1/S requirement is overcome by some unknown factors (e.g.conditions of explants; endogenous cytokinins etc.). Furthermore, ourexperiments indicate, that the roles of cytokinin are related to thepost-translational regulation of the Cdc2MsA/B kinase complex atboth cell cycle transition points in alfalfa leaf protoplast-derivedcells. Finally, as a marker for the transition from the differentiated(G0) stage to the activated (G1) stage, we suggest using the parametersof nuclear morphology (size and ratio ofnucleus/nucleolus).     

Molecular cloning and sequence analysis of mutant alleles of the fission yeast cdc2 protein kinase gene: Implications for cdc2+ protein structure and function

Summary The cdc2 ⁺ gene function plays a central role in the control of the mitotic cell cycle of the fission yeast Schizosaccharomyces pombe. Recessive temperature-sensitive mutations in the cdc2 gene cause cell cycle arrest when shifted to the restrictive temperature, while a second class of mutations within the cdc2 gene causes a premature advancement into mitosis. Previously the cdc2 ⁺ gene has been cloned and has been shown to encode a 34 kDa phosphoprotein with in vitro protein kinase activity. Here we describe the cloning of 11 mutant alleles of the cdc2 gene using two simple methods, one of which is presented here for the first time. We have sequenced these alleles and find a variety of single amino acid substitutions mapping throughtout the cdc2 protein. Analysis of these mutations has identified a number of regions within the cdc2 protein that are important for cdc2 ⁺ activity and regulation. These include regions which may be involved in the interaction of the cdc2 ⁺ gene product with the proteins encoded by the wee1 ⁺, cdc13 ⁺ and suc1 ⁺ genes.     

Multiple cyclin-dependent kinase complexes and phosphatases control G2/M progression in alfalfa cells

Reversible phosphorylation of proteins by kinases and phosphatases plays a key regulatory role in several eukaryotic cellular functions including the control of the division cycle. Increasing numbers of sequence and biochemical data show the involvement of cyclin-dependent kinases (CDKs) and cyclins in regulation of the cell cycle progression in higher plants. The complexity represented by different types of CDKs and cyclins in a single species such as alfalfa, indicates that multicomponent regulatory pathways control G₂/M transition. A set of cdc2-related genes (cdc2Ms A, B, D and F) was expressed in G₂ and M cells. Phosphorylation assays also revealed that at least three kinase complexes (Cdc2Ms A/B, D and F) were successively active in G₂/M cells after synchronization. Interaction between alfalfa mitotic cyclin (Medsa;CycB2;1) and a kinase partner has been reported previously. The present yeast two-hybrid analyses showed differential interaction between defined D-type cyclins and Cdc2Ms kinases functioning in G₂/M phases. Localization of Cdc2Ms F kinase to the preprophase band (PPB), the perinuclear ring in early prophase, the mitotic spindle and the phragmoplast indicated a pivotal role for this kinase in mitotic plant cells. So far limited research efforts have been devoted to the functions of phosphatases in the control of plant cell division. A homologue of dual phosphatase, cdc25, has not been cloned yet from alfalfa; however tyrosine phosphorylation was indicated in the case of Cdc2Ms A kinase and the p^13suc1-bound kinase activity was increased by treatment of this complex with recombinant Drosophila Cdc25. The potential role of serine/threonine phosphatases can be concluded from inhibitor studies based on okadaic acid or endothall. Endothall elevated the kinase activity of p13^suc1-bound fractions in G₂-phase alfalfa cells. These biochemical data are in accordance with observed cytological abnormalities. The present overview with selected original data outlines a conclusion that emphasizes the complexity of G₂/M regulatory events in flowering plants.     

Analysis of gametophytic development in the moss,Physcomitrella patens,using auxin and cytokinin resistant mutants

Mutants altered in their response to auxins and cytokinins have been isolated in the moss Physcomitrella patens either by screening clones from mutagenized spores for growth on high concentrations of cytokinin or auxin, in which case mutants showing altered sensitivities can be recognized 3–4 weeks later, or by non-selective isolation of morphologically abnormal mutants, some of which are found to have altered sensitivities. Most of the mutants obtained selectively are also morphologically abnormal. The mutants are heterogeneous in their responses to auxin and cytokinin, and the behaviour of some is consistent with their being unable to make auxin, while that of others may be due to their being unable to synthesize cytokinin. Physiological analysis of the mutants has shown that both endogenous auxin and cytokinin are likely to play important and interdependent roles in several steps of gametophytic development. Although their morphological abnormalities lead to sterility, genetic analysis of some of the mutants has been possible by polyethyleneglycol induced protoplast fusion.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - NAA 1-naphthalene acetic acid - 2,4D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - IAP 6-( ²isopentenyl) aminopurine - NAR NAA resistant mutants - BAR BAP resistant mutants     

PLANT MITOSIS PROMOTING FACTOR DISASSEMBLES THE MICROTUBULE PREPROPHASE BAND AND ACCELERATES PROPHASE PROGRESSION IN TRADESCANTIA

The regulation of mitosis in higher plant cells has been investigated by microinjecting protein kinase from the metaphase-arresting (met1) mutant ofChlamydomonas. Biochemical characterization of this enzyme complex confirms the presence of a p34^cdc2/cyclin B-like kinase. The enzyme was injected into living stamen hair cells ofTradescantia virginianain which microtubules (MTs) were visualized using fluorescent analogue cytochemistry and confocal laser scanning microscopy. Microinjection of this p34^cdc2/cyclin B-like kinase caused rapid disassembly of the preprophase band of MTs but not of interphase-cortical, spindle or phragmoplast MTs. Effects of the enzyme on the cytomorphology of live prophase cells were also monitored using video microscopy. We found that injection of this enzyme accelerated chromatin condensation and nuclear envelope breakdown. This indicates the presence and function in plants of an enzyme that can initiate nuclear division similar to the maturation or mitosis promoting factor (MPF) of animal cells. These studies provide the first direct evidence that the mitotically-active form of plant MPF can drive disassembly of preprophase band MTs, chromosome condensation and initiation of mitosis in plant cells.     

A phosphorylation-independent role for the yeast cyclin-dependent kinase activating kinase Cak1

Cdc28 is the main cyclin-dependent kinase (CDK) directing the cell cycle in the budding yeast Saccharomyces cerevisiae. Besides cyclin binding, Cdc28 requires phosphorylation by the Cak1 kinase to achieve full activity. We have previously isolated carboxy-terminal cdc28^CST mutants that are temperature sensitive and exhibit high chromosome instability. Both phenotypes are suppressed by high copy Cak1 in a manner that is independent of its catalytic activity and conversely, combination of cdc28^CST and cak1 mutations results in synthetic lethality. Altogether, these results suggest that for the Cdc28 complexes to remain stable and active, an interaction with Cak1 is needed via the carboxyl terminus of Cdc28. We report two-hybrid assay data that support this model, and results that indicate that actively growing yeast cells require an optimum Cdc28:Cak1 ratio. While Cak1 is constitutively active and expressed, dividing cells tightly regulate Cak1 protein levels to ensure presence of adequate levels of Cdc28 CDK activity.