Cytokinin controls the cell cycle at mitosis by stimulating the tyrosine dephosphorylation and activation of p34cdc2-like H1 histone kinase

In excised pith parenchyma from Nicotiana tabacum L. cv. Wisconsin Havana 38, auxin (naphthalene-1-acetic acid) together with cytokinin (6-benzylaminopurine) induced a greater than 40-fold increase in a p34^cdc2-like protein, recoverable in the p13^suc1-binding fraction, that had high H1 histone kinase activity, but enzyme induced without cytokinin was inactive. In suspension-cultured N. plumbaginifolia Viv., cytokinin (kinetin) was stringently required only in late G2 phase of the cell division cycle (cdc) and cells lacking kinetin arrested in G2 phase with inactive p34^cdc2-like H1 histone kinase. Control of the Cdc2 kinase by inhibitory tyrosine phosphorylation was indicated by high phosphotyrosine in the inactive enzyme of arrested pith and suspension cells. Yeast cdc25 phosphatase, which is specific for removal of phosphate from tyrosine at the active site of p34^cdc2 enzyme, was expressed in bacteria and caused extensive in-vitro activation of p13^suc1-purified enzyme from pith and suspension cells cultured without cytokinin. Cytokinin stimulated the removal of phosphate, activation of the enzyme and rapid synchronous entry into mitosis. Therefore, plants can control cell division by tyrosine phosphorylation of Cdc2 but differ from somatic animal cells in coupling this mitotic control to hormonal signals.Abbreviations BAP 6-benzylaminopurine - BrdUrd 5-bromo-2-deoxyuridine - cdc cell division cycle - Cdc25 cdc phospho-protein phosphatase - CKI cyclin dependent kinase inhibitor - 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6 diamidino-2-phenylindole - GST-cdc25 glutathione sulfur transferase-truncated cdc25 fusion - MS Murashige and Skoog (1962) - NAA naphthalene-1-acetic acid - p34^cdc2 34-kDa product of the cdc2 gene     

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.     

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.     

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 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.     

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)     

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.     

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.     

In vitro cold storage of cork oak shoot cultures

Friable callus cultures were initiated from cotyledons and hypocotyls of Opuntia ficus-indica. Explants from cotyledons produced significantly more callus than those from hypocotyls. Optimum callus growth was observed on Murashige & Skoog medium supplemented with 0.9 μM 6-furfurylaminopurine, 2.3 μM 2,4-dichlorophenoxyacetic acid, 1.0 μM 4-amino 3,5,6-trichloropicolinic acid, 400 mg l^-1 casein hydrolysate and 3% sucrose. The same medium without agar was used for establishing cell suspensions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Synthesis of extracellular proteins in embryogenic and non-embryogenic cell cultures of alfalfa

Extracellular proteins, released into the culture medium from alfalfa cells grown in embryogenic and non-embryogenic conditions, were ³⁵S-methionine labelled at different days of culture. SDS-PAGE analysis showed significant differences between the patterns of extracellular proteins secreted into the medium devoid of 2,4-d, in which cells formed somatic embryos, or in presence of 2,4-d, in which undifferentiated cell proliferation took place. Some proteins, evident in 2,4-d-supplied cultures, disappeared when cells were subcultured in the embryogenic conditions. Western analysis with antibodies against the carrot extracellular proteins EP1 and EP2 showed the presence of homologous alfalfa proteins. In 2,4-d depleted alfalfa cells, an EP1-like protein disappeared and another one was reduced, while the presence of the EP2-like protein was, in the same conditions, strongly enhanced.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - EP extracellular proteins - ns-LTP non specific lipid transfer protein - SDS-PAGE Sodium dodecyl sulphate polyacrilamide gel electrophoresis     

Synthesis and accumulation of p34cdc2 and cyclin B in mouse oocytes during acquisition of competence to resume meiosis

This study tests the hypothesis 033 that growing murine oocytes, which are incompetent to resume meiosis, are deficient in their content of p34^cdc2 and/or cyclin B, the two subunits of maturation promoting factor (MPF). Accumulation of the two MPF components occurred in an asynchronous manner in growing oocytes. Cyclin B content reached maximal levels in oocytes that were not yet competent to undergo germinal vesicle breakdown (GVB), the first obvious morphological manifestation of the resumption of meiosis. Thus, the amount of cyclin B is not the limiting factor rendering these growing oocytes incompetent to undergo GVB. In contrast, synthesis and accumulation of p34^cdc2 increased during the period of oocyte growth in vivo when they became competent to undergo GVB. A similar increase in the amount of p34^cdc2 also occurred in cultured granulosa cell-free oocytes despite the lack of oocyte growth, but these cultured oocytes did not become GVB competent. Thus, the accumulation of p34^cdc2 is probably necessary, but not sufficient, for mouse oocytes to become competent to undergo GVB. This accumulation occurs autonomously in oocytes independently of growth or of the participation of follicular somatic cells. © 1995 Wiley-Liss, Inc.     

Plant regeneration from protoplasts isolated from cotyledons of Sesbania bispinosa

Protoplasts were isolated from cotyledons of Sesbania bispinosa (Jacq.) W.F. Wight. In a liquid-over-agar culture system with Murashige and Skoog (MS) medium supplemented with 1 mg l^-1 2,4-dichlorophenoxyacetic acid (2,4-d, 2 mg l^-1 benzyladenine (BA), 1 mg l^-1 glutamine and 0.5 and formed callus. The first division occurred after 3–4 days. Callus formed from the protoplasts differentiated shoots by organogenesis on MS medium with 1 mg l^-1 indolebutyric acid (IBA) and 1 mg l^-1 BA. These shoots developed into complete plantlets when excised and cultured on MS medium with 0.5 mg l^-1 IBA.     

The function of the chicken p34CDC2 protein kinase in fission yeast is cold sensitive for cell cycle progression through the G1 phase and temperature sensitive for traversal of mitosis.

The protein kinase p34^cdc2 is required at the onset of DNA replication and for entry into mitosis. The catalytic subunit and its regulatory proteins, notably the cyclins, are conserved from yeast to man. This suggests that the control mechanisms necessary for progression through the cell cycle in fission yeast are conserved throughout evolution. This work describes the characterization of a fission yeast strain that is dependent for cell cycle progression on the activity of the p34^CDC2 protein kinase from chicken. The response of the chicken p34^CDC2 protein kinase to cell cycle components of fission yeast was examined. Cells expressing the chicken p34^CDC2 protein divide at reduced size at 31° C. Cells are temperature sensitive at 35.5° C and die as a result of mitotic catastrophe. This phenotype can be rescued by delaying cell cycle progression at the G1-S transition by adding low concentrations of hydroxyurea. Schizosaccharomyces pombe cells that are dependent on chicken p34^CDC2 are cold sensitive. At 19° C to 25° C cells arrest in the G1 phase, while traversal of the G2-M transition is not blocked at low temperature. Expression of chicken p34^CDC2 in the cold-sensitive G2-M mutant cdc2A21 suppresses the G1 arrest. Received: 14 October 1998 / Accepted: 15 March 1999     

Evidence For the Presence of A Cdc2-Like Protein Kinase In the Dinoflagellate Crypthecodinium Cohnii

ABSTRACT. The unusual nature of mitosis and ancestral organization of the dinoflagellate nucleus prompted the question of whether the cdc 2-like histone H1 kinase, a presumed ubiquitous cell cycle regulator in eukaryotes, is present in these primitive organisms. Western blotting of Crypthecodinium cohnii protein extracts using antibody against the Pro-Ser-Thr-Ala-Ile-Arg-Glu (=PSTAIRE) amino acid sequence motif, conserved in all cdc 2 homologues known, revealed one prominent band corresponding to a protein with an apparent relative molecular weight ≈ 34,000, identical in mobility to that from HeLa cells and Physarum polycephalum , higher and lower eukaryotic controls, respectively. Incubation of C. cohnii cell lysates with p13 ^{suc 1}-sepharose beads, which preferentially, though not exclusively, bind p34 ^{cdc 2}, resulted in precipitation of a 34-kDa protein which was reactive with anti-PSTAIRE antibody, selectively competed for by the PSTAIRE peptide and able to phosphorylate histone H1 in vitro. We conclude that the dinoflagellate C. cohnii contains a protein very similar to the cdc 2 gene product from fission yeast and its homologues in all eukaryotes studied thus far.