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     

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.     

Purification and biochemical analysis of catalytically active human cdc25C dual specificity phosphatase

We describe a reliable and efficient method for the purification of catalytically active and mutant inactive full-length forms of the human dual specificity phosphatase cdc25C from bacteria. The protocol involves isolating insoluble cdc25C protein in inclusion bodies, solubilization in guanidine HCL, and renaturation through rapid dilution into low salt buffer. After binding renatured proteins to an ion exchange resin, cdc25C elutes in two peaks at 350 and 450 mM NaCl. Analysis by gel exclusion chromatography and enzymatic assays reveals the highest phosphatase activity is associated with the 350 mM NaCl with little or no activity present in the 450 mM peak. Furthermore, active cdc25C has a native molecular mass of 220 kDa consistent with a potential tetrameric complex of the 55-kDa cdc25C protein. Assaying phosphatase activity against artificial substrates pNPP and 3-OMFP reveals a 220 kDa form of the phosphatase is active in a non-phosphorylated state. The protein effectively activates cdk1/cyclin B prokinase complexes in vitro in the absence of cdk1 kinase activity in an orthovanadate sensitive manner but is inactivated by A-kinase phosphorylation. In vitro phosphorylation of purified cdc25C by cdk1/cyclin B1, cdk2/cyclin A2 and cdk2/cyclin E shows that distinct TP/SP mitotic phosphorylation sites on cdc25C are differentially phosphorylated by these 3 cdk/cyclin complexes associated with different levels of cdc25C activation. Finally, we show that endogenous native cdc25C from human cells is present in high molecular weight complexes with other proteins and resolves mostly above 200-kDa. These data show that untagged cdc25C can be purified with a simple protocol as an active dual specificity phosphatase with a native molecular mass consistent with a homo-tetrameric configuration.     

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)     

Elevated Expression of the cdc25A Protein Phosphatase in Colon Cancer

The nuclear protein phosphatase cdc25A has been postulated to be a protooncogene. The total nuclear phosphotyrosyl protein phosphatase (PTP) activity and the expression of cdc25A were compared in normal and cancerous colon epithelial tissue. Nuclei derived from normal mucosal epithelium and tumors were analyzed for phosphotyrosyl protein phosphatase activity using the malachite green assay and a synthetic phosphotyrosyl peptide based on the sequence of cdc2, a known cdc25A phosphotyrosyl protein substrate. Tumorigenesis resulted in elevated nuclear PTP activity (343.0 ± 37.0% of normal epithelial PTP activity) in 52% (29 of 56) of colon tumors. In all cases elevated nuclear PTP activity correlated with an increase in the expression of cdc25A. The changes in PTP activity observed were not due to any increase in the rate of growth of the colonic mucosa as no corresponding changes occurred with PTP activity under conditions of rapid mucosal growth.     

Regulatory properties of neuronal cdc2-like kinase

Neuronal cdc2-like kinase, nclk, is a heterodimer of cyclin dependent protein kinase 5, cdk5, and a 25 kDa subunit derived from a novel, neuron-specific, 35 kDa protein: p35. The characterization and regulation of nclk will be summarized in this minireview. The activity of nclk appears to be governed by highly complex regulatory mechanisms including protein-protien interaction, protein phosphorylation and isoforms. The histone H1 kinase activity of nclk is absolutely dependent of the interaction between the 25 kDa subunit and the catalytic subunit, cdk5. In addition, nclk interacts with other cellular proteins to form macromolecular complexes. The kinase activity of nclk is inhibitedin vitro by the phosphorylation reactions of a weel-like protein tyrosine kinase and a protein serine/threonine kinase from bovine thymus. Northern blot analysis has revealed the existence of two populations of p35 mRNA of 2 and 4 kb. A novel cDNA encoding a p35 homologous protein has been obtained from a human hippocampus library.     

A Novel Neutral Protease from <Emphasis Type="Italic">Thermoactinomyces</Emphasis> Species 27a: Sequencing of the Gene,Purification, and Characterization of the Enzyme

The nucleotide sequence of the previously cloned (Zabolotskaya, M. V., Nosovskaya, E. A., Kaplun, M. A., and Akimkina, T. V. (2001). Mol. Gen. Mikrobiol. Virusol. No 1, 32–34) DNA fragment from Thermoactinomyces sp. 27a (GenBank Accession No. AY280367) containing the metalloproteinase gene was determined. A continuous open reading frame encoding a polypeptide of 673 aa was revealed. Analysis of this sequence demonstrated that the metalloproteinase from Thermoactinomyces sp. 27a is synthesized as a preproprotein and includes a leader peptide (26 aa), N-terminal propeptide (215 aa), mature region (317 aa), and additional C-terminal domain (115 aa). The recombinant enzyme from Thermoactinomyces sp. 27a was expressed in Bacillus subtilis AJ73 cells and purified by anion exchange chromatography to an electrophoretically homogeneous state. The determined N-terminal amino acid sequence of the mature protein was identical to that deduced from the gene. The obtained data suggest that the mature protein should include 432 aa and have a calculated molecular weight of 46,262 Da. However, the molecular weight of the mature protein determined by mass spectrometry was 34,190 ± 70 Da indicating a C-terminal processing. Theproteinase was not inhibited by phenylmethyl sulfonyl fluoride but was inhibited by o-phenanthroline and ethylenediaminetetraacetic acid. The enzyme had maximum activity by azocasein hydrolysis at 55°C and pH 6.5–7.5; it was stable at pH 7.5–8.5 and remained stable at 50°C for several hours. The k_cat/K_m for 3-(2-furyl)acryloyl-glycyl-L-leucine amide hydrolysis was (2.8 ± 0.1) ×10³ M^–1×s^–1.     

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.     

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.     

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.     

Isolation and characterization of a second protein tyrosine phosphatase gene, PTP2, from Saccharomyces cerevisiae.

A putative protein tyrosine phosphatase (PTPase) gene, PTP2, was cloned from Saccharomyces cerevisiae. The complete yeast PTP2 gene encodes a 750-amino acid residue protein with a predicted mass of 86 kDa. The conserved PTPase domain was localized in the C-terminal half of the protein. Amino acid sequence alignment of the yeast PTPase domain with other phosphatases indicated approximately 20-25% sequence identity with the mammalian PTPase and a similar degree of identity with the PTPase encoded by the yeast PTP1 gene. The PTP2 gene is closely linked to the yeast RET1 and STE4 genes and is localized on the right arm of chromosome 15. Gene disruption experiments demonstrated that neither PTP2 alone nor PTP2 in combination with PTP1 was essential for growth under the conditions tested. The ability of PTP2 to complement the cdc25-22 mutant of Schizosaccharomyces pombe was also examined, and unlike the human T-cell PTPase, which was able to complement the cdc25-22 mutant, the S. cerevisiae PTP2 was unable to complement the cdc25-22 mutant of S. pombe.     

Cloning and characterization of a cdc25 phosphatase from mouse lymphocytes

Members of the cdc25 phosphatase family are proposed to function as important regulators of the eukaryotic cell cycle, particularly in the induction of mitotic events. A new cdc25 tyrosine phosphatase, cdc25M1, has been cloned from a mouse pre-B cell cDNA library and characterized. The cdc25M1 protein consists of 465 amino acids with a predicted relative molecular mass (M_r) of 51 750. Over the highly conserved carboxyl terminal region, the amino acid sequence similarity to the human cdc25 C or Hs1 isoform is 89%, while the overall similarity is 67%. The phosphatase active site is located within residues 367–374. Tissue expression of the cdc25M1 was highest in mouse spleen and thymus by northern blot analysis. The cdc25M1 mRNA was detected in a number of cloned mouse lymphocyte cell lines including both CD8⁺ and CD4⁺ cells. cdc25M1 mRNA was shown to be cell cycle-regulated in T cells following interleukin-2 (IL-2)-stimulation. Accumulation of cdc25M1 mRNA occured at 48 h after IL-2 stimulation, when lymphocytes were progressing from S phase to G₂/M phase of the cell cycle. This pattern of expression is in contrast to that observed for other protein tyrosine phosphatases expressed in T lymphocytes including CD45, LRP, SHP, and PEP. The elevation in cdc25M1 mRNA level occurred concomittant to the appearance of the hyperphosphorylated form of p34^cdc2 protein kinase. A purified, bacterial-expressed recombinant cdc25M1 phosphatase domain catalyzed the dephosphorylation of p-nitrophenol phosphate, as well as [³²P-Tyr] and [³²P-Ser/Thr]-containing substrates. Preincubation of p34^cdc2 kinase with cdc25M1 activated its histone H1 kinase activity in vitro. These results suggest that cdc25M1 may be involved in regulating the proliferation of mouse T lymphocytes following cytokine stimulation, through its action on p34^cdc2 kinase.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number L16926.     

Isolation and Characterization of the First Putative Peroxidase Gene from Oilseed Rape (Brassica napus) which is Highly Homologous to HRPC

A new gene, designated as BnPrx (GenBank Accession No. DQ078754), was isolated from oilseed rape (Brassica napus) by SMART Rapid Amplification of cDNA Ends (RACE). The full-length cDNA is 1307 bp long and contains a 1062 bp open reading frame (ORF), which encodes a 354 amino acid peroxidase precursor, with a 31 aa N-terminal signal peptide and a 15 aa C-terminal propeptide. The putative protein has a molecular weight of 38.86 kDa and a calculated pI of 5.85. BnPrx shares high identity with HRPC (89%). BnPrx possesses all active residues and two Ca²⁺ sites present in Horseradish peroxidase isoenzymes C (HRPC) as well as six N-glycosylation sites. The predicted 3-D structure of BnPrx is very similar to that of HRPC. Assisted by genomic walking technology, the genomic DNA of BnPrx was also cloned, consisting of 3 introns and 4 exons. Thirty-two TATA boxes, 18 CAAT boxes and many cis-elements, such as WUN, MeJR, were found in its promoter region. Southern blot analysis indicated that BnPrx belonged to a small gene family. Northern blot analysis revealed that BnPrx was constitutively expressed in all tested tissues, including roots, stems and leaves, with the high expression in leaves and stems. The expression of BnPrx could be induced by methyl jasmonate (MeJA), salicylic acid (SA), cold and H₂O₂. The cloning and characterizing of BnPrx might not only help us understand the physiological function and molecular evolution of the large peroxidase gene family more comprehensively, but also provide an alternative way of seeking a more effective and economical substitute for HRPC.     

Expression,purification, and characterization of recombinant Chinese shrimp crustin-like protein (CruFc) in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A crustin-like protein (CruFc) from Fenneropenaeus chinensis was expressed in Pichia pastoris and then purified to electrophoretic homogeneity on a Sephacryl S-100 column with a band corresponding to the expected one (13 kDa) shown by 15% SDS-PAGE. Western blot indicated that the rCruFc specifically reacted with polyclonal rabbit anti-Fenneropenaeus chinensis CruFc. Production in a 5 l bioreactor gave 237 mg rCruFc/l. Antimicrobial assay revealed that 4 μM rCruFc inhibited growth of Staphylococcus aureus.     

Toxin A secretion in Pseudomonas aeruginosa: the role of the first 30 amino acids of the mature toxin

Toxin A, one of several virulence factors secreted by the gram-negative bacterium Pseudomonas aeruginosa, is synthesized as a 71 kDa precursor with a typical prokaryotic leader peptide (LP), and is secreted as a 68 kDa mature protein. Evidence from a previous study suggested that a signal required for toxin A secretion in P. aeruginosa may reside within the region defined by the toxin A LP and the first 30 amino acids (aa) of mature toxin A. In the present study, we have used exonuclease Ba131 deletion analysis to examine the specific role of the first 30 as in toxin A secretion. Four toxA subclones, which encode products containing the toxin A LP and different segments of the 30-residue region fused to a toxin A carboxy-terminal region, were identified. In addition, a gene fusion encoding a hybrid protein consisting of the LP of P. aeruginosa elastase and the final 305 residues of toxin A, was generated. The cellular location of the toxA subclone products in P. aeruginosa was determined by immunoblotting analysis. Toxin A CRMs (cross-reacting material) encoded by different subclones were detected in different fractions of P. aeruginosa including the periplasm and the supernatant. Results from these studies suggest that (1) mature toxin A contains two separate secretion signals one within the N-terminal region and one within the C-terminal region; and (2) the first 30 residues of the mature toxin A form part of the N-terminal secretion signal.     

p80cdc25 mitotic inducer is the tyrosine phosphatase that activates p34cdc2 kinase in fission yeast.

We have investigated the mechanism by which fission yeast p80cdc25 induces mitosis. The in vivo active domain was localized to the C-terminal 23 kDa of p80cdc25. This domain produced as a bacterial fusion protein (GST-cdc25) caused tyrosyl dephosphorylation and activation of immunoprecipitated p34cdc2. Furthermore, GST-cdc25 dephosphorylated both para-nitrophenyl-phosphate (pNPP) and casein phosphorylated on serine in vitro. Reaction requirements and inhibitor sensitivities were the same as those of phosphotyrosine phosphatases (PTPases). Analysis of cdc25 C-terminal domains from a variety of species revealed a conserved motif having critical residues present at the active site of PTPases. Mutation of the cdc25 Cys480 codon, corresponding to an essential cysteine in the active site of PTPases, abolished the phosphatase activity of GST-cdc25. These data indicate that cdc25 proteins define a novel subclass of eukaryotic PTPases, and strongly argue that cdc25 proteins directly dephosphorylate and activate p34cdc2 kinase to induce M-phase.     

Research note: Identification of a cDNA homologous to the cell-cycle-controlling cdc2 gene in Acroslphonia duriuscula (Acrosiphoniales, Chlorophyta)

Cyclin‐dependent kinases, the most notable of which is cdc2, are key regulators of the cell cycle and are highly conserved in evolution. We have cloned and analyzed one cDNA containing an open reading frame of 337 amino acids from a multinucleate, multicellular green alga, Acrosiphonia duriuscula (Ruprecht) Collins. The deduced protein, named Adcdc2‐1, showed 51% and 54% amino acid sequence identity to yeast cdc2/cdc28 and human cdc2, respectively. Several domains that characterize the cdc2‐related kinases were identified from this sequence, although the PSTAIRE motif was replaced with PPTTIRE. Furthermore, this protein has conserved Tyr and Thr residues that are sites of phosphorylation in cdc2‐related kinases and are important for regulating kinase activity. The present results suggest that the universal cdc2 is conserved in algae with unique structural characteristics.