Requirement for p34cdc2 kinase is restricted to mitosis in the mammalian cdc2 mutant FT210

The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34cdc2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wild-type FM3A cell line each possess at least three distinct histone H1 kinases. H1 kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34cdc2 and the human cyclins A and B1. The results show that p34cdc2 forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the non-permissive temperature in vitro and in vivo. A third H1 kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-like 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34cdc2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34cdc/CDC28 is essential only during the G2-to-M transition in FT210 cells.     

Conversion of differentiation inducer resistance to differentiation inducer sensitivity in erythroleukemia cells.

Hexamethylene bisacetamide (HMBA) is a potent inducer of differentiation of murine erythroleukemia cells (MELC). Commitment, the irreversible initiation of the program of terminal-cell differentiation, is first detected in HMBA-sensitive DS19-SC9 MELC in culture after 10 to 12 h of exposure to HMBA. Vincristine (VC)-resistant MELC derived from the DS19-SC9 MELC line display increased sensitivity to HMBA and become committed with little or no latent period. In the present study, we showed that the MELC line R1, which is resistant to HMBA-mediated differentiation, became sensitive to inducer if selected for a low level of VC resistance (less than 10 ng of VC per ml). Four independently derived VC-resistant cell lines from HMBA-resistant R1 cells, designated R1[VCR]a to R1[VCR]d, acquired sensitivity to HMBA and the accelerated kinetics of commitment that are characteristic of VC-resistant MELC derived from the parental DS19-SC9 cells. The calcium channel blocker verapamil suppresses the VC resistance of R1[VCR] cells but does not alter the accelerated response to HMBA. In R1[VCR] cells there was no detectable increase in the level of the 140-kilodalton P-glycoprotein. Transient inhibition of protein synthesis during the latent period delays inducer-mediated commitment of VC-sensitive DS19-SC9 MELC but does not alter the accelerated commitment kinetics of R1[VCR]a cells. Previously, we have reported evidence that protein kinase C beta (PKC beta) plays a role in HMBA-induced MELC differentiation and that compared with DS19-SC9 cells, R1 cells have a relatively low level and R1[VCR]a cells have a high level of PKC beta. These findings suggest that (i) acquisition of VC resistance overcomes the block acquired by R1 cells to HMBA-mediated differentiation; (ii) the accelerated kinetics of HMBA-induced commitment of VC-resistant MELC is not dependent on the verapamil-sensitive transport channel that is responsible, at least in part, for resistance to VC; (iii) in VC-resistant MELC, there is constitutive expression or accumulation of a protein required for HMBA-induced differentiation; and (iv) an elevated level of PKC beta activity may play a role in the altered response of R1[VCR] and other VC-resistant MELC to HMBA.     

Loss of RCC1, a nuclear DNA-binding protein, uncouples the completion of DNA replication from the activation of cdc2 protein kinase and mitosis.

The temperature-sensitive mutant cell line tsBN2, was derived from the BHK21 cell line and has a point mutation in the RCC1 gene. In tsBN2 cells, the RCC1 protein disappeared after a shift to the non-permissive temperature at any time in the cell cycle. From S phase onwards, once RCC1 function was lost at the non-permissive temperature, p34cdc2 was dephosphorylated and M-phase specific histone H1 kinase was activated. However, in G1 phase, shifting to the non-permissive temperature did not activate p34cdc2 histone H1 kinase. The activation of p34cdc2 histone H1 kinase required protein synthesis in addition to the presence of a complex between p34cdc2 and cyclin B. Upon the loss of RCC1 in S phase of tsBN2 cells and the consequent p34cdc2 histone H1 kinase activation, a normal mitotic cycle is induced, including the formation of a mitotic spindle and subsequent reformation of the interphase-microtubule network. Exit from mitosis was accompanied by the disappearance of cyclin B, and a decrease in p34cdc2 histone H1 kinase activity. The kinetics of p34cdc2 histone H1 kinase activation correlated well with the appearance of premature mitotic cells and was not affected by the presence of a DNA synthesis inhibitor. Thus the normal inhibition of p34cdc2 activation by incompletely replicated DNA is abrogated by the loss of RCC1.     

B-type cyclins regulate G1 progression in fission yeast in opposition to the p25rum1 cdk inhibitor.

The onset of S phase in fission yeast is regulated at Start, the point of commitment to the mitotic cell cycle. The p34cdc2 kinase is essential for G1 progression past Start, but until now its regulation has been poorly understood. Here we show that the cig2/cyc17 B-type cyclin has an important role in G1 progression, and demonstrate that p34cdc2 kinase activity is periodically associated with cig2 in G1. Cells lacking cig2 are defective in G1 progression, and this is particularly clear in small cells that must regulate Start with respect to cell size. We also find that the cig1 B-type cyclin can promote G1 progression. Whilst p25rum1 can inhibit cig2/cdc2 activity in vitro, and may transiently inhibit this complex in vivo, cig1 is regulated independently of p25rum1. Since cig1/cdc2 kinase activity peaks in mitotic cells, and decreases after mitosis with similar kinetics to cdc13-associated kinase activity, we suggest that cig2 is likely to be the principal fission yeast G1 cyclin. cig2 protein levels accumulate in G1 cells, and we propose that p25rum1 may transiently inhibit cig2-associated p34cdc2 activity until the critical cell size required for Start is reached.     

Cell volume changes in relation to the cell cycle of differentiating erythroleukemic cells

Murine erythroleukemic cells (MELC) were synchronized by sequential exposure to thymidine and hydroxyurea. Upon removal from hydroxyurea, cells cultured with or without agents that induce erythroid differentiation, such as hexamethylene bisacetamide (HMBA) or dimethylsulfoxide (Me₂SO), proceed through S, G2 and mitosis with the same kinetics: S phase averages 5 h and G2 plus mitosis, 2 h. Cells cultured with HMBA and Me₂SO remain in the subsequent G1 for 5–7 h, compared with an average of only 3 h for cells cultured without inducer. Modal cell volume doubles as the cells proceed from G1 to G2. During the inducer-mediated prolonged G1, MELC retain a small cell volume. In cultures of non-synchronous MELC, inducers also increase the G1 fraction, as well as the proportion of small cells. An Me₂SO-resistant MELC variant (DR10), cultured with Me₂SO, shows little prolongation of G1 and little difference in the modal cell volume compared with cells without inducer. However, HMBA, which induces differentiation of DR10 cells, prolongs G1 and increases the proportion of small cells. These studies indicate that early changes in cell volume associated with induction of MELC to differentiate, in large part reflect alterations in the cell cycle. Evidence is presented which suggests that only one round of DNA synthesis in the presence of inducer may be necessary to initiate differentiation.     

Cyclin B targets p34cdc2 for tyrosine phosphorylation.

A universal intracellular factor, the 'M phase-promoting factor' (MPF), triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin Bcdc13. In M phase, its activation as an active MPF displaying histone H1 kinase (H1K) originates from the concomitant tyrosine dephosphorylation of the p34cdc2 subunit and the phosphorylation of the cylin Bcdc13 subunit. We have investigated the role of cyclin in the formation of this complex and the tyrosine phosphorylation of p34cdc2, using highly synchronous mitotic sea urchin eggs as a model. As cells leave the S phase and enter the G2 phase, a massive tyrosine phosphorylation of p34cdc2 occurs. This large p34cdc2 tyrosine phosphorylation burst does not arise from a massive increase in p34cdc2 concentration. It even appears to affect only a fraction (non-immunoprecipitable by anti-PSTAIR antibodies) of the total p34cdc2 present in the cell. Several observations point to an extremely close association between accumulation of unphosphorylated cyclin and p34cdc2 tyrosine phosphorylation: (i) both events coincide perfectly during the G2 phase; (ii) both tyrosine-phosphorylated p34cdc2 and cyclin are not immunoprecipitated by anti-PSTAIR antibodies; (iii) accumulation of unphosphorylated cyclin by aphidicolin treatment of the cells, triggers a dramatic accumulation of tyrosine-phosphorylated p34cdc2; and (iv) inhibition of cyclin synthesis by emetine inhibits p34cdc2 tyrosine phosphorylation without affecting the p34cdc2 concentration. These results show that, as it is synthesized, cyclin B binds and recruits p34cdc2 for tyrosine phosphorylation; this inactive complex then requires the completion of DNA replication before it can be turned into fully active MPF. These results fully confirm recent data obtained in vitro with exogenous cyclin added to cycloheximide-treated Xenopus egg extracts.     

Cyclin is a component of the sea urchin egg M-phase specific histone H1 kinase.

A so-called 'growth-associated' or 'M-phase specific' histone H1 kinase (H1K) has been described in a wide variety of eukaryotic cell types; p34cdc2 has previously been shown to be a catalytic subunit of this protein kinase. In fertilized sea urchin eggs the activity of H1K oscillates during the cell division cycle and there is a striking temporal correlation between H1K activation and the accumulation of a phosphorylated form of cyclin. H1K activity declines in parallel with proteolytic cyclin destruction of the end of the first cell cycle. By virtue of the high affinity of the fission yeast p13suc1 for the p34cdc2 protein, H1K strongly binds to p13-Sepharose beads. Cyclin, p34cdc2 and H1K co-purify on this affinity reagent as well as through several conventional chromatographic procedures. Anticyclin antibodies immunoprecipitate the M-phase specific H1K in crude extracts or in purified fractions. Sea urchin eggs appear to contain much less cyclin than p34cdc2, suggesting that p34cdc2 may interact with other proteins. These results demonstrate that cyclin and p34cdc2 are major components of the M-phase specific H1K.     

Expression of the murine homologue of the cell cycle control protein p34cdc2 in T lymphocytes.

The mammalian homologue of the cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a p34cdc2 cyclin-dependent kinase that regulates the cell cycle of a wide variety of cell types. Resting murine T lymphocytes contained no detectable p34cdc2 protein, histone kinase activity, or specific mRNA for the cdc2 gene. Activation of the T cells by immobilized anti-CD3 resulted in the expression of specific mRNA late in the G1 phase of the cell cycle, and p34cdc2 protein was detectable at or near G1/S. At this point in the cell cycle, the protein was phosphorylated at tyrosine and displayed no H1 histone kinase activity. As the cells progressed through the cycle, the amount of specific mRNA and p34cdc2 increased, and H1 histone kinase activity was detectable when the cells were blocked at G2/M by nocodazole. The activation of T cells by phorbol dibutyrate induced the expression of IL-2R but failed to induce the synthesis of IL-2 or the expression of cdc2-specific mRNA. Under these conditions, the activated cells failed to enter the S phase of the cell cycle. Because the presence of IL-2 added exogenously during activation by phorbol dibutyrate resulted in the expression of cdc2-specific mRNA and progression through the cell cycle, either IL-2 or the interaction with IL-2R may be involved in the expression of cdc2 and regulation of the G1/S transition.     

Properties of Saccharomyces cerevisiae wee1 and its differential regulation of p34CDC28 in response to G1 and G2 cyclins.

Wee1 is a protein kinase that negatively regulates p34cdc2 kinase activity. We have identified a Saccharomyces cerevisiae wee1 homolog encoded by the SWE1 gene. SWE1 overexpression arrests cells in G2 with short spindles whereas deletion of SWE1 did not alter the cell cycle but did eliminate the G2 delay observed in mih1- mutants. Swe1 immunoprecipitates were capable of tyrosine phosphorylating and inactivating p34CDC28 complexed with Clb2, a G2-type cyclin, but not p34CDC28 complexed with Cln2, a G1-type cyclin, consistent with the inability of Swe1 overexpression to inhibit the G1/S transition. These results suggest that specific cyclin subunits target p34CDC28 for distinct regulatory controls which may be important for ensuring proper p34CDC28 function during the cell cycle.     

Activation-induced T-cell death is cell cycle dependent and regulated by cyclin B.

Developing thymocytes and some T-cell hybridomas undergo activation-dependent programmed cell death. Although recent studies have identified some critical regulators in programmed cell death, the role of cell cycle regulation in activation-induced cell death in T cells has not been addressed. We demonstrate that synchronized T-cell hybridomas, irrespective of the point in the cell cycle at which they are activated, stop cycling shortly after they reach G2/M. These cells exhibit the diagnostic characteristics of apoptotic cell death. Although p34cdc2 levels are not perturbed after activation of synchronously cycling T cells, cyclin B- and p34cdc2-associated histone H1 kinase activity is persistently elevated. This activation-dependent induction of H1 kinase activity in T cells is associated with a decrease in the phosphotyrosine content of p34cdc2. We also demonstrate that transient inappropriate coexpression of cyclin B with p34cdc2 induces DNA fragmentation in a heterologous cell type. Finally, in T cells, cyclin B-specific antisense oligonucleotides suppress activation-induced cell death but not cell death induced by exposure to dexamethasone. We therefore conclude that a persistent elevation of the level of cyclin B kinase is required for activation-induced programmed T-cell death.     

Isolation of a human cyclin cDNA: evidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdc2

This paper reports the nucleotide and predicted amino acid sequence of a human B-type cyclin. The predicted protein sequence shows strong homology to the other known cyclins in the central third of the protein. We show that the level of cyclin mRNA is regulated during the cell cycle, increasing during G2 phase to four time that present in G1. The protein accumulates steadily during G2 to at least 20 times its level in G1 and is abruptly destroyed at mitosis. In G2/M phase, cyclin is associated with p34cdc2, the human homolog of the fission yeast gene cdc2+, and this complex has histone H1 kinase activity.     

Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity.

The Vpr accessory gene product of human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus is believed to play a role in permitting entry of the viral core into the nucleus of nondividing cells. A second role for Vpr was recently suggested by Rogel et al. (M. E. Rogel, L. I. Wu, and M. Emerman, J. Virol. 69:882-888, 1995), who showed that Vpr prevents the establishment in vitro of chronically infected HIV producer cell lines, apparently by causing infected cells to arrest in the G2/M phase of the cell cycle. In cycling cells, progression from G2 to M phase is driven by activation of the p34cdc2/cyclin B complex, an event caused, in part, by dephosphorylation of two regulatory amino acids of p34cdc2 (Thr-14 and Tyr-15). We show here that Vpr arrests the cell cycle in G2 by preventing the activation of the p34cdc2/cyclin B complex. Vpr expression in cells caused p34cdc2 to remain in the phosphorylated, inactive state, p34cdc2/cyclin B complexes immunoprecipitated from cells expressing Vpr were almost completely inactive in a histone H1 kinase assay. Coexpression of a constitutively active mutant p34cdc2 molecule with Vpr relieved the G2 arrest. These findings strongly suggest that Vpr arrests cells in G2 by preventing the activation of the p34cdc2/cyclin B complex that is required for entry into M phase. In vivo, Vpr might, by preventing p34cdc2 activation, delay or prevent apoptosis of infected cells. This would increase the amount of virus each infected cell produced.     

Differential phosphorylation of vertebrate p34cdc2 kinase at the G1/S and G2/M transitions of the cell cycle: identification of major phosphorylation sites.

The cdc2 kinase is a key regulator of the eukaryotic cell cycle. The activity of its catalytic subunit, p34cdc2, is controlled by cell cycle dependent interactions with other proteins as well as by phosphorylation--dephosphorylation reactions. In this paper, we examine the phosphorylation state of chicken p34cdc2 at various stages of the cell cycle. By peptide mapping, we detect four major phosphopeptides in chicken p34cdc2; three phosphorylation sites are identified as threonine (Thr) 14, tyrosine (Tyr) 15 and serine (Ser) 277. Analysis of synchronized cells demonstrates that phosphorylation of all four sites is cell cycle regulated. Thr 14 and Tyr 15 are phosphorylated maximally during G2 phase but dephosphorylated abruptly at the G2/M transition, concomitant with activation of p34cdc2 kinase. This result suggests that phosphorylation of Thr 14 and/or Tyr 15 inhibits p34cdc2 kinase activity, in line with the location of these residues within the putative ATP binding site of the kinase. During M phase, p34cdc2 is also phosphorylated, but phosphorylation occurs on a threonine residue distinct from Thr 14. Finally, phosphorylation of Ser 277 peaks during G1 phase and drops markedly as cells progress through S phase, raising the possibility that this modification may contribute to control the proposed G1/S function of the vertebrate p34cdc2 kinase.     

cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication.

RPA is a single-stranded DNA binding protein complex purified from human cells and is essential for the initiation and elongation stages of SV40 DNA replication in vitro. In both human and yeast cells, the 34 kDa polypeptide subunit of RPA is phosphorylated in the S and G2 phases of the cell cycle and not in G1. One of the major RPA kinases present in extracts of human cells was purified and shown to be the cyclin B-cdc2 complex. This purified kinase, and a closely related cyclin A associated cdc2-like kinase, phosphorylated RPA p34 on a subset of the chymotryptic peptides that were phosphorylated in vivo at the G1-S transition. Two serines near the N-terminus of RPA p34 were identified as possible sites of phosphorylation by cdc2 kinase. These same serines were necessary for RPA phosphorylation in vivo. The purified cdc2 kinase stimulated SV40 DNA replication in vitro when added to G1 cell extracts. The kinase also stimulated unwinding at the origin of replication, one of the earliest steps in DNA replication requiring RPA, but only in the presence of an additional factor present in G1 cell extracts. Thus, one or more members of the cyclin-cdc2 kinase family may be required for the initiation and maintenance of S phase, in part due to their ability to phosphorylate and activate a cellular DNA replication factor, RPA.     

Platelet-activating factor- and thrombin-induced stimulation of p34cdc2-cyclin histone H1 kinase activity in platelets.

Numerous studies of cell cycle control in dividing cells have pointed to the central role of a 34-kDa histone H1 kinase (p34cdc2) complexed with regulatory subunits known as cyclins. We now report that p34cdc2-cyclin may also participate in signal transduction in nonproliferating, terminally differentiated cells, in this instance during sheep platelet activation. Immunological evidence for the presence of a p34cdc2 cognant in sheep platelet cytosol was obtained with antipeptide antibodies raised against peptide sequences in the conserved PSTAIRE and C-terminus regions of murine cdc2. The immunoreactive 32-kDa protein was adsorbed onto p13suc1-Sepharose, which selectively binds p34cdc2. A 58-kDa protein that also bound to p13suc1-Sepharose was identified as cyclin A on the basis of its size and immunoreactivity with two different anticyclin peptide antibodies. The p34cdc2-cyclin A complex was regulated during platelet activation. Its histone H1 phosphorylating activity was stimulated 2-fold in p13suc1-Sepharose extracts from platelets that had been exposed to platelet-activating factor or thrombin for 1 min prior to harvesting. Our findings imply that the p34cdc2-cyclin complex may serve alternative functions besides control of cell division.     

Biochemical characterization of the p34cdc2 protein kinase component of purified maturation-promoting factor from Xenopus eggs

Genetic studies in the fission yeast Schizosaccharomyces pombe and biochemical data in oocytes and eggs of Xenopus laevis have implicated the product of the cdc2+ gene as critical for the G2 to M transition in the cell cycle. The product of the cdc2+ gene is a 34-kDa serine/threonine protein kinase, designated p34cdc2, that is a component of purified maturation-promoting factor (MPF) and also of purified mammalian growth-associated histone H1 kinase. The biochemical properties of p34cdc2 H1 kinase activity in the MPF complex were studied. Phosphorylation of the p45cyclin component in the MPF complex by p34cdc2 exhibited kinetics consistent with an intramolecular reaction. On glycerol gradient centrifugation, MPF kinase against several substrates sedimented with an apparent Mr = 45,000-55,000. p34cdc2 was found to utilize ATP, GTP, and adenosine 5'-O-(3-thiotriphosphate) with apparent Km values of 75, 700, and 250 microM, respectively. The kinase activity was inhibited by beta-glycerophosphate, NaF, and zinc, whereas p-nitrophenyl phosphate was slightly stimulatory. The relative rates of phosphorylation of various substrates by MPF and growth-associated H1 kinase were similar. These findings should prove useful in further work on the regulation of MPF kinase activity and characterization of its substrates.