Alternative fates of keratinocytes transduced by human papillomavirus type 18 E7 during squamous differentiation

The human papillomavirus type 18 (HPV-18) E7 protein promotes S-phase reentry in postmitotic, differentiated keratinocytes in squamous epithelium to facilitate vegetative viral DNA amplification. To examine the nature and fate of the differentiated cells that reenter S phase, organotypic cultures of primary human keratinocytes transduced with HPV-18 E7 were pulse-chase-pulse-labeled with (3)H-thymidine ((3)H-TdR) and bromodeoxyuridine (BrdU). The kinetics of the appearance of doubly labeled suprabasal cells demonstrate that E7 expression did not promote prolonged S phase. Rather, there was a considerable lag before a small percentage of the cells reentered another round of S phase. Fluorescence in situ hybridization analysis, indeed, revealed a small fraction of the cells with more than 4n chromosomes in the differentiated strata. Differentiated cells positive for (3)H-TdR, BrdU, or both often had enlarged nuclei or were binucleated. These results suggest that S phase is not followed by cell division, although nuclear division may occur. Interestingly, a significant fraction of differentiated cells that entered S phase subsequently accumulated p27kip1 protein with a kinetics preceding the accumulation of cyclin E. We conclude that E7-transduced, differentiated keratinocytes that enter S phase have two alternative fates: (i) a low percentage of cells undergoes endoreduplication, achieving higher than 4n ploidy, and (ii) a high percentage of cells accumulates the p27kip1, cyclin E, and p21cip1 proteins, resulting in arrest and preventing further S-phase reentry.     

Two different protein kinases act on a different time schedule as glial filament kinases during mitosis.

Glial fibrillary acidic protein (GFAP) is a component of glial filaments specific to astroglia. We now report the spatial and temporal distributions of four phosphorylated sites in the GFAP molecule during mitosis of astroglial cells, determined by antibodies which can distinguish phosphorylated epitopes from non-phosphorylated-epitopes. Immunofluorescence microscopy showed that the Ser8 residues in the entire cytoplasmic glial filament system are initially phosphorylated when the cells enter mitosis. In cytokinesis, the phosphoSer8 residues become dephosphorylated, whereas Thr7, Ser13 and Ser34 in glial filaments at the cleavage furrow become the preferred sites of phosphorylation. The cdc2 kinase purified from mitotic cells can phosphorylate GFAP at Ser8 but not at Thr7, Ser13 or Ser34, in vitro. These results suggest that cdc2 kinase acts as a glial filament kinase only at the G2-M phase transition while other glial filament kinases are probably activated at the cleavage furrow before final separation of the daughter cells.     

Chk1 phosphorylation at Ser286 and Ser301 occurs with both stalled DNA replication and damage checkpoint stimulation

We previously reported Chk1 to be phosphorylated at Ser286 and Ser301 by cyclin-dependent kinase (Cdk) 1 during mitosis [T. Shiromizu et al., Genes Cells 11 (2006) 477-485]. Here, we demonstrated that Chk1-Ser286 and -Ser301 phosphorylation also occurs in hydroxyurea (HU)-treated or ultraviolet (UV)-irradiated cells. Unlike the mitosis case, however, Chk1 was phosphorylated not only at Ser286 and Ser301 but also at Ser317 and Ser345 in the checkpoint response. Treatment with Cdk inhibitors diminished Chk1 phosphorylation at Ser286 and Ser301 but not at Ser317 and Ser345 with the latter. In vitro analyses revealed Ser286 and Ser301 on Chk1 to serve as two major phosphorylation sites for Cdk2. Immunoprecipitation analyses further demonstrated that Ser286/Ser301 and Ser317/Ser345 phosphorylation occur in the same Chk1 molecule during the checkpoint response. In addition, Ser286/Ser301 phosphorylation by Cdk2 was observed in Chk1 mutated to Ala at Ser317 and Ser345 (S317A/S345A), as well as Ser317/Ser345 phosphorylation by ATR was in S286A/S301A. Therefore, Chk1 phosphorylation in the checkpoint response is regulated not only by ATR but also by Cdk2.     

Delaying the onset of M phase in NIH 3T3 cells blocked in early S phase occurs via accumulating cyclin B1 and tyrosine-phosphorylated p34cdc2 in the nucleus

An affinity-purified antibody (anti-Cdc2C) raised against the carboxy terminal sequence LDNQIKKM of p34^cdc2 uncovered in NIH 3T3 cells a protein subpopulation, the location and the level of accumulation of which evolve during progression through the cell cycle: it first emerges inside the nucleus in late G1/early S phase and continues to build up principally in this location throughout S phase; a cytoplasmic expression then becomes apparent near the end of S phase, develops during G2 and sometimes prevails over the nuclear expression; it finally relocates to the nucleus in early prophase. We propose that a major part of this subpopulation would represent p34^cdc2 molecules existing inside a complex with cyclin B1. NIH 3T3 cells arrested in early S phase with aphidicolin do not commit prematurely to mitosis which indicates that the regulatory pathway involved in preserving the temporal order of S and M phases is functioning in these conditions. Conjugated Western blot analysis and immunofluorescence microscopy showed that cyclin A, cyclin B1 and tyrosine-phosphorylated p34^cdc2 continue to build up predominantly in the nucleus of the arrested cells. After release from the block, the cells rapidly reenter S and G2 phases and, concomitantly, cyclin B1 and tyrosine-phosphorylated p34^cdc2 relocate to the cytoplasm before redistributing again in the nucleus in early prophase. These data would suggest that delaying the onset of M phase in NIH 3T3 cells in which the rate of DNA replication is reduced, is first ensured by a mechanism that prevents the cytoplasmic relocation of inactive p34^cdc2/cyclin B1 complexes continually forming in the nucleus once the G1 period of mitotic cyclin instability is over.     

The multiple roles of cyclin E1 in controlling cell cycle progression and cellular morphology of Trypanosoma brucei

Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases. Previous RNA interference (RNAi) experiments in Trypanosoma brucei indicated that cyclin E1, cdc2-related kinase (CRK)1 and CRK2 are involved in regulating G1/S transition, whereas cyclin B2 and CRK3 play a pivotal role in controlling the G2/M checkpoint. To search for potential interactions between the other cyclins and CRKs that may not have been revealed by the RNAi assays, we used the yeast two-hybrid system and an in vitro glutathione-S-transferase pulldown assay and observed interactions between cyclin E1 and CRK1, CRK2 and CRK3. Cyclins E1-E4 are homologues of yeast Pho80 cyclin. But yeast complementation assays indicated that none of them possesses a Pho80-like function. Analysis of cyclin E1+CRK1 and cyclin E1+CRK2 double knockdowns in the procyclic form of T. brucei indicated that the cells were arrested more extensively in the G1 phase beyond the cumulative effect of individual knockdowns. But BrdU incorporation was impaired significantly only in cyclin E1+CRK1-depleted cells, whereas a higher percentage of cyclin E1+CRK2 knockdown cells assumed a grossly elongated posterior end morphology. A double knockdown of cyclin E1 and CRK3 arrested cells in G2/M much more efficiently than if only CRK3 was depleted. Taken together, these data suggest multiple functions of cyclin E1: it forms a complex with CRK1 in promoting G1/S phase transition; it forms a complex with CRK2 in controlling the posterior morphogenesis during G1/S transition; and it forms a complex with CRK3 in promoting passage across the G2/M checkpoint in the trypanosome.     

Abrogation of growth arrest signals by human papillomavirus type 16 E7 is mediated by sequences required for transformation.

Cells arrest in the G1 or G0 phase of the cell cycle in response to a variety of negative growth signals that induce arrest by different molecular pathways. The ability of human papillomavirus (HPV) oncogenes to bypass these signals and allow cells to progress into the S phase probably contributes to the neoplastic potential of the virus. The E7 protein of HPV-16 was able to disrupt the response of epithelial cells to three different negative growth arrest signals: quiescence imposed upon suprabasal epithelial cells, G1 arrest induced by DNA damage, and inhibition of DNA synthesis caused by treatment with transforming growth factor beta. The same set of mutated E7 proteins was able to abrogate all three growth arrest signals. Mutant proteins that failed to abrogate growth arrest signals were transformation deficient and included E7 proteins that bound retinoblastoma protein in vitro. In contrast, HPV-16 E6 was able to bypass only DNA damage-induced G1 arrest, not suprabasal quiescence or transforming growth factor beta-induced arrest. The E6 and E7 proteins from the low-risk virus HPV-6 were not able to bypass any of the growth arrest signals.     

Conditionally activated E7 proteins of high-risk and low-risk human papillomaviruses induce S phase in postmitotic, differentiated human keratinocytes

The productive program of human papillomaviruses (HPVs) in epithelia is tightly linked to squamous differentiation. The E7 proteins of high-risk HPV genotypes efficiently inactivate the pRB family of proteins that control the cell cycle, triggering S phase in suprabasal keratinocytes. This ability has until now not been demonstrated for the low-risk HPV-6 or HPV-11 E7 proteins. An inducible system in which HPV-16 E7 is fused to the ligand binding domain of the human estrogen receptor (ER) was described by Smith-McCune et al. (K. Smith-McCune, D. Kalman, C. Robbins, S. Shivakumar, L. Yuschenkoff, and J. M. Bishop, Proc. Natl. Acad. Sci. USA 96:6999-7004, 1999). In the absence of hormone, E7ER is cytoplasmic, and upon addition of 17beta-estradiol, it translocates to the nucleus. Using organotypic epithelial raft cultures developed from primary human keratinocytes, we show that 16E7ER promotes either S-phase reentry or p21cip1 accumulation in differentiated keratinocytes in a stochastic manner as early as 6 h postinduction with 17beta-estradiol. A vector expressing the ER moiety alone had no effect. These observations prove unequivocally that the E7 protein drives S-phase reentry in postmitotic, differentiated keratinocytes rather than preventing S-phase exit while the cells ascend through the epithelium. HPV-11 E7ER and, much less efficiently, HPV-6 E7ER also promoted S-phase reentry by differentiated cells upon exposure to 17beta-estradiol. S-phase induction required the consensus pRB binding motif. We propose that the elevated nuclear levels of the low-risk HPV E7 protein afforded by the inducible system account for the positive results. These observations are entirely consistent with the fact that low-risk HPV genotypes replicate in the differentiated strata in patient specimens, as do the high-risk HPVs.     

Human papillomavirus type 16 mutant E7 protein induces oncogenic transformation via up-regulation of cyclin A and cdc25A

A new mutant human papiUomavirus type 16 E7 gene, termed HPV16 HBE7, was isolated from cervical carcinoma biopsy samples from patients in an area with high incidence of cervical cancer (Hubei province, China). A previous study showed that the HPVI6 HBE7 protein was primarily cytoplasmic while wild-type HPV16 E7 protein, termed HPV16 WET, was concentrated in the nucleus. With the aim of studying the biological functions of HPV16 HBE7, the transforming potential of HPV16 HBE7 in NIH/3T3 cells was detected through observation of cell morphology, cell proliferation assay and anchorage-independent growth assay. The effect of HPVI6 HBE7 on cell cycle was examined by flow cytometry. Dual-luciferase reporter assay and RT-PCR were used to investigate the influence of HPVI6 HBE7 protein on the expression of regulation factors associated with GI/S checkpoint. The results showed that HPV16 HBE7 protein, as well as HPV16 WE7 protein, held transformation activity. NIH/3T3 cells expressing HPV16 HBE7 could easily transition from G1 phase into S phase and expressed high level of cyclin A and cdc25A. These results indicated HPV16 mutant E7 protein, located in the cytoplasm, induces oncogenic transformation of NIH/3T3 cells via up-regulation of cyclin A and cdc25A.     

Recognition of phosphodegron motifs in human cyclin E by the SCF(Fbw7) ubiquitin ligase

Turnover of cyclin E is controlled by SCF(Fbw7). Three isoforms of Fbw7 are produced by alternative splicing. Whereas Fbw7alpha and -gamma are nuclear and the beta-isoform is cytoplasmic in 293T cells, all three isoforms induce cyclin E destruction in an in vivo degradation assay. Cyclin E is phosphorylated on Thr(62), Ser(88), Ser(372), Thr(380), and Ser(384) in vivo. To examine the roles of phosphorylation in cyclin E turnover, a series of alanine point mutations in each of these sites were analyzed for Fbw7-driven degradation. As expected, mutation of the previously characterized residue Thr(380) to alanine led to profound defects of cyclin E turnover, and largely abolished association with Fbw7. Mutation of Thr(62) to alanine led to a dramatic reduction in the extent of Thr(380) phosphorylation, suggesting an indirect effect of this mutation on cyclin E turnover. Nevertheless, phosphopeptides centered at Thr(62) associated with Fbw7, and residual binding of cyclin E(T380A) to Fbw7 was abolished upon mutation of Thr(62), suggesting a minor role for this residue in direct association with Fbw7. Mutation of Ser(384) to alanine also rendered cyclin E resistant to degradation by Fbw7, with the largest effects being observed with Fbw7beta. Cyclin E(S384A) associated more weakly with Fbw7alpha and -beta isoforms but was not defective in Thr(380) phosphorylation. Analysis of the localization of cyclin E mutant proteins indicated selective accumulation of cyclin E(S384A) in the nucleus, which may contribute to the inability of cytoplasmic Fbw7beta to promote turnover of this cyclin E mutant protein.     

Geminin is bound to chromatin in G2/M phase to promote proper cytokinesis

Previous studies suggested that geminin plays a vital role in both origin assembly and DNA re-replication during S-phase; however, no data to support a role for geminin in G2/M cells have been described. Here it is shown that in G2/M-phase, geminin participates in the promotion of proper cytokinesis. This claim can be supported through a series of observations. First, geminin in G2/M is loaded onto chromatin after it is tyrosine phosphorylated. It is unlike S-phase geminin that resides in the nuclear soluble fraction, where it is exclusively S/T phosphorylated. Secondly, on chromatin, geminin gets S/T phosphorylated in late G1; this modification causes the release of geminin from the chromatin. Cyclins bind and phosphorylate geminin in a sequential, cell cycle-dependent manner. These modifications correlated well with geminin departure from the chromatin. This suggests that cyclin functions to either release geminin from chromatin or at least keep it at bay until late S-phase. Thirdly, depletion of geminin from a diploid mammary epithelial cell line (HME) causes cells to arrest in late G2/M-phase. Massive serine-10 phosphorylated histone H3 staining and survivin localization to mid-body were observed; this suggests that they could be arrested in either mitosis or at cytokinesis. Finally, while in the absence of geminin, cyclin B1, chk1 and cdc7 are all over expressed. This paper will demonstrate that only cdc7 is important in maintaining the cytokinesis arrest in the absence of geminin. Only double depletion of geminin and cdc7 induce apoptosis. Our results taken together show, for the first time, that phosphorylation-induction activates oscillation of geminin between both nuclear soluble and chromatin compartments. Chromatin-bound geminin species functions to initiate or maintain proper cytokineses. In the absence of geminin, cells arrest in cytokinesis; this defines a novel checkpoint, monitored by cdc7, rather than cyclin B1 or chk1.     

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.     

Analysis of the p53-mediated G1 growth arrest pathway in cells expressing the human papillomavirus type 16 E7 oncoprotein.

Cells expressing human papillomavirus type 16 (HPV-16) E7, similar to those which express HPV-16 E6, are resistant to a p53-mediated G1 growth arrest. We examined the p53-mediated DNA damage response pathway in E7-expressing cells to determine the mechanism by which E7-containing cells continue to cycle. In response to DNA damage, no dramatic difference was detected in G1- or S-phase cyclin or cyclin-dependent kinase (Cdk) levels when E7-expressing cells were compared to the parental cell line, RKO. Furthermore, Cdk2 kinase activity was inhibited in both RKO cells and E7-expressing cells, while Cdk2 remained active in E6-expressing cells. However, the steady-state levels of pRB and p107 protein were substantially lower in E7-expressing cells than in the parental RKO cells or E6-expressing cells. There was no reduction in pRB mRNA levels, but the half-life of pRB in E7-expressing cells was markedly shorter. Infection of primary human foreskin keratinocytes with recombinant retroviruses expressing HPV-16 E7 resulted in a decrease in pRB protein levels, indicating this phenomenon is a consequence of E7 expression, not of immortalization or transformation. These data strongly suggest E7 interferes with the stability of pRB and p107 protein. We propose that the removal of these components of the p53-mediated G1 growth arrest pathway in E7-expressing cells contributes to the ability of E7 to overcome a p53-mediated G1 growth arrest.     

The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2.

Cyclin D-Cdk4/6 and cyclin A/E-Cdk2 are suggested to be involved in phosphorylation of the retinoblastoma protein (pRB) during the G1/S transition of the cell cycle. However, it is unclear why several Cdks are needed and how they are different from one another. We found that the consensus amino acid sequence for phosphorylation by cyclin D1-Cdk4 is different from S/T-P-X-K/R, which is the consensus sequence for phosphorylation by cyclin A/E-Cdk2 using various synthetic peptides as substrates. Cyclin D1-Cdk4 efficiently phosphorylated the G1 peptide, RPPTLS780PIPHIPR that contained a part of the sequence of pRB, while cyclins E-Cdk2 and A-Cdk2 did not. To determine the phosphorylation state of pRB in vitro and in vivo, we raised the specific antibody against phospho-Ser780 in pRB. We confirmed that cyclin D1-Cdk4, but not cyclin E-Cdk2, phosphorylated Ser780 in recombinant pRB. The Ser780 in pRB was phosphorylated in the G1 phase in a cell cycle-dependent manner. Furthermore, we found that pRB phosphorylated at Ser780 cannot bind to E2F-1 in vivo. Our data show that cyclin D1-Cdk4 and cyclin A/E Cdk2 phosphorylate different sites of pRB in vivo.     

Human Parvovirus B19 Induces Cell Cycle Arrest at G2 Phase with Accumulation of Mitotic Cyclins

Human parvovirus B19 infects specifically erythroid progenitor cells, which causes transient aplastic crises and hemolytic anemias. Here, we demonstrate that erythroblastoid UT7/Epo cells infected with B19 virus fall into growth arrest with 4N DNA, indicating G(2)/M arrest. These B19 virus-infected cells displayed accumulation of cyclin A, cyclin B1, and phosphorylated cdc2 and were accompanied by an up-regulation in the kinase activity of the cdc2-cyclin B1 complex, similar to that in cells treated with the mitotic inhibitor. However, degradation of nuclear lamina and phosphorylation of histone H3 and H1 were not seen in B19 virus-infected cells, indicating that the infected cells do not enter the M phase. Accumulation of cyclin B1 was persistently localized in the cytoplasm, but not in the nucleus, suggesting that B19 virus infection of erythroid cells raises suppression of nuclear import of cyclin B1, resulting in cell cycle arrest at the G(2) phase. The B19 virus-induced G(2)/M arrest may be the critical event in the damage of erythroid progenitor cells seen in patients with B19 virus infection.     

Casein kinase II phosphorylates p34cdc2 kinase in G1 phase of the HeLa cell division cycle.

The activity of p34cdc2 kinase is regulated in the phases of vertebrate cell cycle by mechanisms of phosphorylation and dephosphorylation. In this paper, we demonstrate that casein kinase II (CKII) phosphorylates p34cdc2 in vivo and in vitro at Ser39 during the G1 phase of HeLa cell division cycle. Human p34cdc2 shows a typical phosphorylation sequence motif site for CKII at Ser39 (ES39EEE). In our experiments, either p34cdc2 expressed and purified from bacteria or p34cdc2 immunoprecipitated from HeLa cells enriched in G1 by elutriation were substrates for in vitro phosphorylation by CKII. Phosphoamino acid analysis, N-chlorosuccinimide mapping, and two-dimensional tryptic mapping of p34cdc2 phosphorylated in vitro were performed to determine the phosphorylation site. A synthetic peptide spanning residues 33-50 of human p34cdc2, including the CKII site, was used to map the site. In addition, phosphorylation at Ser39 also occurs in vivo, since p34cdc2 is phosphorylated during G1 on serine, and its two-dimensional tryptic map shows two phosphopeptides that comigrate exactly with the synthetic peptides used as standard.     

SAPK/JNK regulates cdc2/cyclin B kinase through phosphorylation and inhibition of cdc25c

Cells undergo M phase arrest in response to stresses like UV irradiation or DNA damage. Stress-activated protein kinase (SAPK, also known as c-Jun N-terminal kinase, JNK) is activated by such stress stimuli. We addressed the potential effects of SAPK activation on cell cycle regulatory proteins. Activation of SAPK strongly correlated with inhibition of cdc2/cyclin B kinase, an important regulator of G2/M phase. SAPK directly phosphorylated the cdc2 regulator, cdc25c, in vitro on serine 168 (S168). This residue was highly phosphorylated in vivo in response to stress stimuli. cdc25c phosphorylated on S168 in cells lacks phosphatase activity, and expression of a S168A mutant of cdc25c reversed the inhibition of cdc2/cyclin B kinase activity by cell stress. Antibodies directed against phosphorylated S168 detect increased phosphorylation of S168 after cell stress. We conclude that SAPK regulates cdc2/cyclin B kinase following stress events by a novel mechanism involving inhibitory phosphorylation of the cdc2-activating phosphatase cdc25c on S168.     

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.     

The human polo-like kinase, PLK, regulates cdc2/cyclin B through phosphorylation and activation of the cdc25C phosphatase

Entry into mitosis by mammalian cells is triggered by the activation of the cdc2/cyclin B holoenzyme. This is accomplished by the specific dephosphorylation of key residues by the cdc25C phosphatase. The polo-like kinases are a family of serine/threonine kinases which are also implicated in the control of mitotic events, but their exact regulatory mechanism is not known. Recently, a Xenopus homologue, PLX1, was reported to phosphorylate and activate cdc25, leading to activation of cdc2/cyclin B. Jurkat T leukemia cells were chemically arrested and used to verify that PLK protein expression and its phosphorylation state is regulated with respect to cell cycle phase (i.e., protein is undetectable at G1/S, accumulates at S phase and is modified at G2/M). Herein, we show for the first time that endogenous human PLK protein immunoprecipitated from the G2/M-arrested Jurkat cells directly phosphorylates human cdc25C. In addition, we demonstrate that recombinant human (rh) PLK also phosphorylates rhcdc25C in a time- and concentration-dependent manner. Phosphorylation of endogenous cdc25C and recombinant cdc25C by PLK resulted in the activation of the phosphatase as assessed by dephosphorylation of cdc2/cyclin B. These data are the first to demonstrate that human PLK is capable of phosphorylating and positively regulating human cdc25C activity, allowing cdc25C to dephosphorylate inactive cdc2/cyclin B. As this event is required for cell cycle progression, we define at least one key regulatory mode of action for human PLK in the initiation of mitosis.     

Autophosphorylated residues involved in the regulation of human chk2 in vitro

Human checkpoint kinase 2 is a major actor in checkpoint activation through phosphorylation by ataxia telangiectasia mutated in response to DNA double-strand breaks. In the absence of de novo DNA damage, its autoactivation, reported in the event of increased Cds1/checkpoint kinase 2 (Chk2) expression, has been attributed to oligomerization. Here we report a study performed on autoactivated recombinant Chk2 proteins that aims to correlate kinase activity and phosphorylation status. Using a fluorescence-based technique to assay human checkpoint kinase 2 catalytic activity, slight differences in the ability to phosphorylate Cdc25C were observed, depending on the recombinant system used. Using mass spectrometry, the phosphorylation sites were mapped to identify sites potentially involved in the kinase activity. Five phosphorylated positions, at Ser120, Ser260, Thr225, Ser379 and Ser435, were found to be common to bacteria and insect cells expression systems. They were present in addition to the six known phosphorylation sites induced by ionizing radiation (Thr68, Thr432, Thr387, Ser516, Ser33/35 and Ser19) detected by immunoblotting. After phosphatase treatment, Chk2 regained activity via autorephosphorylation. The determination of the five common sites and ionizing-radiation-inducible positions as rephosphorylated confirms that they are potential positive regulators of Chk2 kinase activity. For Escherichia coli's most highly phosphorylated 6His-Chk2, 13 additional phosphorylation sites were assigned, including 7 novel sites on top of recently reported phosphorylation sites.