Cdc25M2 activation of cyclin-dependent kinases by dephosphorylation of threonine-14 and tyrosine-15

Recent evidence has suggested that human cyclin-dependent kinase 2 (CDK2) is an essential regulator of cell cycle progression through S phase. CDK2 is known to complex with at least two distinct human cyclins, E and A. The kinase activity of these complexes peaks in G1 and S phase, respectively. The vertebrate CDC2/cyclin B1 complex is an essential regulator of the onset of mitosis and is inhibited by phosphorylation of CDC2 on Thr-14 and Tyr-15. In vitro, CDC2/cyclin B1 is activated by treatment with the members of the Cdc25 family of phosphatases. We found that, like CDC2, CDK2 is also phosphorylated on Thr-14 and Tyr-15 and that treatment of cyclin A or cyclin E immunoprecipitates with bacterially expressed Cdc25M2 (the mouse homolog of human CDC25B) increased the histone H1 kinase activity of these immune complexes 5- to 10-fold. Tryptic peptide mapping demonstrated that Cdc25M2 treatment of cyclin A or cyclin B1 immune complexes resulted in the specific dephosphorylation of Thr-14 and Tyr-15 on CDK2 or CDC2, respectively. Thus, we have confirmed that Cdc25 family members comprise a class of dual-specificity phosphatases. Furthermore, our data suggest that the phosphorylation and dephosphorylation of CDKs on Thr-14 and Tyr-15 may regulate not only the G2/M transition but also other transitions in the cell cycle and that individual cdc25 family members may regulate distinct cell cycle checkpoints.     

Control of cell division in eucaryotes

In eucaryotes, M-phase promoting factor (MPF) triggers meiosis in germ cells and mitosis in somatic cells. MPF is composed of two proteins of which one is homologous with the protein kinase encoded by gene cdc2 of Schizosaccharomyces pombe (p34cdc2) and the other is a cyclin whose concentration oscillates during the cell cycle. Inactivation of p34cdc2 (MPF) requires cyclin degradation, which occurs during the metaphase-anaphase transition of the M-phase. Cyclin degradation is not only associated with cell cycle progression, but is also required for this event. At the G2/M transition, p34cdc2 protein kinase is activated and catalyzes phosphorylation of numerous key proteins, thus enabling cell changes to occur. p34cdc2 undergoes multiple-site phosphorylation in a cell cycle-dependent manner. At onset of mitosis, the protein phosphatase cdc25 catalyzes dephosphorylation of the p34cdc2 kinase at the threonine 14 and tyrosine 15 sites. This event may be the rate-limiting step controlling onset of mitosis in cells of vertebrates. A second protein kinase, encoded by the proto-oncogene c-mos, acts as a cytostatic factor preventing cyclin degradation and keeping unfertilized eggs from progressing beyond the second meiotic metaphase.     

Phosphorylation of the regulatory subunit of type II beta cAMP-dependent protein kinase by cyclin B/p34cdc2 kinase impairs its binding to microtubule-associated protein 2

Subcellular localization of type II cAMP-dependent protein kinase is determined by the interactions of the regulatory subunit (RII) with specific RII-anchoring proteins. By using truncated NH2-terminal RII beta fusion proteins expressed in Escherichia coli and the mitotic protein kinase p34cdc2 isolated from HeLa cells or starfish oocytes, we investigated the in vitro phosphorylation of RII beta by these kinases. The putative site for phosphorylation by the mitotic kinases is Thr-69 in the NH2-terminal domain of RII beta. This phosphorylation site matches the consensus sequence X(T/S)PX(K/R) for p34cdc2 recognition and belongs to a well-conserved sequence found in all RII beta sequences known to date. In contrast to phosphorylation by casein kinase II or the cAMP-dependent protein kinase catalytic subunit, phosphorylation of RII beta by mitotic kinases impaired its interaction with a well-known RII-anchoring protein, the neuronal microtubule-associated protein 2. The potential regulatory significance of the phosphorylation of this site on the interaction with microtubule-associated protein 2 and other RII-anchoring proteins and the physiological relevance of this cyclin B/p34cdc2 kinase-catalyzed modification of RII beta (or phosphorylation by other proline-directed protein kinases) are discussed.     

The fission yeast SPB component Cut12 links bipolar spindle formation to mitotic control

During fission yeast mitosis, the duplicated spindle pole bodies (SPBs) nucleate microtubule arrays that interdigitate to form the mitotic spindle. cut12.1 mutants form a monopolar mitotic spindle, chromosome segregation fails, and the mutant undergoes a lethal cytokinesis. The cut12(+) gene encodes a novel 62-kD protein with two predicted coiled coil regions, and one consensus phosphorylation site for p34(cdc2) and two for MAP kinase. Cut12 is localized to the SPB throughout the cell cycle, predominantly around the inner face of the interphase SPB, adjacent to the nucleus. cut12(+) is allelic to stf1(+); stf1.1 is a gain-of-function mutation bypassing the requirement for the Cdc25 tyrosine phosphatase, which normally dephosphorylates and activates the p34(cdc2)/cyclin B kinase to promote the onset of mitosis. Expressing a cut12(+) cDNA carrying the stf1.1 mutation also suppressed cdc25.22. The spindle defect in cut12.1 is exacerbated by the cdc25.22 mutation, and stf1.1 cells formed defective spindles in a cdc25.22 background at high temperatures. We propose that Cut12 may be a regulator or substrate of the p34(cdc2) mitotic kinase.     

A dual-specificity phosphatase Cdc25B is an unstable protein and triggers p34(cdc2)/cyclin B activation in hamster BHK21 cells arrested with hydroxyurea

By incubating at 30 degrees C in the presence of an energy source, p34(cdc2)/cyclin B was activated in the extract prepared from a temperature-sensitive mutant, tsBN2, which prematurely enters mitosis at 40 degrees C, the nonpermissive temperature (Nishimoto, T. , E. Eilen, and C. Basilico. 1978. Cell. 15:475-483), and wild-type cells of the hamster BHK21 cell line arrested in S phase, without protein synthesis. Such an in vitro activation of p34(cdc2)/cyclin B, however, did not occur in the extract prepared from cells pretreated with protein synthesis inhibitor cycloheximide, although this extract still retained the ability to inhibit p34(cdc2)/cyclin B activation. When tsBN2 cells arrested in S phase were incubated at 40 degrees C in the presence of cycloheximide, Cdc25B, but not Cdc25A and C, among a family of dual-specificity phosphatases, Cdc25, was lost coincidentally with the lack of the activation of p34(cdc2)/cyclin B. Consistently, the immunodepletion of Cdc25B from the extract inhibited the activation of p34(cdc2)/cyclin B. Cdc25B was found to be unstable (half-life < 30 min). Cdc25B, but not Cdc25C, immunoprecipitated from the extract directly activated the p34(cdc2)/cyclin B of cycloheximide-treated cells as well as that of nontreated cells, although Cdc25C immunoprecipitated from the extract of mitotic cells activated the p34(cdc2)/cyclin B within the extract of cycloheximide-treated cells. Our data suggest that Cdc25B made an initial activation of p34(cdc2)/cyclin B, which initiates mitosis through the activation of Cdc25C.     

The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2

Activation of the cyclin-dependent protein kinases p34cdc2 and p33cdk2 requires binding with a cyclin partner and phosphorylation on the first threonine residue in the sequence THEVVTLWYRAPE. We present evidence that this threonine residue, number 160 in p33cdk2, can be specifically phosphorylated by a cdc2-related protein kinase from Xenopus oocytes called p40MO15. Binding to cyclin A and phosphorylation of this threonine are both required to activate fully the histone H1 kinase activity of p33cdk2. In cell extracts, a portion of p40MO15 is found in a high molecular weight complex that is considerably more active than a lower molecular weight form. Wild-type MO15 protein expressed in bacteria does not possess kinase activity, but acquires p33cdk2-T160 kinase activity after incubation with cell extract and ATP. We conclude that p40MO15 corresponds to CAK (cdc2/cdk2 activating kinase) and speculate that, like p33cdk2 and p34cdc2, p40MO15 requires activation by phosphorylation and association with a companion subunit.     

A rate limiting function of cdc25A for S phase entry inversely correlates with tyrosine dephosphorylation of Cdk2

The cdc25A phosphatase removes inhibitory phosphates from threonine-14 and tyrosine-15 of cyclin dependent kinase-2 (cdk2) in vitro, and it is therefore widely assumed that cdc25A positively regulates cyclin E- and A-associated cdk2 activity at the G1 to S phase transition of the mammalian cell division cycle. Human cdc25A was introduced into mouse NIH3T3 fibroblasts co-expressing a form of the colony-stimulating factor-1 (CSF-1) receptor that is partially defective in transducing mitogenic signals. Cdc25A enabled these cells to form colonies in semisolid medium containing serum plus human recombinant CSF-1 in a manner reminiscent of cells rescued by c-myc. However, cdc25A-rescued cells could not proliferate in chemically defined medium containing CSF-1 and continued to require c-myc function for S phase entry. When contact-inhibited cells overexpressing cdc25A were dispersed and stimulated to synchronously enter the cell division cycle, they entered S phase 2-3 h earlier than their parental untransfected counterparts. Shortening of G1 phase temporally correlated with more rapid degradation of the cdk inhibitor p27Kip1 and with premature activation of cyclin A-dependent cdk2. Paradoxically, tyrosine phosphorylation of cdk2 increased considerably as cells entered S phase, and cdc25A overexpression potentiated rather than diminished this effect. At face value, these results are inconsistent with the hypothesis that cdc25A acts directly on cdk2 to activate its S phase promoting function.     

Cell cycle-dependent cytotoxicity, G2/M phase arrest, and disruption of p34cdc2/cyclin B1 activity induced by doxorubicin in synchronized P388 cells

We studied the effect of doxorubicin (Dox) on cell cycle progression and its correlation with DNA damage and cytotoxicity in p53-mutant P388 cells. P388 cells synchronized in S and G2/M phases were > 3-fold more sensitive to Dox than were cells in G1 phase (Dox ID50 = 0.50 +/- 0.16 microM in cells synchronized in S phase versus 1.64 +/- 0.12 microM in asynchronized cells; drug exposure, 1 hr). Treatment of synchronized cells in early S phase with 1 microM Dox (2 x ID50) for 1 hr induced a marked cell arrest at G2/M phase at 6-12 hr after drug incubation. We then studied the effect of Dox on the p34cdc2/cyclin B1 complex because it plays a key role in regulating G2/M phase transition. In untreated control P388 cells, p34cdc2 kinase localizes in the nucleus and cytoplasms, particularly in the centrosomes, and p34cdc2 kinase activity is dependent on cell cycle progression, with the enzyme activity increasing steadily from G1/S to G2/M and markedly declining thereafter. Treatment of synchronized P388 cells in early S phase with 1 microM Dox for 1 hr did not affect the pattern of subcellular distribution of the enzyme but completely abrogated its function for > or = 10 hr. In a cell-free system, Dox did not inhibit p34cdc2 kinase activity, indicating that is has no direct effect on the enzyme function. In whole cells, Dox treatment prevented p34cdc2 kinase dephosphorylation without altering its synthesis, and this effect was due to neither down-regulation of cdc25C nor inhibition of protein-tyrosine phosphatase activity. In contrast, Dox treatment was found to induced cyclin B1 accumulation as a result of stimulating its synthesis and inhibiting its degradation. A good correlation was found between extent of DNA double-strand breaks and p34cdc2 kinase activity inhibition. Our results suggest that anthracycline-induced cytotoxicity is cell cycle dependent and is mediated, at least in part, by disturbance of the regulation of p34cdc2/cyclin B1 complex, thus leading to G2/M phase arrest.     

Characterization of the in vitro reconstituted cyclin A or B1-dependent cdk2 and cdc2 kinase activities

Human cyclins A and B1 were assembled with the cdk2 or cdc2 protein to reconstitute their respective kinase activities in vitro. Both cyclins complemented either cdk2 or cdc2, yielding kinase activities that supported the phosphorylation of histone H1. Activation of cdk2-catalyzed H1 kinase activity by cyclin A required a 10-min preincubation of the two components, whereas cdc2 kinase supported phosphate incorporation without a detectable time lag upon the addition of cyclin B1, suggesting a slower association rate of cdk2 with cyclin A compared with cdc2 and cyclin B1. Both cdk2 and cyclin A, as well as cdc2 and cyclin B1, formed stable complexes in the absence of ATP and substrate that could be isolated after glycerol gradient centrifugation. Incubation of the isolated complexes with ATP and histone H1 supported the phosphorylation of the substrate. Cyclin A-activated cdk2 or cdc2 phosphorylated p107, a pRB-related cellular protein, 10 times more effectively than the cyclin B1-complexed kinases. This was most likely due to a direct association of cyclin A with p107 (Ewen, M. E., Faha, B., Harlow, E., and Livingston, D. (1992) Science 255, 85-87; Faha, B., Ewen, M. E., Tsai, L.-H., Livingston, D., and Harlow, E. (1992) Science 255, 87-90). The reconstituted cdc2-cyclin B1 complex incorporated 4-5-fold more phosphate into the p34 subunit of the three-subunit (p70, p34, and p14) human single-stranded DNA-binding protein (also called RP-A), a DNA replication and DNA repair factor, than cdc2-cyclin A. No detectable phosphorylation of the p34 protein was observed with cdk2 complexed with either cyclin B1 or A. These data indicate that both cyclins as well as the catalytic subunits are important factors in controlling the rate of phosphorylation of a given substrate. The cyclin-activated cdc2 family kinases may target their cellular substrates through cyclin-mediated protein-protein interactions.     

Effects of phosphate on in vitro 2-cell block of AKR/N mouse embryos based on changes in cdc2 kinase activity and phosphorylation states

This study demonstrated the effects of phosphate on the 2-cell block of AKR/N mouse embryos at the molecular level and focused on changes in the kinase activity and the phosphorylation state of cdc2, which is shown to regulate the cell division cycle. Removal of phosphate from the culture medium dramatically increased developmental rates to the 4-cell (91.8%) and blastocyst (42.6%) stages compared with those of embryos cultured in 1.17 mM phosphate (3.3% and 0%, respectively). The rate of development to the 4-cell stage was significantly inhibited by 0.001 mM phosphate (p < 0.05), and no morula formation was observed at 1.0 mM. The patterns of cdc2 kinase activity during the first cell cycle in AKR/N embryos were similar to those of control MCH embryos, showing the highest activity at M phase and low activity during the interphase. The phosphorylated form of cdc2 increased during the interphase, indicating that the synthesis of cyclin B and accumulation of inactive pre-maturation-promoting factor (pre-MPF) as well as abrupt dephosphorylation of cdc2 at the first cleavage correlated with the activation of cdc2 kinase. When phosphate was absent, the activation pattern of cdc2 kinase during the second cell cycle in AKR/N embryos was similar to that in the first cell cycle. On the other hand, no dephosphorylation of cdc2 was observed and the kinase activity remained at a low level until 56 h after insemination in the presence of phosphate, although an increase in phosphorylated cdc2 was observed as in the phosphate-free group. Treatment of AKR/N embryos arrested at the 2-cell stage with okadaic acid resulted in the dephosphorylation and activation of cdc2, confirming the presence of a sufficient amount of pre-MPF. These results show that phosphate has a deteriorative effect on the in vitro development of AKR/N embryos and suggest that this effect was not on the synthesis of cyclin B but on the dephosphorylation of phosphorylated cdc2.     

Proteolysis and tyrosine phosphorylation of p34cdc2/cyclin B. The role of MCM2 and initiation of DNA replication to allow tyrosine phosphorylation of p34cdc2

Previously, it has been shown that Aspergillus cells lacking the function of nimQ and the anaphase-promoting complex (APC) component bimEAPC1 enter mitosis without replicating DNA. Here nimQ is shown to encode an MCM2 homologue. Although mutation of nimQMCM2 inhibits initiation of DNA replication, a few cells do enter mitosis. Cells arrested at G1/S by lack of nimQMCM2 contain p34(cdc2)/cyclin B, but p34(cdc2) remains tyrosine dephosphorylated, even after DNA damage. However, arrest of DNA replication using hydroxyurea followed by inactivation of nimQMCM2 and bimEAPC1 does not abrogate the S phase arrest checkpoint over mitosis. nimQMCM2, likely via initiation of DNA replication, is therefore required to trigger tyrosine phosphorylation of p34(cdc2) during the G1 to S transition, which may occur by inactivation of nimTcdc25. Cells lacking both nimQMCM2 and bimEAPC1 are deficient in the S phase arrest checkpoint over mitosis because they lack both tyrosine phosphorylation of p34(cdc2) and the function of bimEAPC1. Initiation of DNA replication, which requires nimQMCM2, is apparently critical to switch mitotic regulation from the APC to include tyrosine phosphorylation of p34(cdc2) at G1/S. We also show that cells arrested at G1/S due to lack of nimQMCM2 continue to replicate spindle pole bodies in the absence of DNA replication and can undergo anaphase in the absence of APC function.     

The G2/M DNA damage checkpoint inhibits mitosis through Tyr15 phosphorylation of p34cdc2 in Aspergillus nidulans

It is possible to cause G2 arrest in Aspergillus nidulans by inactivating either p34cdc2 or NIMA. We therefore investigated the negative control of these two mitosis-promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DNA. However, non-dividing quiescent conidiospores of the Tyr15 mutant strain were not sensitive to DNA damage. The UV and MMS sensitivity of cells unable to tyrosine phosphorylate p34cdc2 is therefore caused by defects in DNA damage checkpoint regulation over mitosis. Both the nimA5 and nimT23 temperature-sensitive mutations cause an arrest in G2 at 42 degrees C. Addition of MMS to nimT23 G2-arrested cells caused a marked delay in their entry into mitosis upon downshift to 32 degrees C and this delay was correlated with a long delay in the dephosphorylation and activation of p34cdc2. Addition of MMS to nimA5 G2-arrested cells caused inactivation of the H1 kinase activity of p34cdc2 due to an increase in its Tyr15 phosphorylation level and delayed entry into mitosis upon return to 32 degrees C. However, if Tyr15 phosphorylation of p34cdc2 was prevented then its H1 kinase activity was not inactivated upon MMS addition to nimA5 G2-arrested cells and they rapidly progressed into a lethal mitosis upon release to 32 degrees C. Thus, Tyr15 phosphorylation of p34cdc2 in G2 arrests initiation of mitosis after DNA damage in A. nidulans.     

Activation of CDC 25 phosphatase and CDC 2 kinase involved in GL331-induced apoptosis

CDC 25 is a dual phosphatase responsible for dephosphorylation and, thus, activation of CDC 2 kinase in G2. Abnormal activation of cyclin B-associated CDC 2 kinase has been implicated in apoptosis induced by cancer chemotherapeutic agents such as paclitaxel (Taxol) and etoposide (VP-16). In this study, we found that the CDC 2 kinase could be transiently activated when nasopharyngeal carcinoma NPC-TW01 cells were treated for 3 h with a new anticancer agent, GL331. GL331 treatment also induced a concomitant increase in CDC 25A phosphatase activity and a reduced level of Tyr-15-phosphorylated CDC 2 in NPC-TW01 cells. Furthermore, subsequent apoptotic DNA fragmentation induced by GL331 could be interrupted by treatment of the cells with the cyclin B1-specific antisense oligonucleotides, suggesting that abnormal activation of cyclin B1-associated CDC 2 kinase and CDC 25A phosphatase was involved in GL331-induced apoptosis. Raf-1 has been shown to associate with CDC 25A and, thus, to stimulate its phosphatase activity. Our results revealed that GL331 could facilitate the association of CDC 25A with Raf-1, resulting in the cascade of CDC 25A phosphatase activation and CDC 2 kinase activation, as well as related signaling pathways, and ultimately causing apoptosis in cancer cells.     

Characterization of the interactions between human cdc25C, cdks, cyclins and cdk-cyclin complexes

We have overexpressed and purified human dual-specificity phosphatase cdc25C from a prokaryotic expression system at high levels and in a soluble, active form, and have studied and quantified its potential to interact with cdks, cyclins and preformed cdk-cyclin complexes by fluorescence spectroscopy and size-exclusion chromatography. Our data indicate that human cdc25C forms stable complexes, through hydrophobic contacts, with cdk and cyclin monomers, as well as with preformed cdk-cyclin complexes. In vitro, cdc25C interacts with cyclin monomers with high affinity, with tenfold less affinity with cdks, and with intermediate affinity with cdk-cyclin complexes. Moreover, changes observed in the intrinsic fluorescence of cdks, cyclins and cdk-cyclin complexes upon interaction with cdc25C are indicative of concomitant conformational changes within cdks and cyclins. From our results, we propose that in vitro, in the presence of monomeric cdks and cyclins, cdc25C forms stable ternary complexes, first through a high affinity interaction with a cyclin, which may then help target cdc25C towards a cdk. We discuss the biological relevance of our results and propose that a similar, two-step mechanism of interaction between cdc25C and cdk-cyclin complexes may occur in vivo.     

Evidence for an age-related dysfunction in the antiproliferative response to transforming growth factor-beta in vascular smooth muscle cells

The p34cdc2 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 p34cdc2, as well as several of the proteins that interact with and regulate p34cdc2 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 p34cdc2 protein is entirely absent and is replaced by its human functional homologue p34CDC2. We have used this strain to analyse aspects of the function of the human p34CDC2 protein genetically. We show that the function of the human p34CDC2 protein in fission yeast cells is dependent upon the action of the protein tyrosine phosphatase p80cdc25, that it responds to altered levels of both the mitotic inhibitor p107wee1 and the p34cdc2-binding protein p13suc1, and is lethal in combination with the mutant B-type cyclin p56cdc13-117. In addition, we demonstrate that the human p34CDC2 protein is proficient for fission yeast meiosis, and examine the behaviour of two mutant p34CDC2 proteins in fission yeast.     

Hierarchical phosphorylation at N-terminal transformation-sensitive sites in c-Myc protein is regulated by mitogens and in mitosis

The N-terminal domain of the c-Myc protein has been reported to be critical for both the transactivation and biological functions of the c-Myc proteins. Through detailed phosphopeptide mapping analyses, we demonstrate that there is a cluster of four regulated and complex phosphorylation events on the N-terminal domain of Myc proteins, including Thr-58, Ser-62, and Ser-71. An apparent enhancement of Ser-62 phosphorylation occurs on v-Myc proteins having a mutation at Thr-58 which has previously been correlated with increased transforming ability. In contrast, phosphorylation of Thr-58 in cells is dependent on a prior phosphorylation of Ser-62. Hierarchical phosphorylation of c-Myc is also observed in vitro with a specific glycogen synthase kinase 3 alpha, unlike the promiscuous phosphorylation observed with other glycogen synthase kinase 3 alpha and 3 beta preparations. Although both p42 mitogen-activated protein kinase and cdc2 kinase specifically phosphorylate Ser-62 in vitro and cellular phosphorylation of Thr-58/Ser-62 is stimulated by mitogens, other in vivo experiments do not support a role for these kinases in the phosphorylation of Myc proteins. Unexpectedly, both the Thr-58 and Ser-62 phosphorylation events, but not other N-terminal phosphorylation events, can occur in the cytoplasm, suggesting that translocation of the c-Myc proteins to the nucleus is not required for phosphorylation at these sites. In addition, there appears to be an unusual block to the phosphorylation of Ser-62 during mitosis. Finally, although the enhanced transforming properties of Myc proteins correlates with the loss of phosphorylation at Thr-58 and an enhancement of Ser-62 phosphorylation, these phosphorylation events do not alter the ability of c-Myc to transactivate through the CACGTG Myc/Max binding site.     

Interrelationships between strains of Salmonella enteritidis belonging to phage types 4, 7, 7a, 8, 13, 13a, 23, 24 and 30

Cyclin A is a nuclear protein which is part of a kinase complex with either p34cdc2 or p33cdk2. Cyclin A is required in higher eukaryotic cells at the G1/S and the G2/M transitions. To examine the relationship between cyclin A and DNA replication, we simultaneously labeled exponentially growing HeLa cells for the distribution of cyclin A and proliferating cell nuclear antigen (PCNA). We have now demonstrated, by means of immunoelectron microscopy, that cyclin A is located at the sites of DNA replication visualized by both BrdU and PCNA labeling. Thus cyclin A may play a significant role in the phosphorylation of proteins at or near the sites of DNA replication.     

G1 cyclin-dependent activation of p34CDC28 (Cdc28p) in vitro

In Saccharomyces cerevisiae, transient accumulation of G1 cyclin/p34CDC28 (Cdc28p) complexes induces cells to traverse the cell cycle Start checkpoint and commit to a round of cell division. To investigate posttranslational controls that modulate Cdc28p activity during the G1 phase, we have reconstituted cyclin-dependent activation of Cdc28p in a cyclin-depleted G1 extract. A glutathione S-transferase-G1 cyclin chimera (GST-Cln2p) efficiently binds to and activates Cdc28p as a histone H1 kinase. Activation of Cdc28p by GST-Cln2p requires ATP, crude yeast cytosol, and the conserved Thr-169 residue that serves in other organisms as a substrate for phosphorylation by cyclin-dependent protein kinase-activating kinase. This assay may be useful for distinguishing genes that promote directly the posttranslational assembly of active Cln2p/Cdc28p kinase complexes from those that stimulate the accumulation of active complexes via a positive-feedback loop that governs synthesis of G1 cyclins.     

Effect of suramin on p34cdc2 kinase in vitro and in extracts from human H69 cells: evidence for a double mechanism of action

We examined the effect of suramin, an anticancer agent and a functional analog of naturally occuring glycosaminoglycans, on p34cdc2 kinase. We find that suramin strongly inhibits the catalytic activity of purified p34cdc2 kinase (IC50 approximately 4 microM), whereas it only weakly inhibits the p13-agarose precipitated kinase activity from nuclear and cytoplasmic extracts of the asynchronous H69 human small cell lung cancer cells. We also find that the tyrosine phosphorylation of p34cdc2 kinase in the nuclear extract is increased about twice when the extracts are preincubated with 50 microM of suramin prior to the p13-agarose precipitation. We propose that this increase might result from the inhibitory effect of suramin towards p34cdc2-specific tyrosine phosphatases. These results suggest both a direct and an indirect effect of suramin on p34cdc2 kinase. We also find that heparin is a potent inhibitor of purified cdc2 kinase (IC50 approximately 3.5 micrograms/ml). Therefore, glycosaminoglycans might be physiological regulators of p34cdc2 kinase in vivo.