Cyclin A in cell cycle control and cancer.

Cyclin A is particularly interesting among the cyclin family because it can activate two different cyclin-dependent kinases (CDKs) and functions in both S phase and mitosis. An embryonic form of cyclin A that is only essential for spermatogenesis is also present in some organisms. In S phase, phosphorylation of components of the DNA replication machinery such as CDC6 by cyclin A-CDK is believed to be important for initiation of DNA replication and to restrict the initiation to only once per cell cycle. In mitosis, the precise role of cyclin A is still obscure, but it may contribute to the control of cyclin B stability. Cyclin A starts to accumulate during S phase and is abruptly destroyed before metaphase. The synthesis of cyclin A is mainly controlled at the transcription level, involving E2F and other transcription factors. Removal of cyclin A is carried out by ubiquitin-mediated proteolysis, but whether the same anaphase-promoting complex/cyclosome targeting subunits are used as for cyclin B is debatable. Consistent with its role as a key cell cycle regulator, expression of cyclin A is found to be elevated in a variety of tumors.

Cleavage of cyclin A at R70/R71 by the bacterial protease OmpT.

Previous work has shown that cyclin A can be cleaved at Arg-70/Arg-71 by a proteolytic activity present in an in vitro-coupled transcription/translation system by using rabbit reticulocyte lysate programmed by plasmid DNA encoding p27(KIP1), a cyclin-dependent kinase inhibitor, but not by plasmid DNAs encoding other cyclin-dependent kinases inhibitors. Here we report that cyclin A is also cleaved by translation product programmed by plasmid DNA encoding cyclin B. Several findings indicate that the cleavage activity in this assay is provided by the bacterial protease OmpT, which cofractionates with cyclin B and p27(KIP1) plasmid DNAs and is thus carried over into the coupled in vitro transcription/translation reactions. (i) Cleavage activity appeared even when transcription or translation of the cyclin B or p27(KIP1) was blocked. (ii) Activity resembling OmpT, a serine protease that cleaves between dibasic residues, routinely copurifies with p27(KIP1) and cyclin B plasmid DNAs. (iii) Both cyclin A cleavage activity and OmpT activity are heat stable, resistant to denaturation, and inhibited by Zn(2+), Cu(2+), or benzamidine. (iv) Cyclin A cleavage activity is detected when using lysates or DNAs prepared from Escherichia coli strains that contained OmpT but not with strains lacking OmpT. (v) Purified OmpT enzyme itself cleaves cyclin A at R70/R71. These data indicate that OmpT can be present in certain DNA preparations obtained by using standard plasmid purification protocols, and its presence can potentially affect the outcome and interpretation of studies carried out using in vitro-translated proteins.

On the concentrations of cyclins and cyclin-dependent kinases in extracts of cultured human cells.

Cyclins and cyclin-dependent kinases (CDKs) are key regulators of the human cell cycle. Here we have directly measured the concentrations of the G(1) and G(2) cyclins and their CDK partners in highly synchronized human cervical carcinoma cells (HeLa). To determine the exact concentrations of cyclins and CDKs in the cell extracts, we developed a relatively simple method that combined the use of (35)S-labeled standards produced in rabbit reticulocyte lysates and immunoblotting with specific antibodies. Using this approach, we formally demonstrated that CDC2 and CDK2 are in excess of their cyclin partners. We found that the concentrations of cyclin A2 and cyclin B1 (at their peak levels in the G(2) phase) were about 30-fold less than that of their partner CDC2. The peak levels of cyclin A2 and cyclin E1, at the G(2) phase and G(1) phase, respectively, were only about 8-fold less than that of their partner CDK2. These ratios are in good agreement with size fractionation analysis of the relative amount of monomeric and complexed forms of CDC2 and CDK2 in the cell. All the cyclin A2 and cyclin E1 are in complexes with CDC2 and CDK2, but there are some indications that a significant portion of cyclin B1 may not be in complex with CDC2. Furthermore, we also demonstrated that the concentration of the CDK inhibitor p21(CIP1/WAF1) induced after DNA damage is sufficient to overcome the cyclin-CDK2 complexes in MCF-7 cells. These direct quantitations formally confirmed the long-held presumption that CDKs are in excess of the cyclins in the cell. Moreover, similar approaches can be used to measure the concentration of any protein in cell-free extracts.

Degradation of cyclin A does not require its phosphorylation by CDC2 and cyclin-dependent kinase 2.

Many cyclins are degraded by the ubiquitination/proteasome pathways involving the anaphase-promoting complex and SCF complexes. These degradations are frequently dependent on phosphorylation by cyclin-dependent kinases (CDKs), providing a self-limiting mechanism for CDK activity. Here we present evidence from in vitro and in vivo assay systems that the degradation of human cyclin A can be inhibited by kinase-inactive mutants of CDK2 and CDC2. One obvious interpretation of these results is that like other cyclins, CDK-dependent phosphorylation of the cyclin A may be involved in cyclin A degradation. Our data indicated that CDK2 can phosphorylate cyclin A on Ser-154. Site-directed mutagenesis of Ser-154 abolished the phosphorylation by recombinant CDK2 in vitro and the majority of cyclin A phosphorylation in the cell. Activation of CDK2 and binding to SKP2 or p27(KIP1) were not affected by the phosphorylation of Ser-154. Surprising, in marked contrast to cyclin E, where phosphorylation of Thr-380 by CDK2 is required for proteolysis, degradation of cyclin A was not affected by Ser-154 phosphorylation. It is likely that the stabilization of cyclin A by the kinase-inactive CDKs was mainly due to a cell cycle effect. These data suggest an important difference between the regulation of cyclin A and cyclin E.

The spindle checkpoint in the dinoflagellate Crypthecodinium cohnii.

Dinoflagellates are a major group of organisms with an extranuclear spindle. As the purpose of the spindle checkpoint is to ensure proper alignment of the chromosomes on the spindle, dinoflagellate cell cycle control may be compromised to accomodate the extranuclear spindle. In the present study, we demonstrated that nocodazole reversibly prolonged the G2 + M phase of the dinoflagellate cell cycle, in both metaphase and anaphase. The regulation of the spindle checkpoint involves the activation and inhibition of the anaphase promoting complex (APC), which in turn degrades specific cell cycle regulators in the metaphase to anaphase transition. In Crypthecodinium cohnii, nocodazole was also able to induce a prolongation of the degradation of mitotic cyclins and a delay in the inactivation of p13(suc1)-associated histone kinase activities. In addition, cell extracts prepared from C. cohnii in G1 phase and G2/M phase (or nocodazole treated) were able to activate and inhibit, respectively, the degradation of exogenous human cyclin B1 in vitro. The present study thus demonstrated the presence of the spindle checkpoint and APC-mediated cyclin degradation in dinoflagellates. This is discussed in relation to a possible role of the nuclear membrane in mitosis in dinoflagellates.

G1 versus G2 cell cycle arrest after adriamycin-induced damage in mouse Swiss3T3 cells.

Cell cycle arrest after different types of DNA damage can occur in either G1 phase or G2 phase of the cell cycle, involving the distinct mechanisms of p53/p21(Cip1/Waf1) induction, and phosphorylation of Cdc2, respectively. Treatment of asynchronously growing Swiss3T3 cells with the chemotherapeutic drug adriamycin induced a predominantly G2 cell cycle arrest. Here we investigate why Swiss3T3 cells were arrested in G2 phase and not in G1 phase after adriamycin-induced damage. We show that adriamycin was capable of inducing a G1 cell cycle arrest, both during the G0-G1 transition and during the G1 phase of the normal cell cycle. In G0 cells, adriamycin induced a prolonged cell cycle arrest. However, adriamycin caused only a transient cell cycle delay when added to cells at later time points during G0-G1 transition or at the G1 phase of normal cell cycle. The G1 arrest correlated with the induction of p53 and p21(Cip1/Waf1), and the exit from the arrest correlated with the decline of their expression. In contrast to the G1 arrest, adriamycin-induced G2 arrest was relatively tight and correlated with the Thr-14/Tyr-15 phosphorylation of cyclin B-Cdc2 complexes. The relative stringency of the G1 versus G2 cell cycle arrest may explain the predominance of G2 arrest after adriamycin treatment in mammalian cells.

MDM2 and MDMX inhibit the transcriptional activity of ectopically expressed SMAD proteins.

Transforming growth factor-beta (TGF-beta) inhibits cell proliferation in many cell types, and acquisition of TGF-beta resistance has been linked to tumorigenesis. One class of proteins that plays a key role in the TGF-beta signal transduction pathway is the SMAD protein family. MDM2, a key negative regulator of p53, has recently been shown to suppress TGF-beta-induced growth arrest in a p53-independent manner. Here we show that MDM2 and the structurally related protein MDMX can inhibit the transcriptional activity of ectopically expressed SMAD1, SMAD2, SMAD3, and SMAD4. Immunofluorescence staining indicated that ectopically expressed SMAD4 was present in both the cytoplasm and nucleus, and MDM2 and NIDMX were localized mainly to the nucleus and cytoplasm, respectively. When SMAD4 was coexpressed with either MDM2 or MDMX, nuclear accumulation of SMAD4 was strikingly inhibited. We have no evidence that SMAD4 binds directly to MDM2 or MDMX; hence, the inactivation and nuclear exclusion of SMAD4 by MDM2/MDMX may involve other indirect mechanisms.

Regulation of cyclin A-Cdk2 by SCF component Skp1 and F-box protein Skp2.

Cyclin A-Cdk2 complexes bind to Skp1 and Skp2 during S phase, but the function of Skp1 and Skp2 is unclear. Skp1, together with F-box proteins like Skp2, are part of ubiquitin-ligase E3 complexes that target many cell cycle regulators for ubiquitination-mediated proteolysis. In this study, we investigated the potential regulation of cyclin A-Cdk2 activity by Skp1 and Skp2. We found that Skp2 can inhibit the kinase activity of cyclin A-Cdk2 in vitro, both by direct inhibition of cyclin A-Cdk2 and by inhibition of the activation of Cdk2 by cyclin-dependent kinase (CDK)-activating kinase phosphorylation. Only the kinase activity of Cdk2, not of that of Cdc2 or Cdk5, is reduced by Skp2. Skp2 is phosphorylated by cyclin A-Cdk2 on residue Ser76, but nonphosphorylatable mutants of Skp2 can still inhibit the kinase activity of cyclin A-Cdk2 toward histone H1. The F box of Skp2 is required for binding to Skp1, and both the N-terminal and C-terminal regions of Skp2 are involved in binding to cyclin A-Cdk2. Furthermore, Skp2 and the CDK inhibitor p21(Cip1/WAF1) bind to cyclin A-Cdk2 in a mutually exclusive manner. Overexpression of Skp2, but not Skp1, in mammalian cells causes a G1/S cell cycle arrest.

Characterization of the cullin and F-box protein partner Skp1.

Skp1 interacts with cullins, F-box containing proteins, and forms a complex with cyclin A-Cdk2 in mammalian cells. Skp1 is also involved in diverse biological processes like degradation of key cell cycle regulators, glucose sensing, and kinetochore function. However, little is known about the structure and exact function of Skp1. Here we characterized the interaction between Skp1 and the F-box protein Skp2. We show that Skp1 can bind to Skp2 in vitro using recombinant proteins, and in vivo using the yeast two-hybrid system. Deletion analysis of Skp1 indicated that most of the Skp1 protein is required for binding to Skp2. In mammalian cell extracts, a large portion of Skp1 appears to associate with proteins other than Skp2. Biochemical analysis indicated that Skp1 is likely to be a flexible, non-spherical protein, and is capable of forming dimers.