Destruction Box Research Articles

The effects of radiation on the expression of a newly cloned and characterized rat cyclin B mRNA

Purpose : To evaluate the hypothesis that the radiation induced G2 delay in the cell cycle is associated with radiation induced effects on cyclin B expression in a rodent cell system. Methods and Materials : Two rodent, rat and Chinese hamster, cyclin B cDNAs were cloned and characterized. The two rodent species were 85% and 86% identical, respectively, when compared to the human cyclin B, indicating that they are the rodent homologues of cyclin B. 3.7 cells (rat embryo cells transformed by H-ras and v-myc) were synchronized and then irradiated. Flow cytometry and Northern blots were performed to evaluate the effects of radiation on cyclin expression in relation to phase of the cell cycle. Results : Examination of the rodent cyclin B sequences revealed only two regions with significant divergence to the human sequence, one in the lysine rich region adjacent to the cyclin destruction box, which is the putative site for ubiquitination, and one at the C terminal end. Although many of the amino acids diverged in the lysine rich region, the positions of the lysines themselves were virtually invariant suggesting their potential importance in ubiquitination. Both rodent species were also noted to have a PEST-like sequence which occurs in the human, but not in non-mammalian cyclins cloned to date and could also potentially contribute to rapid destruction. The rat and Chinese hamster mRNAs contain much longer 3′ untranslated regions than the published human sequence with multiple AUUUA and AUUU motifs which are seen in other mRNAs with rapid turnover times. This feature has not been previously found in cyclin mRNAs. In addition we have found that in the 3′ region of the rodent cDNAs we find two potential polyadenylation sites suggesting that this gene may have several transcripts. Our studies suggest that multiple mechanisms of control of mammalian cyclin B destruction exist, both at the mRNA and protein level. Evidence is also provided that the levels of rat cyclin B mRNA peaks during G2/M. Irradiation is shown to induce a G2 delay in synchronized 3.7 cells, compared to unirradiated controls, and the delay is temporally related to decreased levels of cyclin B mRNA expression. Since the G2 delay induced by ionizing radiation may contribute to the ability of cells to survive irradiation, cyclin B expression may be a key component in the determination of sensitivity or resistance to radiation therapy. Conclusion : The isolation and characterization of two rodent cyclin B's confirm that multiple mechanisms of control of mammalian cyclin B destruction exist. Our studies show that rat cyclin B expression is influenced by radiation and is temporally related to the delay in the G2 phase induced by radiation.

Isolation of a mitotic-like cyclin homologue from the protozoan Trypanosoma brucei

Activation of the p34 cdc2 protein kinase (PK) at different stages of the eukaryotic cell cycle is controlled by interaction with regulatory proteins known as cyclins (CYCs). Using a probe obtained by PCR amplification, we have isolated from the protozoan, Trypanosoma brucei, a cDNA clone encoding a CYC homologue. The amino acid sequence deduced for this gene ( CYC1) shares structural homology with A- and B-type CYCs of other organisms, including a motif, the destruction box, which has been related to the rapid turnover of these CYC proteins in mitosis. When expressed in fission yeast, CYC1 is able to rescue the defect of a temperature-sensitive cdc13 mutant, demonstrating that it is functional as a cell-cycle regulator. In trypanosome cells, CYC1 associates with a 34-kDa protein that cross-reacts with a monoclonal antibody against the conserved ‘PSTAIR’ epitope of p34 cdc2 , and the complex displays histone H1 PK activity. Furthermore, when trypanosome cells are synchronized by hydroxyurea treatment, CYC1 accumulates as cells progress towards mitosis. These observations, taken together, suggest that CYC1 is a component of the active PK complex required for the control of trypanosome mitosis.

Identification of the domains in cyclin A required for binding to, and activation of, p34cdc2 and p32cdk2 protein kinase subunits.

The binding of cyclin A to p34cdc2 and p32cdk2 and the protein kinase activity of the complexes has been measured by cell-free translation of the corresponding mRNA in extracts of frog eggs, followed by immunoprecipitation. A variety of mutant cyclin A molecules have been constructed and tested in this assay. Small deletions and point mutations of highly conserved residues in the 100-residue "cyclin box" abolish binding and activation of both p34cdc2 and p32cdk2. By contrast, large deletions at the N-terminus have no effect on kinase binding and activation, until they remove residues beyond 161, where the first conserved amino acids are found in all known examples of cyclin A. At the C-terminus, removal of 14 or more amino acids abolishes activity. We also demonstrate that deletion of, or point mutations, in the cyclin A homologue of the 10-residue "destruction box," previously described in cyclin B (Glotzer et al., 1991) abolish cyclin proteolysis at the transition from M-phase to interphase.

Cyclin A-Cys41 does not undergo cell cycle-dependent degradation in Xenopus extracts

Truncated cyclin A and cyclin B lacking the N-terminal domain comprising the ‘destruction box’ escape from proteolysis and arrest cells at metaphase. Mutation of a conserved arginine residue or the destruction domain makes cyclin B resistant to proteolysis, Here we show that mutation of the same residue also makes cyclin A resistant to proteolysis. in either of two situations in Which the cyclin degradation pathway is turned on: (i) in Xenopus extracts of activated eggs where the degradation pathway has been permanently turned on by adding a recombinant undegradable cyclin B in which the arginine residue of the destruction box has been substituted by alanine; (ii) in extracts of metaphase II-arrested oocytes after Ca 2+-dependent inactivation of the cytostatic factor (CSF).

Degradation of the proto-oncogene product p39mos is not necessary for cyclin proteolysis and exit from meiotic metaphase: requirement for a Ca(2+)-calmodulin dependent event.

Exit from M phase, which requires cyclin degradation, is prevented from occurring in unfertilized eggs of vertebrates arrested at second meiotic metaphase due to a cytostatic factor recently identified as p39mos, the product of the proto-oncogene c-mos. Calpain can destroy both p39mos and cyclin in vitro in extracts prepared from metaphase-arrested Xenopus eggs, but only when free Ca2+ concentration is raised to the millimolar range. When free Ca2+ concentration is raised for only 30 s to the micromolar range, as occurs in physiological conditions after fertilization, cyclin degradation is induced, byt p39mos is not degraded. Cyclin proteolysis at micromolar free Ca2+, is not inhibited by calpastatin, and therefore does not involve calpain. A cyclin mutant modified in the destruction box is found to be resistant at micromolar, but not millimolar free Ca2+, suggesting that the ubiquitin pathway mediates cyclin degradation at micromolar Ca2+ concentration whereas calpain is involved at the millimolar level. A synthetic peptide which binds Ca(2+)-calmodulin with high affinity suppresses cyclin degradation at micromolar but not millimolar free Ca2+, and this only when it is present in the extract during the first 30 s after raising free Ca2+ concentration. The inhibition of the cyclin degradation pathway by the Ca(2+)-calmodulin binding peptide can be overcome by adding calmodulin. These results strongly suggest that a Ca(2+)-calmodulin process is required as an early event following fertilization to release the cyclin degradation pathway from inhibition in metaphase-arrested eggs. In contrast, p39mos degradation is not required.

A fission yeast B-type cyclin functioning early in the cell cycle

We have cloned a fission yeast gene, cig1 +, encoding a 48 kd product that is most similar to cyclin B proteins. The cig1 + protein has a “cyclin box” ∼40% identical to B-type cyclins of other species, but lacks the “destruction box” required for proteolysis of mitotic cyclins. Deletion of cig1 + had no observable effect on cell viability or progression through G2 or M phase, but instead caused a marked lag in the progression from G1 to S phase. G1 constituted ∼70% of the cell cycle in cig1 deletion strains, as compared with <10% in cit1 + strains. Constitutive cig1 + overexpression was lethal, causing cessation of growth and arrest in G1. Expression of cig1 + failed to rescue an S. cerevisiae strain lacking CLN Start cyclins. Thus, cig1 1 identifies a new class of B-type cyclin acting in G1 or S phase that appears to be functionally distinct from all previously described cyclin proteins.