BIOLOGICAL OVERVIEW

Cyclin B, along with its dimerization partner cdc2, plays a significant role in cell division in Drosophila, but this fact is easy to overlook for several reasons. First, mutational studies can be misleading, with respect to the vital role played by Cyclin B during cell division. A biological fail safe mechanism appears to exist between Cyclin B and Cyclin A: a single mutational deficiency in either of these two cyclins involved in the G2-M transition can be compensated for by the unmutated cyclin. One might reasonably suppose that because Cyclin B is thus replaceable, it is less than essential to the process. The compensation is not 100% effective however; mitotic spindles are abnormal and progression through mitosis is delayed in cyclin B deficient embryos (Knoblich, 1993).

Two other factors mitigate against finding a substantial role for Cyclin B in Drosophila development: first, maternal cyclins are involved in development through mitosis 14, at which time they are degraded. Second, and occuring after this time, the dynamics of String protein limit the rate of mitosis. Despite these limiting factors, Cyclins A and B are produced in G2 both before and after cell cycle 14.

The strongest evidence that Cyclin B is crucial to mitosis is provided by the measurement of cyclin levels throughout the process. During cycles 8-13 a progressive increase in the degradation of cyclins at mitosis leads to increasing oscillations of cdc2 kinase activity. Quantitative measurements indicate that less than 10% of Cyclin A and B are degraded at mitosis 5, and that about 30%, 60%, and 80% are degraded at mitoses 8, 11 and 13 respectively. It appears that cyclin synthesis is limiting for mitoses 10-13 (Edgar, 1994).

During interphase 14, programmed degradation of maternal String protein leads to inhibitory phosphorylation of cdc2 and cell cycle arrest. Subsequently, mitoses 14-16 are triggered by pulses of zygotic string transcription. The use of an N-terminally truncated Cyclin B in Drosophila provides evidence for the role of Cyclin B in timing mitosis. Cyclin A is degraded during metaphase and Cyclin B degradation occurs at approximately the metaphase-anaphase transition (Whitfield, 1990). The N-terminally truncated Cyclin B fails to degrade due to absence of a 'destruction box' in the truncated N-terminal region. The truncated Cyclin B results in mitotic delay at late anaphase (Rimmington, 1994). The gene fizzy, homologous to cdc20 in S. cerevisiae, is required for metaphase-anaphase transition in Drosophila, and is required for normal cyclin A and B degradation. It is suggested that fizzy functions to promote the ubiquitin-dependent proteolytic events that occur during mitosis (Dawson, 1995).

Looking to yeast and to vertebrates for clues as to the role of Cyclin B in mitosis, one finds a bewildering array of information. There appears to be a connection between G1-S cyclins and G2-M cyclins. Cdk2 kinase, the partner for G1-S cyclins, is a positive regulator of cdc2-Cyclin B complexes in Xenopus. When cdk2 kinase activity is inhibited by the cdk-specific inhibitor, a block to mitosis occurs, and inactive cdc2-cyclin B accumulates (Guadagno, 1996). Eukaryotic cells have evolved regulatory mechanisms to ensure the strict alternation of DNA replication and mitosis. Cdc2/cyclin B has a role in preventing re-replication of the genome before mitosis. A Xenopus homolog of S. pombe, cdc21, exhibits cell-cycle dependent chromatin binding and phosphorylation in association with S-phase control. Cdc21 remains bound to chromatin during the initiation of DNA replication and is displaced with the progression of the replication fork. Cytoplasmic cdc21 remains underphosphorylated but at the beginning of mitosis the entire pool of cdc21 is hyperphosphorylated, possibly by the cdc2/cyclin B kinase. These properties identify Xenopus cdc21 as a possible component of the DNA licensing factor (Coué, 1996). For more information about licensing factor see the DNA replication site.

Several targets of cdc2-Cyclin B kinase have been identified. The dimer cdc2-Cyclin B targets Wee1 kinase. Wee1 then inhibits cell division by phosphorylating cdc2. In each cell cycle, the mouse Wee1 kinase is phosphorylated at M-phase resulting in inactivation of Wee kinase. The N-terminal domain or entire molecule is extensively phosphorylated by the cdc2-Cyclin B dimer (Honda, 1995). Drosophila Cyclin B has a consensus cAMP-dependent protein kinase site. Evidence from Xenopus suggests that cyclin degradation and exit from mitosis requires Cyclin B/cdc2-dependent activation of the cAMP-PKA pathway. The concentration of cAMP and the activity of PKA decrease at the onset of mitosis and increase at the transition between mitosis and interphase (Grieco, 1996). Proteins of the mitotic apparatus are direct targets of cdc2-Cyclin B dimer.

A human homolog of Xenopus Eg5, (a kinesin-related motor protein) is implicated in the assembly and dynamics of the mitotic spindle. An evolutionarily conserved cdc2 phosphorylation site in HsEg5 is phosphorylated specifically during mitosis in HeLa cells and in vitro by p34cdc2/Cyclin B. Phosphorylation controls the association of this motor protein with the spindle apparatus (Blangy, 1995).

Cdc2/Cyclin B targets the ubiquitin proteins involved in cyclin destruction. Two specific components are required for the ubiquitination of mitotic cyclins: E2-C, a cyclin-selective ubiquitin carrier protein that is constitutively active during the cell cycle, and E3-C, a cyclin-selective ubiquitin ligase (termed the cyclosome) that purifies as part of an approximately 1500-kDa complex and is active only near the end of mitosis. The cyclosome has been separated from its ultimate upstream activator, cdc2, that activates the cyclosome complex by means of phosphorlyation (Lahav-Baratz, 1995).

The cdc2/Cyclin B heterodimer was first characterized as maturation promoting factor, or MPF. Unfertilized frog eggs manifest cytoplasmic activity that can induce immature oocytes to undergo meiotic maturation. MPF activity drives the events of early mitosis such as nuclear breakdown, chromosome condensation and spindle formation by phosphorylating cellular substrates. While cdc2 (the catalytic subunit of the MPF heterodimer) is required to drive the events of early mitosis, it must be inactivated to allow the events of late mitosis to proceed. The metaphase-anaphase transition is a checkpoint during mitosis. At this juncture the cell is able to monitor the integrity of its spindle before proceeding to inactivate MPF and initiate chromosome separation (Murray, 1992 and references). What is the link between phosphorlyation and this checkpoint? A common mitotic error is the attachment of a chromosome to only one spindle pole rather than to both poles. Even a single such chromosomal error delays anaphase in cells. Recently it has been shown that when the tension associated with proper attachment is absent the kinetochore becomes phosphorylated and anaphase is delayed. It has been proposed that the kinetochore protein dephosphorylation caused by tension is the all-clear signal to the checkpoint. The involvement of Cyclin B/cdc2 in the events surrounding this mitotic checkpoint have yet to be documented, but the fact that Cyclin B/cdc2 is degraded at the metaphase-anaphase transition, suggests that MPF is the direct target of this checkpoint (Nicklas, 1995 and Dawson, 1995).

GENE STRUCTURE

Bases in 5' UTR -123 or more

Bases in 3' UTR - 776

PROTEIN STRUCTURE

Amino Acids - 530

Structural Domains

Within a central region spanning 206 amino acid residues, the Drosophila Cyclins A and B share 35% identity, whereas clam Cyclin A and Drosophila Cyclin A share 53% identity as do clam Cyclin B and Drosophila Cyclin B. In particular, the Drosophila Cyclin B sequence contains a consensus cAMP-dependent protein kinase site, a feature common to all other Cyclin B sequences (Whitfield, 1990).

date revised: 21 APR 97 

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