 |
|
 |
|
 |
| milestone 20
Disappearing act
We have known since 1971 (Milestone 7) that the activity of 'maturation-promoting factor' — later identified as cyclin B- CDC2 — oscillates throughout the cell cycle, but the controller of these oscillations took much longer to track down. The discovery that proteolysis is a key regulator of cyclin B levels, mediated by a huge ubiquitin ligase complex that we now call the anaphase-promoting complex or cyclosome (APC/C), changed our view of the cell cycle, but also had enormous implications for understanding the regulation of cellular processes by ubiquitin-mediated proteolysis.
The catalyst to the discovery and characterization of the APC/C was a 1991 Nature paper by Michael Glotzer, Andrew Murray and Marc Kirschner. They had been studying cyclin B synthesis and degradation in frog eggs, but because degradation was transient, it was difficult to analyse at the molecular level. They cracked this nut by adding a truncated cyclin B, which lacked the first 90 amino acids (cycΔ90) to crude extracts of interphase frog eggs. This generated 'mitotic' extracts that consistently degraded exogenously added cyclins. They already knew that amino acids 13-90 were necessary for cyclin B degradation, but could these residues target other proteins for destruction? A fusion protein of these residues combined with protein A (13-91prA) was degraded in the mitotic extracts, but not in interphase extracts. Sequence analysis of cyclins showed a conserved nine-residue region that they dubbed the 'destruction box', and mutation analysis showed that it was the destruction box that conferred M-phase-specific destructablility on cyclin B. During these experiments, the authors noticed that, on polyacrylamide gels, one of their degradable mutants was converted to a ladder of higher molecular weight forms before it was destroyed. Each rung of the ladder represented an increment of about 7 kDa — the molecular weight of ubiquitin. Could ubiquitylation be responsible for targeting cyclin B for destruction? At the time, ubiquitylation was thought to be responsible only for the destruction of misfolded proteins, but Glotzer and colleagues showed that radioiodinated ubiquitin was added to 13-91prA. Ubiquitylation was M-phase dependent and was blocked by mutations in the destruction box that prevented degradation. But it was hard to tell whether this was true regulation of cyclin B levels, or merely destruction of misfolded cyclins. The Kirschner lab had no means of blocking ubiquitylation to see what effect this would have on cyclin B levels, but their kinetic studies indicated that all of the cyclin that was degraded passed through a ubiquitylated intermediate. Avram Hershko and colleagues later used methyl-ubiquitin to block cyclin degradation in clam extracts, confirming the kinetic results. But what wasn't known was what conferred cell-cycle regulation on ubiquitylation of cyclin B. Glotzer et al. speculated that 'there is a form of [ubiquitin-conjugating enzymes] specific for cyclin'. The APC/C was born conceptually — but what was its identity?
Ubiquitylation is a three-step process involving ubiquitin-activating (E1), -conjugating (E2) and -ligating (E3) enzymes. In theory, any of these processes could be regulated in a cell-cycle-specific manner. In 1995, Randall King, a graduate student in Marc Kirchner's lab, isolated two activities from their frog-egg extracts: an E2 and a large (20S) complex containing an E3 activity. Only this complex, which they termed the anaphase-promoting complex, could reconstitute destruction of cyclin B in interphase extracts. At the same time, Hershko and colleagues had also isolated a complex, which they called the cyclosome, from their clam extracts. We now know that the APC and the cyclosome are one and the same. But is the onset of anaphase purely down to destruction of cyclin B? In 1993, Sandra Holloway and colleagues, working in Andrew Murray's lab, showed that was, in fact, more complex: in frog-egg extracts that could make spindles, an amino-terminal fragment of cyclin B that blocks cyclin B's destruction had no effect on the onset of anaphase, but methyl-ubiquitin did. "A simple resolution to this paradox", they proposed, "is that chromosome segregation requires the ubiquitination and degradation of a protein that is not cyclin but is recognized by some of the same proteins that recognize cyclin and target its degradation". This put the APC — still nameless and unidentified in 1993 — at the centre stage in the onset of anaphase, leaving cyclin B to exit stage left. The next act was to uncover the identity of the other proteolytic target. An obvious candidate was Pds1, an anaphase-inhibiting protein from budding yeast that was discovered by Orna Cohen-Fix and co-workers in Doug Koshland's lab. Evidence that Pds1 might be a substrate of the APC/C began to accumulate when Hiro Funabiki, in Mitsuhiro Yanagida's lab, found that destruction of Cut2, the fission-yeast orthologue of Pds1, was necessary for anaphase to occur. Rafal Ciosk and colleagues, working in Kim Nasmyth's lab, then worked out the details of the mechanism: the destruction of Pds1 releases a protease, Esp1, that degrades a component of cohesin — the multiprotein complex that holds sister chromatids together (Milestone 23). And that was just the beginning: most, if not all, of the APC's subunits have now been identified and their individual functions are being unravelled. But although cyclin B was one of the first proteins to be recognized as a regulated target of ubiquitylation — a process that we now know to be as important as phosphorylation in cellular regulation — the enzyme that catalyses this process is still holding on to some of its secrets.
|
