Regulation of mitosis : molecular analysis of the anaphase-promoting complex

Regulation of mitosis molecular analysis of the anaphase-promoting complex. Thesis by Pia-Marie Jorgensen, Department of Cell and Molecular Biology, Division of Molecular Genetics, Karolinska Institutet, S-171 77 Stockholm Cell division is achieved by the progression through a series of events known as the cell cycle. To make sure that the original cell is copied with high fidelity, an elaborate control system using so called checkpoints is employed, preventing cell cycle events to occur prematurely or in the wrong order. Chromosome segregation is an important process in mitosis and must be performed correctly to ensure that the two resulting daughter cells have the same DNA content. Missegregation of chromosomes results in aneuploidy, something that is frequently found in cancers, suggesting that the machinery surveying the chromosome segregation process has somehow been compromised during the development of these tumours. One of the cell cycle checkpoints, the mitotic spindle checkpoint, has also been shown to be defective in cancers with chromosomal instability. The spindle checkpoint, which monitors the metaphase to anaphase transition, involves e.g. the Mad and Bub proteins and makes sure that each chromosome is attached and the spindle is functional before giving the signal to separate the sister chromatids. This signal is relayed to a complex called the Anaphase Promoting Complex or Cyclosome, APC/C. The APC/C is a ubiquitin ligase, E3, which targets cell cycle regulatory proteins for degradation by the proteasome, thereby allowing progression through the cell cycle. The APC/C is regulated by phosphorylation and by two proteins, Cdc20 and Cdh1, which affect APC/C activity in early mitosis and late mitosis/G1 respectively. The Cdc20 is also known to interact with the spindle assembly checkpoint through a protein called Mad2. The mammalian APC/C consists of at least eleven subunits, for most of which the specific function is still unknown. Apc2 contains a cullin domain and interacts with another subunit, Apc11, which has a RING-H2 finger domain. These two might based on their similarity to components of another ubiquitin ligase complex important in cell cycle regulation, the SCF (Skp1-cullin-F-box protein), be responsible for the interaction with the rest of the ubiquitination system. The function of the other APC/C subunits remains obscure. Some contain the protein-protein interaction tetratricopeptide (TPR) motif. Two of the subunits, Apc3 and Apc6, have been localised to the centrosome and mitotic spindle. The largest subunit, Apc1, shares a motif of unknown function with two subunits of the 19S cap complex of the proteasome. In the articles upon which this thesis is based we have now shown that the Apc1 protein is a centromereassociated protein in mammalian cells, and that it exists in two forms: a soluble form associated with other components of the APC/C and a centromere-bound form, which may or may not be associated with other APC/C components. We have further shown that both the Apc1 and Apc3 are bound to isolated mitotic chromosomes, suggesting that more APC/C components than can be observed by immunofluorescence are present on mitotic chromosomes. The Apc1 and Apc3 proteins are also likely targets for APC/C regulatory phosphorylation recognised by the 3F3/2 antibody at the centromere. The Apc2 and Apc8 proteins are shown to localise to the centrosome in both interphase and mitotic cells, confirming the presence of the APC/C at this cellular location. Polo-like kinase (Plk) is shown to specifically phosphorylate components of the APC/C and activate it, whereas protein kinase A (PKA) phosphorylation suppresses APC/C activity. PKA is superior to Plk in regulation of the APC/C. We have also shown that APC/C subunits are expressed in most tissues and cell types at fairly constant levels relative to each other, suggesting that they always perform their functions as part of a complex. Their presence in non-dividing cells also suggests the possibility of additional APC/C functions outside the cell cycle.