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Paul R Clarke
Professor of Cancer Cell Biology
Cell Cycle and Apoptosis Group
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E-mail: p.r.clarke@dundee.ac.uk Tel. 01382 425580 (35580 internal)
The control of cell proliferation and cell survival is critical for the normal development and tissue homeostasis of multicellular organisms. Defects in these processes underlie a number of major human diseases, in particular cancer, which is associated with loss of controls over cell division and evasion of cell death by apoptosis. In order to understand how cancer develops and how it may be treated more effectively, we need to understand the molecular mechanisms controlling these processes. Our goal is to understand what determines determine the balance between cell cycle controls and the induction of apoptosis in response to stress signals, DNA damage and during cell division.
We are working on two main aspects of the control of the cell division cycle and apoptosis:–
1. Regulation and functions of Ran GTPase. Ran is a member of the Ras superfamily that plays important roles at several stages of the cell division cycle in all eukaryotic cells. During interphase, Ran is concentrated in the nucleus, mainly in the GTP-bound form, and plays an important role in controlling the direction of transport of proteins and RNA between the nucleus and cytoplasm. During mitosis, when the nuclear envelope breaks down in vertebrate cells, Ran-GTP plays roles in the stabilisation of microtubules nucleated at centrosomes and in the organisation of the mitotic spindle. At the end of mitosis, Ran directs the rebinding of precursor vesicles to the chromatin and controls the reassembly of the nuclear envelope. We are investigating the control of Ran and its nucleotide exchange factor RCC1 during the cell cycle, and their potential roles in cancer.
Figure 1 shows the localisation of a Ran mutant expressed in live cells as a fusion with green fluorescent protein (green). Chromatin is labeled by a histone-red fluorescent protein fusion. The GFP-Ran mutant is dispersed in the cytoplasm and is concentrated at the nuclear pore complexes within the nuclear envelope. Image by Dr James Hutchins.
2. Control of apoptosis. We are investigating the control of apoptosis in response to stress signals, DNA damage and during cell division. Apoptosis involves caspases, a family of proteases that cleave key proteins to bring about the biochemical and morphological changes associated with apoptosis (Fig.2). Activation of the initiator caspase-9 involves proteolytic processing of the proenzyme by Apaf-1 and is stimulated by cytochrome c released from mitochondria. Caspase-9 then activates the effector enzymes caspase-3 and -7. We are studying the regulation of caspase-9 activation by Bcl-2 family proteins and by protein kinases. We have found that caspase-9 is phosphorylated and inhibited by multiple protein kinase signalling pathways, which are activated by survival factors and oncogene products that suppress apoptosis.
Figure 2 shows the induction of apoptosis in mitotic cells in response to the microtubule poison nocodazole. Immunofluorescence microscopy of active caspase-3 (indicating apoptosis) and the mitotic marker H3 pS10. DNA was visualized using DAPI. The histogram shows the number of cells staining for both active caspase-3 and H3 pS10 as a percentage of the total number of active caspase-3 positive cells following nocodazole treatment for the times indicated. Caspase-3 activation in mitotic cells is accelerated in T125A-M2.3 cells in which the phosphorylation of caspase-9 has been abolished. See Allan & Clarke (2007) Mol Cell 26, 301-310.
Our work is supported by Cancer Research UK, Medical Research Council, Biotechnology and Biological Sciences research Council and the Association for International Cancer Research. Paul Clarke is a Royal Society Wolfson Research Merit Award holder.
Selected Publications
- Zhang, C. and Clarke, P.R. (2000) Chromatin-independent
nuclear envelope assembly induced by Ran GTPase in Xenopus
egg extracts. Science 288, 1429-1432. Abstract or Full Text available.
- Moore, W.J., Zhang, C. and Clarke, P.R. (2002) Targeting
of RCC1 to chromosomes is required for proper mitotic
spindle assembly in human cells. Curr. Biol. 12, 1442-1447.
- Allan, L.A., Morrice, N, Brady, S., Magee, G., Pathak,
S. and Clarke, P.R. (2003) Inhibition of caspase-9 by
phosphorylation at Thr125 by ERK MAPK. Nature Cell Biol.
5, 647-654.
- Hutchins, J.R.A., Moore, W.J., Hood, F.E., Wilson, J.S.J., Andrews, P.D., Swedlow, J.R. and Clarke, P.R. (2004) Phosphorylation regulates the dynamic interaction of RCC1 with chromosomes during mitosis. Curr. Biol. 14, 1099-1104.
- Brady, S.C., Allan, L.A. and Clarke, P.R. (2005) Regulation of caspase-9 through phosphorylation by protein kinase C zeta in response to hyperosmotic stress. Mol. Cell. Biol. 25, 10543-10555.
- Allan, L.A. and Clarke, P.R. (2007) Phosphorylation of caspase-9 by CDK1/cyclin B1 protects cells against apoptosis. Mol. Cell 26, 301-310.
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