Principal Supervisor: Dr. Ildiko Gyory - Department of Molecular and Cell Biology
Co-supervisor: Professor Martin Dyer
PhD project title: Regulation of Immune Development by intra-cellular compartmentalisation of Cell Cycle Regulatory Proteins
University of Registration: University of Leicester
Cyclin dependent kinases, along with cyclins, are major control switches for the cell cycle. The D-type cyclins form a complex with and functions as a regulatory subunit of cyclin dependent kinases 4 and 6 (CDK4 or CDK6), whose activtiy is required for cell cycle G1/S transition. In every cell type, at least one of the 3 D cyclins is expressed (CCND1, CCND2 and CCND3).
In B lymphocytes Cyclin D3 (CCND3) is the dominant subtype and it determines the balance between proliferation and differentiation, which is crucial to immune system development. Deregulated CCND3 expression often results in lymphomas and leukaemias, which underscores its importance.
Temporal control of subcellular localization and proteolysis is critical for maintenance of CCND3 homeostasis. One important mechanisms to increase stability of CCND3 is nuclear retention, since its degradation occurs in the cytoplasm. A conserved residue near the C terminus of the molecule, which is phosphorylated by the kinases p38/SAPK or GSK-3depending on the cell type or upstream signalling) mediates its association with the nuclear exportin CRM1. Recently, several other kinases were reported to play similar roles but their substrate recognition site is unknown and phosphorylation independent mechanisms could also be involved.
In contrast, we have found that in cell lines of early B cell origin CCND3 predominantly locates to the cytoplasm and chemical inhibition of nuclear export leads to rapid degradation of both the cytoplasmic and the nuclear pools, in sharp contrast to the expected nuclear retention. Overexpression of CCND3 in these cells increase growth rate, but mutation in the GSK3recognition site has no additional effect.
The major aim of this project is to understand how the post-transcriptional regulation of CCND3 is changing over the course of B lymphocyte development. Clearly, during early stages of development, the protein is predominantly cytoplasmic and its nuclear import is tightly regulated while at later stages this shifts to a mostly nuclear presence and the export becomes the regulated process.
To identify the mechanisms, we will apply a reverse Cancer Genetic approach. The large database of mutations from lymphoma patients that we have at hand could serve as a functional toolbox to identify the major regulatory sites. The mutations cluster around the C-terminus and expected to result in amino acid substitutions that increase the stability of the CCND3 protein.
A recurring point-mutation of CCND3 that is found in lymphomas was already shown to affect the GSK-3B recognition site and thereby the stability of CCND3, proving the principle.
The second major aim of the project is to understand the cytoplasmic retention mechanisms and function (CDK4/6 independent cytoplasmic role?) of CCND3 during early B lymphocyte development.
To achieve these we will have the following specific approaches:
- Identification of nuclear and cytoplasmic association partners of CCND3 in cells representing prototypes of B lymphocyte differentiation stages
- Studying nuclear-cytoplasmic trafficking by chemical inhibition of the CRM1 transporter and the known kinases that regulate shuttling
- Investigation of hyper-proliferative phenotype caused by cytoplasmic over-expression of CCND3 in early B cells
- Generation of knock-in cell lines of late B lymphoid origin that reproduce tumour-specific mutations by the cutting edge CRISPR/Cas9 technology to study nuclear-cytoplasmic shuttling and the stability of CCND3
- Identification of chemical inhibitors of CCND3 stability to which the mutant cell lines retain sensitivity in order to decipher the hierarchy of regulatory pathways
In conclusion, regulated intra-cellular compartmentalisation of CCND3 is necessary to normal cell division and proliferation of B lymphocytes. Deregulated nuclear-cytoplasmic shuttling lead to cancer and the prospective student will identify the underlying molecular mechanisms of compartmentalisation.
BBSRC Strategic Research Priority: Molecules, cells and systems
Techniques that will be undertaken during the project:
- CRISPR/Cas9 mediated knock in of specific mutations in cell lines
- MALDI-TOF proteomics of protein complexes
- Standard molecular techniques such as molecular cloning and protein detection by Western blotting, co-immunoprecipitation, flow cytometry, apoptosis and cell proliferation assays, confocal microscopy, live cell imaging
Contact: Dr Ildiko Gyory, University of Leicester