2012PateljtPhD.pdf (8.33 MB)
Characterising SUN1 Mitotic Phosphorylation and its Effects on LINC Complex Interactions
thesisposted on 2013-02-05, 16:34 authored by Jennifer Tulsi Patel
Mammalian inner nuclear membrane (INM) proteins, SUN1 and SUN2, interact with nesprins located on the outer nuclear membrane to form the linker of nucleoskeleton to cytoskeleton (LINC) complex. The complex acts as a molecular bridge connecting the cytoskeleton to the nuclear lamina. At its nucleoplasmic N-terminus, SUN1 interacts with lamin A/C, emerin and small nesprin isoforms, strengthening LINC complex anchorage at the NE and contributing to attachment of the nuclear lamina to the NE. At the onset of mitosis, the nuclear envelope (NE) disassembles to allow chromosome segregation. Nuclear envelope breakdown (NEBD) is induced by partial disassembly of nuclear pore complexes and depolymerisation of the nuclear lamina, which weakens the NE allowing microtubule-dependent forces to physically tear open the NE. This process is initiated by phosphorylation of NPC components, the nuclear lamina and INM proteins by mitotic kinases. This thesis aimed to determine whether SUN1 undergoes mitotic phosphorylation and to assess how phosphorylation impacts upon LINC complex interactions. I found that SUN1 is phosphorylated specifically during mitosis at serines 48, 138 and 333. Furthermore, CDK1 is responsible for phosphorylation of S48 and S333, whilst PLK1 phosphorylates S138. Use of phospho-deficient mutants and kinase inhibitors supports these findings and indicates that additional kinases, particularly Aurora A, may also phosphorylate SUN1. Importantly, I showed loss of SUN1 interaction with N-terminal but not C-terminal binding partners in mitotic extracts. A triple phospho-mimetic mutant also displayed increased solubility and reduced retention at the NE. My data demonstrate that mitotic SUN1 phosphorylation does not disrupt the LINC complex itself but is required for its dissociation from the nuclear lamina, presumably promoting NEBD. In addition, by implementing an immunoprecipitation-mass spectrometry approach, I identified numerous potential binding partners for SUN1. My data suggest that SUN1 may be involved in many other protein networks beyond the LINC complex.
Date of award2013-01-01
Awarding institutionUniversity of Leicester