Analysis of the role of rab11-FIP3 phosphorylation during cytokinesis

QR Microbiology

Collins, Louise L. ; Gould, Gwyn W.

University:University of Glasgow

Department:Institute of Molecular Cell and Systems Biology

关键词: cytokinesis, membrane trafficking, Rab11-FIP3, phosphorylation;   

Others  :  http://theses.gla.ac.uk/1682/1/2010collinsphd.pdf

来源: University of Glasgow

【 摘 要 】

Cytokinesis involves abscission of the intracellular bridge between two daughter cells, thus completing mitosis. Membrane traffic is at the heart of mammalian cytokinesis. Rab11-FIP3 (also referred to as FIP3) in complex with Rab11 plays a key role in the delivery and targeting of recycling endosomes to the furrow; this is essential for completion of cytokinesis. FIP3 undergoes spatial and temporal dynamics during mitosis. During metaphase and early anaphase, FIP3 is largely cytosolic, with some localised to endosomal membrane structures. During late anaphase, following furrow initiation, FIP3 localises to the centrosome. At late cytokinesis, FIP3 relocates to the cleavage furrow and midbody. On separation of the daughter cells, FIP3 returns to the centrosome. The regulatory mechanisms governing the dynamics of FIP3 during mitosis are unknown. This work aims to determine if FIP3 can be phosphorylated by the cell-cycle kinases which regulate mitosis, and whether this has an impact on the spatial and temporal dynamics of FIP3. In vitro phosphorylation assays show that FIP3 can be phosphorylated by cyclin B-CDK1, Plk1, Aurora A and weakly by Aurora B. A proteomic approach revealed that, within the limits of our experimental approach, only cyclin B-CDK1 phosphorylated FIP3 significantly, at serine 102. Interestingly, proteomic analysis of FIP3 immunoprecipitated from metaphase cells has identified serine 102, 281, 348 and 451 as sites of potential phosphorylation. Data from this and a collaborating lab offers the hypothesis that FIP3 is phosphorylated during the early stages of the cell-cycle, and that dephosphorylation of FIP3 is the trigger for the association of FIP3 with membranes. A phospho-specific antibody to serine 102 (pS102) detects CDK1 phosphorylated FIP3. Serine 102 is phosphorylated in metaphase and becomes dephosphorylated as the cell progresses through to telophase. Further analysis reveals that cytosolic levels of pS102 peak in metaphase and decrease towards telophase to negligible levels. pS102 is absent in the membrane fraction. This work suggests that FIP3 may be directly phosphorylated by CDK1, at serine 102, in early mitosis. Kinase inhibition studies show that inhibition of CDK1, by the inhibitor BMI-1026, results in a mis-localisation of GFP-FIP3 in HeLa cells. This could also be interpreted as a delay in cytokinesis, since CDK1 inhibition resulted in more cells in telophase displaying GFP-FIP3 in a localisation characteristic of an earlier stage of telophase, compared to the controls. The role of phosphorylation at serines 102, 281, 348 and 451 of FIP3 was investigated by creating phospho-null and phospho-mimetic mutants in the context of GFP-FIP3. It would appear that when mutated singly, the potential phospho-sites of serines 102, 281, 348 and 451 (phospho-mimetic mutant only for serine 451) have no significant effect on the localisation of FIP3 during mitosis, nor do they affect cytokinesis. A phospho-null mutation of serine 451 resulted in poor expression of the protein and an unhealthy population of cells. In summary, the spatial and temporal dynamics of FIP3 may be regulated by phosphorylation. We hypothesise that during prometaphase and metaphase FIP3 is phosphorylated, preventing its association with endosomes. FIP3 is dephosphorylated in late anaphase, allowing it to associate with endosomes and subsequently traffic to the furrow and midbody. We suggest that CDK1 phosphorylates FIP3 in the early stages of mitosis, at serine 102. In conclusion, membrane traffic is central to mammalian cytokinesis and data from this thesis suggests that it may be regulated by the cell-cycle kinases.