UCL logo

UCL Discovery

UCL home » Library Services » Electronic resources » UCL Discovery

Cell Cycle Specific Recruitment of PKCε

Crossland, VM; (2012) Cell Cycle Specific Recruitment of PKCε. Doctoral thesis , UCL (University College London). Green open access

Crossland Thesis July 2012.pdf
Available under License : See the attached licence file.

Download (38MB)
[img] Excel Spreadsheet (Supplementary data)

Download (116kB)


Protein kinase C (PKC) comprises a family of serine/threonine kinases which play central roles in intracellular signal transduction typically triggered by recruitment to membraneous compartments. The epsilon isoform of PKC (PKCå) has been shown to localize to cell-cell contacts and to the cytokinetic furrow/midbody, indicative of a role in the cell cycle. Both recruitment patterns can be visualized under conditions of PKCå inhibition, which is selectively achieved using a gatekeeper mutant (PKCå-M486A) and the inhibitor NaPP1. Initial studies indicated that interphase and mitotic cells were not distinguished in their capacity for PKCå-M486A recruitment as evidenced by optical trapping experiments. I therefore assessed whether recruitment to the furrow/midbody is a general property reflecting the juxtaposition of two membranes and a cell-cell contact response. I have successfully used fluorescence recovery after photobleaching (FRAP) to distinguish between the localization at the furrow/midbody from that at cell-cell contacts by measuring PKCå-M486A turnover at these two compartments. It is demonstrated that PKCå-M486A has a slower turnover at the furrow/midbody. The distinct kinetic behaviour of PKCå M486A at the furrow/midbody is indicative of other factors contributing to recruitment and/or retention. Sites and domains within PKCå-M486A were therefore assessed for their involvement in this process using a combination of mutagenesis and confocal microscopy. Through these studies I have identified a short motif in the regulatory domain of PKCå-M486A, the inter C1 domain (IC1D), that is in part required for the accumulation of PKCå-M486A at the furrow/midbody. The deletion of this domain (PKCå-ÄIC1D-M486A) prevents the kinase being recruited to the furrow/midbody despite, the recruitment and furrow/midbody localization of the co-expressed PKCå-M486A. Given that the IC1D was previously identified as an actin-binding region, I have assessed the relationship between actin and PKCå-M486A recruitment by manipulating actin polymerization. Using latrunculinA, an inhibitor of F-actin assembly, I have shown that PKCå-M86A and RhoA colocalize and are stabilized in the same compartment in conditions where F-actin is depolymerized. Importantly, the behaviour is observed for both active and inactive PKCε-M486A. This condition may be analogous to a stage in midbody biogenesis and may be evidence of the requirement of F-actin for normal PKCε and RhoA behaviour in cytokinesis. These data show some progress towards understanding the unique behaviour of PKCε at the furrow/midbody and indicate a complex relationship between PKCε, actin and RhoA.

Type: Thesis (Doctoral)
Title: Cell Cycle Specific Recruitment of PKCε
Open access status: An open access version is available from UCL Discovery
Language: English
UCL classification: UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
URI: http://discovery.ucl.ac.uk/id/eprint/1352790
Downloads since deposit
Download activity - last month
Download activity - last 12 months
Downloads by country - last 12 months

Archive Staff Only

View Item View Item