Linch, M.D. (2012) Protein kinase C iota in mammalian cell polarity and cancer. Doctoral thesis, UCL (University College London).
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Protein Kinase C iota (PKCɩ) is a serine/threonine kinase that is involved in epithelial polarity and malignancy where polarity is typically abrogated. The aim of this thesis was to characterise the role of PKCɩ in oncogenic signaling in the context of polarity loss. This work set out to identify activators, binding partners and downstream effectors of PKCɩ with a view to informing rational therapeutic design. Madin Darby Canine Kidney (MDCK) cells, grown as polarised cysts containing a central lumen in Matrigel, developed a multiple-lumen phenotype on perturbation of PKCɩ, suggesting a threshold requirement for normal lumenogenesis. When transfected with oncogenic Ras, MDCK cells formed large spheres lacking a lumen and polarity. Upon PKCɩ inhibition with siRNA or small molecules, polarity was partially rescued indicating that PKCɩ lies downstream of Ras in this polarity loss pathway. Structural studies have enabled two distinct approaches to identify effectors of PKCɩ. A novel protein-protein interaction motif (RIPR) within PKCɩ was identified and shown here to be responsible for interaction with a subset of target proteins, including LLGL2. MDCK cysts expressing a PKCɩ mutated in this motif developed a multiple-lumen phenotype. In a second approach, use was made of an inhibitor bound complex of PKCɩ to design a drug insensitive mutant. Expression of the resistant mutant in HEK293 cells led to resistance to inhibitor induced dephosphorylation of LLGL2 and protected MDCK cells from developing inhibitor induced multiple lumens and loss of polarity. This tool has been used to screen for PKCɩ substrates. In summary, PKCɩ lies downstream of H-Ras in MDCK polarity signalling. The site at which PKCɩ binds LLGL2 is defined and shown to contribute to normal lumenogenesis. A drug insensitive PKCɩ mutant is described that can be utilised for substrate and biomarker identification.
|Title:||Protein kinase C iota in mammalian cell polarity and cancer|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences|
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