Zhang, Y; (2017) Differentiating protein kinase C iota functions through multiple PKCι-partner complexes. Doctoral thesis , UCL (University College London).

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Abstract

Atypical protein kinase C iota (PKCι) is a serine/threonine kinase that has been implicated in both epithelial polarity and oncogenic transformation. Spatio-temporal regulation of PKCι activity and accessibility to its binding partners/substrates are therefore essential. This thesis set out to understand how PKCι exerts its multiple functions through coupling with different partner complexes. Previously we identified a dibasic motif (the RIPR motif) within PKCι, which is required for the association of a sub-set of PKCι binding partners. Proteomic screens have identified RIPR-dependent PKCι interacting proteins, amongst which two protein families, MTA and FARP, were chosen for further investigation. Metastasis-associated (MTA) family proteins are components of the histone deacetylase and nucleosome remodelling complex (NuRD). One of the family members, MTA2, localises to the mitotic spindles and the cell cortex, suggesting it has novel functions. Knockdown of MTA2 causes morphogenesis defects in Caco2 3D cyst assays, phenocoping the loss of both aPKCs (PKCι and PKCζ). Furthermore, PKCι-MTA2 association shows cell density dependency and differential localisations inside and outside the nucleus, suggesting the PKCι-MTA2 complex may relay cortical/cytoplasmic information to the nucleus. FERM, RhoGEF and pleckstrin domain-containing protein 1 and 2 (FARP1, 2) are Cdc42/Rac1 guanine nucleotide exchange factors (GEFs) and are robust hits as RIPR-dependent PKCι interacting proteins. Knockdown of FARP1 or FARP2 phenocopies aPKC (PKCι and PKCζ) loss in Caco2 3D cyst assays. Furthermore, FARP2 is shown to be a Cdc42 GEF and phosphorylation of FARP2 on the identified PKCι sites is required for junctional formation of Caco2 cells in 2D culture. Comparison of multiple PKCι-partner complexes has implied the existence of a PKCι monomer/dimer/oligomer equilibrium, which can both determine and be modulated through partner binding. PKCι RIPR motif seems to contribute to this equilibrium, as some partners show indirect RIPR dependency. Biochemical studies of PKCι complexes with MTA2, FARP1, FARP2 and other known PKCι binding partners Llgl2, Par3 and Par6 provide evidence for this model. The indication is that the oligomerisation status of PKCι contributes to its multifunctionality, which is modulated by coupling with distinct partner proteins.

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