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Abstract
Cyclic-AMP dependent protein kinase A (PKA) regulates traffic of multiple proteins at different stages along the constitutive secretory pathway. PKA effects are regulated by protein phosphatases, which reverse the actions of PKA by dephosphorylation of PKA-substrates. Localization of specific PKA effects is mediated by the binding of A-kinase anchoring proteins (AKAPs). Apolipoprotein E (apoE) is an important regulator of lipid metabolism and atherosclerosis, and represents a large proportion of total protein constitutively secreted from macrophages. The signalling and trafficking pathways regulating secretion of apoE are unknown. Catalase is a peroxisomal enzyme which contributes to defence against hydrogen peroxide (H2O2). The primary hypothesis of this thesis is PKA and related protein phosphatase pathways are involved in the regulation of apoE secretion. The secondary hypothesis is that these pathways also regulate cellular clearance of H2O2.
In Chapter Three, I have investigated the role of PKA in apoE secretion from primary human macrophages. Structurally distinct inhibitors of PKA (H89, KT5720, inhibitory peptide PKI14-22) all decreased basal secretion of apoE by between 50-80% whereas apoE mRNA or cellular protein are unaffected. Disruption of PKA-AKAP anchoring also significantly inhibited apoE secretion from human macrophages. Secretion of apoE was not immediately stimulated by PKA activity, suggesting that although PKA activity may be permissive for apoE secretion, it is in itself insufficient to stimulate apoE secretion above basal levels. Data from confocal microscopy and live cell imaging revealed PKA inhibition paralysed apoE vesicular movement from and to the plasma membrane.
In Chapter Four, I investigated the effects of protein phosphatase 2B (PP2B) inhibition on apoE secretion by cyclosporin A (CsA). This was found to dose- and time-dependently inhibit secretion of apoE from primary human macrophages and increased cellular accumulation of apoE without affecting apoE mRNA levels. The role of PP2B in regulating apoE secretion was confirmed by using additional peptide and chemical inhibitors of PP2B. This effect was independent of the known inhibition of ABCA1 by CsA. Live cell imaging and confocal microscopy all demonstrated that inhibition of PP2B did not affect the apparent cellular distribution of apoE. Biochemical and microscopy studies indicated distinct mechanisms for PKA and PP2B regulation of apoE secretion.
Chapter Five identified PKA-anchoring AKAPs in human macrophages, and investigated AKAP220 expression and its role in PKA-dependent processes relevant to atherosclerosis. AKAP220 protein was absent in human monocytes but was detectable after their differentiation into macrophages, with stable expression during late stages of maturation. It was also present in Chinese Hamster Ovary cells (CHO) cells. AKAP220 silencing had no effects on lipoprotein cholesteryl ester accumulation, total cellular apoE levels, apoE secretion or cholesterol efflux from human macrophages. Confocal microscopy in CHO cells revealed peroxisomal localisation of AKAP220. Catalase activity was confirmed to be PKA-regulated process, and AKAP220 was found to be a negative regulator of catalase activity, such that cell lysate catalase activity increased during AKAP220 silencing. AKAP220 silencing also decreased basal secretion of H2O2, detected using a sensitive and specific Amplex® Red assay kit from intact CHO monolayers.
In conclusion, this thesis has provided evidence that apoE secretion occurs via PKA- and PP2B-dependent pathways in human macrophages, and has identified the A-kinase anchoring protein AKAP220 as a regulator of cellular H2O2 clearance. These results will provide a basis for future investigations into the roles of PKA-related pathways in apoE secretion and catalase activity.