Abstract
Cellular processes such as proliferation, apoptosis and differentiation are the basis for normal cellular behavior and are regulated by a number of extracellular stimuli such as hormones, cytokines and growth factors. Via binding to (transmembrane) receptors these factors lead to regulation of intracellular signaling pathways ultimately resulting in gene expression.
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When cells malfunction in one or more processes or when the balance between different processes is disturbed this can lead to abnormal cell behavior and pathological phenotypes such as cancer, obesity, diabetes and premature aging. This malfunctioning can arise when the activity of proteins involved in the pathways is changed due to altered expression, or mutations. The various extracellular factors can activate different intracellular pathways that in turn can converge or diverge between one another. One of the major kinases involved in insulin and other growth factor signaling is phosphoinositide-3-kinase (PI3K). This lipid kinase can recruit protein kinase B (PKB) to the plasma membrane where it is phosphorylated by other kinases. Upon activation PKB in turn can phosphorylate a variety of proteins in a so-called PKB motif (R-X-R-X-X-S/T), thereby regulating their activity. These PKB substrates act on cellular process such as proliferation, cell survival, protein translation but also on for instance transcription. Transcription factors that are described to be phosphorylated by PKB are members of the Forkhead box O (FOXO). Upon phosphorylation these factors are excluded from the nucleus and subsequently inactivated.
In Chapter 2 the identification of a novel interaction partner for PKB is described. In a binding screen the plakin family member periplakin was found to interact with PKB. The interacting regions have been narrowed down to nine amino acids within the PH domain of PKB, and the carboxyterminus of periplakin. Periplakin is present in distinct cellular compartments such as nucleus, plasma membrane, mitochondria and the intermediate filament network through direct binding to vimentin. Binding of PKB to periplakin does not affect PKB activation but can inhibit PKB-mediated phosphorylation and subsequent inactivation of FOXO transcription factors.
Chapter 3 describes an in silico approach to identify novel substrates for PKB and target genes for FOXO factors. To identify downstream targets of PKB, proteins containing the consensus PKB motif (RXRXXS/T) were selected. This selection was narrowed down on the basis of their known function, and evolutionary conservation of the motifs was assessed. Two potential PKB substrates selected for further analysis are Pit-1 and Prop-1. To identify novel FOXO target genes, microarray analysis was performed using a cell line containing an inducible active FOXO3a. Results from this array were compared with other relevant microarrays and putative target genes were checked for the presence of FOXO binding elements in the promoter region.
In Chapter 4, the analysis of Pit-1 and Prop-1 as putative PKB substrates is described. Pit-1 and Prop-1 are pituirary-specific homeodomain transcription factors involved in the production of growth hormone and other pituitary-specific hormones. Both proteins contain PKB consensus motifs and are indeed phosphorylated in vitro by PKB. In vivo phosphorylation however is not observed neither are any changes in localization or transcriptional activity.
Chapter 5 deals with the validation of a novel FOXO target gene encoding for caveolin-1. Caveolin-1 expression is induced upon FOXO activation and downregulated by insulin-dependent PI3K/PKB signaling on both mRNA and protein levels. Regulation of caveolin-1 transcription occurs by direct binding of FOXO to binding elements in the caveolin-1 promoter region. The regulation of caveolin-1 expression results in functional attenuation of EGF signaling.
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