CC2D1A (coiled-coil and C2 domain containing protein 1A), also known as Freud-1, has been identified as a transcriptional repressor of the serotonin receptor 5-HT1A, a regulator of endosomal budding and an activator of NF-KB signaling. It also acts as a scaffold that promotes activity of the PI3K/Akt pathway upon stimulation by the epidermal growth factor (EGF). Moreover, several studies highlight naturally occurring mutations of CC2D1A in humans that produce varying degrees of intellectual disorder and autism.
Use of the Cre-LoxP system to conditionally knockout CC2D1A in mice has provided promising results regarding its effect on 5-HT1A expression and behaviour. This thesis aims to extend the use of this knockout model by studying cell signaling activity in mouse embryonic fibroblasts (MEFs), derived from the CC2D1Aflx/flx transgenic line, that have been treated with a commercially available Cre recombinase to completely knock out CC2D1A. I hypothesize that CC2D1A directly regulates EGF receptor activity and that its Cre-mediated knock down in vitro will entirely block cell signaling pathways activated by the EGF receptor.
Western blot analysis demonstrated that, after Cre-mediated CC2D1A knockout, Akt and Erk1/2 phosphorylation were still maintained upon EGF treatment. In addition, overexpressing Freud-1 via transfection had no effect on cell signaling compared to the wild-type control. Analysis of recombinant Freud-1 constructs reveal that a C-terminal truncation enhances its ability to bind to PIP2 and PIP3 – phospholipids essential to the Akt pathway. In addition, immunocytochemistry analysis demonstrates a responsiveness of CC2D1A to EGF treatment. Altogether, these data highlight a unique and effective way in carrying out gene knockout in vitro while also emphasizing the need to further investigate CC2D1A’s importance in regulating cell signaling pathways and functional compensation by other homologous proteins
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/33400 |
| Date | January 2015 |
| Creators | Hashim, Irshaad |
| Contributors | Albert, Paul |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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