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Signalling proteins are intrinsic to all biological processes and interact with each other in tightly regulated and orchestrated signalling complexes and pathways. Characterization of protein binding can help to elucidate protein function within signalling pathways. This information is vital for researchers to gain a more comprehensive knowledge of cellular networks which can then be used to develop new therapeutic strategies for disease. However, studying protein-protein interactions (PPIs) can be challenging as the interactions can be extremely transient downstream of specific environmental cues. There are many powerful techniques currently available to identify and confirm PPIs. Choosing the most appropriate range of techniques merits serious consideration. The aim of this review is to provide a starting point for researchers embarking on a PPI study. We provide an overview and point of reference for some of the many methods available to identify interactions from in silico analysis and large scale screening tools through to the methods used to validate potential PPIs. We discuss the advantages and disadvantages of each method and we also provide a workflow chart to highlight the main experimental questions to consider when planning cell lysis to maximize experimental success.

Despite extensive efforts, Protein Kinase Cs (PKCs) have proven to be an intractable target in cancer therapies. Traditionally it was accepted that PKCs act as tumour promoters, however new research suggests that PKCs may play an important role in the suppression of cancer. A challenge in targeting PKCs is the limited data available in patient samples. One of the PKC isozymes, PKC gamma, is thought to be present only in the brain and has been largely neglected in the context of cancer. Analysis of gene expression levels of PKC gamma in patient matched normal and colon cancer tissue samples revealed an up-regulation of the gene in the cancer tissue of 54% of the patients examined. Mechanistically we demonstrate that a reduction in the levels of PKC gamma in the colon cancer cells inhibits cell migration and foci formation. Further to this, we observe an increase in cell adhesion and proliferation following the reduction of PKC gamma levels in the cell. Thus, PKC gamma plays a key role in colon cancer; making it an important isozyme that needs to be reconsidered in the context of cancer therapies.

. (2017) RACK1 stabilises the activity of PP2A to regulate the transformed phenotype in mammary epithelial cells. Cellular Signalling, 35, pp. 290-300. (doi:10.1016Signalling, 35, pp. 290-300. (doi:10. /j.cellsig.2016 This is the author's final accepted version.There may be differences between this version and the published version. You are advised to consult the publisher's version if you wish to cite from it.http://eprints.gla.ac.uk/128660/ RACK1/PP2A complex is required for cell adhesion, proliferation, migration, invasion.Potential role for the RACK1/PP2A complex in the progression of breast cancer. KeywordsBreast Cancer, RACK1, PP2A, Protein-Protein Interactions. ABSTRACTConflicting reports implicate the scaffolding protein RACK1 in the progression of breast cancer. RACK1 has been identified as a key regulator downstream of growth factor and adhesion signalling and as a direct binding partner of PP2A. Our objective was to further characterise the interaction between PP2A and RACK1 and to advance our understanding of this complex in breast cancer cells. We examined how the PP2A holoenzyme is assembled on the RACK1 scaffold in MCF-7 cells. We used immobilized peptide arrays representing the entire PP2A-catalytic subunit to identify candidate amino acids on the C subunit of PP2A that might be involved in binding of RACK1. We identified the RACK1 interaction sites on PP2A. Stable cell lines expressing PP2A with FR69/70AA, R214A and Y218F substitutions were generated and it was confirmed that the RACK1/PP2A interaction is essential to stabilize PP2A activity. We used Real-Time Cell Analysis and a series of assays to demonstrate that disruption of the RACK1/PP2A complex also reduces the adhesion, proliferation, migration and invasion of breast cancer cells and plays a role in maintenance of the cancer phenotype. This work has significantly advanced our understanding of the RACK1/PP2A complex and suggests a pro-carcinogenic role for the RACK1/PP2A interaction. This work suggests that approaches to target the RACK1/PP2A complex are a viable option to regulate PP2A activity and identifies a novel potential therapeutic target in the treatment of breast cancer.

RACK1 (Receptor for Activated C Kinases) is a scaffolding protein with 7 WD repeats that interacts with the Insulin-like Growth Factor I receptor (IGF-IR), integrins, and other signalling proteins. RACK1 functions as a scaffolding protein and regulator of many key biological processes and it is highly expressed in most tissue. We recently reported that RACK1 is essential for IGF-I-mediated regulation of PP2A activity and AGAP2 activity at focal adhesions. Downstream of IGF-1R signaling in cancer cells, the scaffolding properties of RACK1 are altered providing distinct migratory advantages to the cell and suggesting that RACK1 is an important regulator of IGF-I signalling in cancer progression. It is important to further characterise the role of RACK1 in cancer and to delineate the molecular mechanisms by which it regulates key signalling pathways. We have identified Protein Kinase A (PKA) as RACK1 interacting protein. PKA is a complex, multicomponent enzyme which is a fundamental protein that functions in cell survival, proliferation, and cytoskeletal remodelling among others. Our hypothesis is that RACK1 regulates the PKA axis in cancer cells leading to Akt and MAP Kinase activation downstream of IGF-1R signalling. We have demonstrated that RACK1 is required to mediate PKA activity in colon cancer cells. Although we show that PKA expression is not altered in colon cancer tissue, we believe that PKA activity is altered significantly in the disease state. Using PKA inhibitors, we have shown that PKA regulates IGF-I-mediated MAP Kinase activation in colon cancer cell lines. In conclusion, this research project will help elucidate the role of PKA in colon cancer. Inhibiting or promoting specific protein interactions with RACK1 will provide very novel therapeutic opportunities and anti-cancer drug targets. Citation Format: Sheri L. Hayes, Catríona M. Dowling, John C. Coffey, Patrick A. Kiely. RACK1 regulates PKA activity to drive tumorigenesis in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2382. doi:10.1158/1538-7445.AM2017-2382