Frödin Group – University of Copenhagen

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Frödin Group

We are interested in determining how protein kinase cascades activated by extracellular signals regulate normal cell functions and how dysregulation of these protein kinases contributes to cancer, diabetes and other important diseases.

The crucial role of abnormal protein phosphorylation in human disease is underscored by the fact that protein kinases are now the largest group of drug targets in the pharmaceutical industry.

Our main expertise is protein kinase cascades downstream of the PI-3 kinase and RAS proto-oncogenes, which are activated by growth factors, insulin and, many regulatory peptides and neurotransmitters. However, we also perform “kinome-wide” screens to reveal roles of other of the 500 human or 240 Drosophila protein kinases in processes important to cancer or other diseases.

In our research, we employ a wide array of techniques including functional, large-scale RNA interference screens in various cell culture model systems, biochemical and molecular cellular analysis, genome-wide approaches like Solexa/Illumina tag sequencing for mRNA expression profiling or high-resolution mapping of histone modifications important for epigenetic control of gene expression as well as gene-manipulation in mice.

 


 

Examples of current projects:

Induction of a motile, invasive phenotype in epithelial cells by RAS

The protooncogene RAS is capable of inducing a motile, invasive, fibroblastic phenotype in epithelial cells. This biological phenomenon, which may be referred to as the epithelial-to-mesenchymal transition (EMT), is essential for embryonic development and in its dysregulated form of importance for cancer metastasis and various fibrotic diseases. Our group has identified a RAS-activated protein kinase, which is necessary and sufficient for induction of a motile, invasive phenotype in epithelial cells (Doehn et al, 2008, submitted). Moreover, by Solexa/Illumina gene expression profiling and RNA interference experiments, we have demonstrated that this kinase induces its dramatic effects by stimulating the expression of a gene cluster which coordinately promotes motility and invasive capacities of epithelial cells. We are now extending these studies by identifying more RAS-activated kinases or kinase-dependent mechanisms required for induction of a motile, invasive phenotype in epithelial cells by using genome-wide RNA interference screens and/or phosphoproteomics.

Epigenetics and protein kinase cascades

The relatively new field of Epigenetics studies how a multitude of diverse proteins controls the structure of DNA to regulate gene expression. Typically, these proteins either confer covalent modifications (methylation, acetylation etc) onto histones or bind these modifications to exert their regulation. Epigenetic mechanisms are fundamental for normal development and cell function and diseases like cancer. We investigate how extracellular signals control epigenetic regulation of gene expression and cell function via protein kinase cascades. Specifically, we aim to identify the major kinases and the regulatory phosphorylation events/mechanisms, whereby these kinases control eg histone methylation patterns in chromatin and gene expression. We study various cellular models. As one example, we wish to investigate how RAS-activated protein kinases exert epigenetic control of gene expression in epithelial cells and how this contributes to induction of an invasive, metastatic phenotype in carcinoma cells.

Identification of “missing” kinases in important disease signalling pathways

Our laboratory has recently developed a screening strategy to identify new kinases in signalling pathways important to human disease. The screens are performed in Drosophila cells, taking advantage of the strict conservation of the major kinase pathways in this model organism and its lack of genetic redundancy, making screens much more efficient compared to human cells. Briefly, we have generated a double stranded RNA library against all 240 Drosophila kinases. We are now performing RNA interference screens to identify “missing” kinases in growth factor/insulin and oncogene signalling networks relevant to diabetes, cancer, hypertension or cardiac hypertrophy. Once a new kinase component has been identified, we proceed by characterizing it in human cells, evaluating whether it may be a new disease gene or possible drug target.

Generation of novel, genetic mouse models for diseases associated with protein kinase cascades

In a longer-term project, we are generating gene-targeted mice with knocking of activating mutations in either of the genes for AKT (also known as PKB), S6K, SGK RSK and MSK. These mice will represent novel genetic, gain-of-function mouse models that we will use to demonstrate and characterize the role of the kinases in cancer, diabetes, hypertension, chronic inflammation, cardiac hypertrophy and tuberous sclerosis.

 

Til toppen Selected recent publications

1. Engelholm LH, Riaz A, Serra D, Dagnæs-Hansen F, Johansen JV, Santoni-Rugiu E, Hansen SH, Niola F and Frödin M (2017). “CRISPR/Cas9 engineering of adult mouse liver demonstrates that the Dnajb1-Prkaca gene fusion is sufficient to induce tumors resembling fibrolamellar hepatocellular carcinoma”. Gastroenterology, 153:1662-1673

2. Lonowski LA, Narimatsu Y, Riaz A, Delay CE, Yang Z, Niola F, Duda K, Ober EA, Clausen H, Wandall HH, Hansen S, Bennett EP and Frödin M (2017). “Genome editing using FACS enrichment of nuclease expressing cells and Indel Detection by Amplicon Analysis (IDAA)”. Nature Protocols, 12:581-603.

3. Niola F and Frödin M (2017). “Generation of model cell lines using ssODN knockin donors and FACS-based genome editing”. In “Genome editing: from TALENs, ZFNs and CRISPRs to molecular surgery”, edited by Krishnarao Appasani, Cambridge University Press, in the press

4. Frank SR, Kollmann CP, van Lidth de Jeude J, Thiagarajah JR, Engelholm LH, Frödin M and Hansen SH (2017). “The focal adhesion-associated proteins DOCK5 and GIT2 comprise a rheostat in control of epithelial invasion”. Oncogene, 36:1816-28

5. Duda K, Lonowski LA, Kofoed-Nielsen, M., Ibarra A, Delay C, Kang Q, Yang Z, Pruett-Miller SM, Bennett EP, Wandal HH, Davis GD, Hansen SH and Frödin M (2014). ”High-efficiency genome editing via 2A-coupled co-expression of zinc finger nucleases or CRISPR/Cas nickase pairs”. Nucleic Acids Research, 42:e84.

6. Serafimova IM, Pufall MA, Krishnan S, Duda K, Cohen MS, Miller RM, McFarland JM, Maglathlin RL, Frödin M and Taunton J (2012). “Reversible covalent targeting of noncatalytic cysteines with chemically tuned electrophiles”. Nature Chemical Biology, 8:471-6, doi: 10.1038/nchembio.925.

7. Frank SR, Bell JH, Frödin M and Hansen SH (2012). “A bPix-PAK2 complex confers protection against Scrib-dependent and cadherin-mediated apoptosis”. Current Biology, 22:1747-1754.

8. Duda K and Frödin M (2012). “Stimuli that activate MSK in cells and the molecular mechanism of activation”. In “MSKs”, edited by J Simon C Arthur, Madame Curie Bioscience Database, Landes Biosciences.

9. Chen F, Pruett-Miller, SM, Huang Y, Gjoka M, Duda K, Taunton J, Collingwood TN, Frödin M and Davis GD (2011) “High frequency genome editing using ssODNs and zinc finger nucleases”. Nature Methods, 8:753-755

10. Doehn U, Hauge C, Frank SR, Jensen CJ, Duda K, Nielsen JV, Cohen MS, Johansen JV, Winther BR, Lund LR, Winther O, Taunton J, Hansen SH and Frödin M (2009) “RSK is a principal effector of the RAS-ERK pathway to induce a pro-motile/invasive gene program and phenotype in epithelial cells”. Molecular Cell, 35:511-522

11. Hauge C, Antal TL, Hirschberg D, Doehn U, Thorup K, Idrissova L, Hansen K, Jensen ON, Jørgensen TJD, Biondi RM and Frödin M (2007) “Mechanism for activation of the growth factor-activated AGC kinases by turn motif phosphorylation”. EMBO Journal, 26:2251-61

12. Frödin M (2007) “A RSK kinase inhibitor reporting its selectivity in vivo”. Nature Chemical Biology, 3:138-9.