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 expertice 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 knockin 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.
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 J. 26(9)2251-61
Frodin M. (2007). A RSK kinase inhibitor reporting its selectivity in vivo. Nat Chem Biol. 3(3):138-9.
Duemmler, B.A., Hauge, C., Silber, J., Yntema, H.G., Kruse, L.S., Kofoed, B., Hemmings, B.A., Alessi, D.R. and Frodin M. (2005). Functional characterization of human RSK4, a new 90 kDa ribosomal S6 kinase, reveals constitutive activation in most cell types. J. Biol. Chem., 280:13304-13314
Frodin, M., Antal, T.L., Dümmler, B.A., Jensen, C.J., Deak, M., Gammeltoft, S. and Biondi, R. (2002). A phosphoserine/threonine binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation. EMBO Journal, 21:5396-5407.
Frodin, M., Jensen, C.J., Merienne, K. and Gammeltoft, S. (2000). A phosphoserine-regulated docking site in the protein kinase RSK2 that recruits and activates PDK1. EMBO Journal, 19:2924-2934.