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Background Research |
| Professor Dario Alessi |
| E: d.r.alessi@dundee.ac.uk |
| T: 44 1382 385602 |
| F: 44 1382 223778 |
Professor Dario Alessi FRS, FRSE - Honorary Programme Leader
Research
Understanding the functions of protein kinases mutated in inherited disorders and exploiting this information to develop improved treatments for disease
Protein kinases are the largest family of enzymes encoded by the human genome and their role is to catalyse the covalent attachment of phosphate to specific amino acid residues in target proteins. This modifies the functions of the target proteins in almost all conceivable way and hence the physiological processes in which they participate.
Recent clinical studies have identified alterations in genes that encode enzymes called "kinases", which cause diseases such cancer, hypertension and Parkinson’s disease (Fig 1). Little is known about how these “kinases” are regulated and how they operate in normal cells. Our research is aimed at understanding the regulation and function of these enzymes and how their alteration leads to disease. This information could lead to new fundamental understanding of the causes of disease that might be exploited to develop new approaches to treat these diseases. Our laboratory utilizes the state of the art biochemistry, mouse genetics-physiology, mass spectrometry and signal transduction technology to address these questions.
(Figure 1)
One kinase our laboratory is very interested in is LKB1, which is mutated in some cancers. A highlight of our recent research has been the discovery of how the protein kinase LKB1 works. We found that, to be active, LKB1 must exist as a complex with two other proteins, termed STRAD and MO25 [1]. We have recently solved the crystal structure of the STRAD and MO25 complex [2]. This allows LKB1 to activate AMPK, another protein kinase that is the major sensor of the energy status of living cells. By creating a strain of mice that do not express LKB1 in muscle, we were able to demonstrate that LKB1 and AMPK regulate the uptake of glucose into muscle during exercise. We have also demonstrated that LKB1 not only activates AMPK, but 12 other related protein kinases (Fig 2). These include protein kinases that control cell polarity and have been implicated in producing “neurofibrillary tangles”, which are the deposits found in the brains of patients with Alzheimer’s disease. We have recently been able to demonstrate that drugs such as clinically approved metformin as well as phenformin and A769662 that induce LKB1 to activate AMPK markedly suppress tumour formation in cancer prone PTEN+/- mice [3]. This suggests these drugs could be used in the clinic to prevent or treat tumours. Further work into whether these drugs would have benefit for the treatment of cancer and if they could be used in combination with other inhibitors of signal transduction is a key future research goal of our laboratory. Our future studies will also focus on defining the cellular functions of the protein kinases that are activated by LKB1, which are poorly understood at present. We will also investigate how these enzymes are regulated by an unusual form of ubiquitination that we have recently discovered [4].
(Figure 2)
We are also dissecting the roles the WNK1 and WNK4 gene that is mutated in patients with Gordon’s syndrome, an inherited hypertension syndrome [5]. We have recently discovered that WNK1 is activated in response to hyperosmotic stress and phosphorylate and activate two other closely related protein kinases termed SPAK and OSR1 [6]. Fascinatingly, SPAK and OSR1 are activated in cells in response to hyperosmotic stress and phosphorylate and regulate the activity of ion co-transporters such as NCC that is the drug target for commonly deployed thiazide hypertension drugs [7] (Fig 3). It is possible that mutations in WNK1 and WNK4 induce hypertension by activating the SPAK/OSR1 kinases leading to the stimulation of NCC ion cotransporter activity and kidney salt retention. We are further investigating this by characterizing the phenotype of mice deficient in components of the WNK pathway. We would also like to identify novel cellular substrates for the SPAK and OSR1 protein kinases and elucidate the molecular mechanism by which WNK1 can sense hyperosmotic stress.
(Figure 3)
Recent findings demonstrate that autosomal dominant point mutations within the gene encoding for the Leucine Rich Repeat protein Kinase-2 (LRRK2), predispose humans to develop Parkinson’s disease (PD). Mutations in LRRK2 are frequent, accounting for ~5% of familial PD, and are also found in sporadic PD (Fig 4). Little is known about how LRRK2 is regulated, what are its substrates and how mutations cause PD. The most prevalent mutant form of LRRK2 comprises an amino acid substitution of Gly2019 that enhances LRRK2 kinase activity 3-fold indicating that LRRK2 inhibitors might have potential for the treatment of Parkinson’s disease [8]. We wish to study LRRK2 expression, activity and investigate which agonists and signalling pathways control its functions. We will undertake screens to identify LRRK2 substrates as well as binding partners and establish the relevance of these in controlling function. We also plan to develop mass spectrometry technologies to study global phosphorylation of proteins in cell and tissue extracts to identify LRRK2 target proteins whose phosphorylation is affected by manipulations of LRRK2 expression.
We are also interested in understanding the regulation and function of another protein kinase of unknown function termed TTBK2 [9]. Mutations in TTBK2 cause the Spinocerebellar ataxia Type 11 movement disorder.
(Figure 4)
In the majority of cancers the mTOR signalling pathway is elevated that contributes to the growth and survival of cancer cells (Fig 5). We are actively involved in studying the regulation and function of the different mTOR complexes and how cancer causing mutations leads to their activation [10, 11]. We are also interested in developing mass spectroscopy methodology so that the effects that PI 3-kinase, PDK1, Akt, MEK and mTOR inhibitors and/or activating AMPK have on the global phosphorylation of proteins in cancer cells as well as cancer prone mice can be assessed.
(Figure 5)
All of the AMPK-related protein kinases activated by LKB1, including SIK (salt-induced kinase), MARK (microtubule-affinity-regulating kinase) and BRSK (brain-specific kinase) isoforms, possess a ubiquitin-associated (UBA) domain or a domain that is likely to be structurally related to this domain immediately C-terminal to the kinase catalytic domain. These are the only protein kinases in the human genome that possess a UBA domain. Our studies suggest that the UBA domain is likely to play crucial roles in controlling the activity AMPK-related kinases and its integrity is also required for LKB1 to phosphorylate and activate these enzymes [5].
We have recently observed that AMPK, as well as many of the AMPK-related kinases, such as NUAK1 and MARK4, are polyubiquitylated in vivo. Topological analysis revealed that ubiquitin monomers attached to NUAK1 and MARK4 are linked by Lys29 and/or Lys33 rather than the more common Lys48/Lys63 [6]. We have also found that MARK4 and NUAK1 interact with the deubiquitylating enzyme USP9X and provided evidence they are deubiquitylated by USP9X. One hypothesis that we would like to investigate is whether polyubiquitin chains attached to NUAK1 or MARK4, can interact with the UBA domain of these kinases leading to the inhibition of these enzymes (Figure 2). We are also keen to investigate how AMPK family kinases are regulated by these unusual Lys29/Lys33-linked polyubiquitin chains and whether these chains play scaffolding roles to recruit and colocalise other substrates or regulators. We would also like to how USP9X is regulated and to establish the identity of the ubiquitin ligases that introduce the Lys29 and Lys33-conjugated ubiquitin chains to NUAK1 and MARK4.
References
[1] Alessi, D. R., Sakamoto, K. and Bayascas, J. R. (2006) LKB1-Dependent Signaling Pathways. Annu Rev Biochem 75, 137-63 Abstract
[2] Zeqiraj, E., Filippi, B. M., Goldie, S., Navratilova, I., Boudeau, J., Deak, M., Alessi, D. R. and van Aalten, D. M. (2009) ATP and MO25alpha regulate the conformational state of the STRADalpha pseudokinase and activation of the LKB1 tumour suppressor. PLoS Biol 7, e1000126 Abstract
[3] Huang, X., Wullschleger, S., Shpiro, N., McGuire, V. A., Sakamoto, K., Woods, Y. L., McBurnie, W., Fleming, S. and Alessi, D. R. (2008) Important role of the LKB1-AMPK pathway in suppressing tumorigenesis in PTEN-deficient mice. Biochem J 412, 211-221 Abstract
[4] Al-Hakim, A. K., Zagorska, A., Chapman, L., Deak, M., Peggie, M. and Alessi, D. R. (2008) Control of AMPK-related kinases by USP9X and atypical Lys(29)/Lys(33)-linked polyubiquitin chains. Biochem J 411, 249-260 Abstract
[5] Richardson, C. and Alessi, D. R. (2008) The regulation of salt transport and blood pressure by the WNK-SPAK/OSR1 signalling pathway. J Cell Sci 121, 3293-3304 Abstract
[6] Zagorska, A., Pozo-Guisado, E., Boudeau, J., Vitari, A. C., Rafiqi, F. H., Thastrup, J., Deak, M., Campbell, D. G., Morrice, N. A., Prescott, A. R. and Alessi, D. R. (2007) Regulation of activity and localization of the WNK1 protein kinase by hyperosmotic stress. J Cell Biol 176, 89-100 Abstract
[7] Richardson, C., Rafiqi, F. H., Karlsson, H. K., Moleleki, N., Vandewalle, A., Campbell, D. G., Morrice, N. A. and Alessi, D. R. (2008) Activation of the thiazide-sensitive Na+-Cl- cotransporter by the WNK-regulated kinases SPAK and OSR1. J Cell Sci 121, 675-684 Abstract
[8] Jaleel, M., Nichols, R. J., Deak, M., Campbell, D. G., Gillardon, F., Knebel, A. and Alessi, D. R. (2007) LRRK2 phosphorylates moesin at threonine-558: characterization of how Parkinson's disease mutants affect kinase activity. Biochem J 405, 307-317 Abstract
[9] Houlden, H., Johnson, J., Gardner-Thorpe, C., Lashley, T., Hernandez, D., Worth, P., Singleton, A. B., Hilton, D. A., Holton, J., Revesz, T., Davis, M. B., Giunti, P. and Wood, N. W. (2007) Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11. Nat Genet 39, 1434-1436
[10] Garcia-Martinez, J. M. and Alessi, D. R. (2008) mTOR complex-2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum and glucocorticoid induced protein kinase-1 (SGK1). Biochem J 416, 375-85 Abstract
[11] Garcia-Martinez, J. M., Moran, J., Clarke, R. G., Gray, A., Cosulich, S. C., Chresta, C. M. and Alessi, D. R. (2009) Ku-0063794 is a specific inhibitor of the mammalian target Abstract
University of Dundee