University of Dundee

Professor Dario Alessi FRS, FRSE

Understanding signalling pathways mutated in inherited disorders
Position: 
Professor of Signal Transduction and Director of MRC Protein Phosphorylation and Ubiquitylation Unit (MRC-PPU) and the Division of Signal Transduction Therapy Unit (DSTT)
Address: 
College of Life Sciences, University of Dundee, Dundee
Full Telephone: 
+44 (0) 1382 3888058, int ext 88058

Research

Overview

My laboratory focuses on unravelling the roles that protein phosphorylation or ubiquitylation pathway components that emerge from the genetic analysis of human disease play.

We currently focus on understanding signalling pathways associated with neurodegenerative movement disorders (LRRK2, Fbxo7, PINK1, Parkin, TTBK2), hyperstension (WNK1, WNK4, SPAK, OSR1, Cullin3 and KLHL3) and cancer (PDK1, Akt, SGK, mTOR, LKB1, ARK5/NUAK1 and AMPK).

The aim of our research is to work out how these pathways are organised, how they recognise signals, how the signal moves down the pathway to elicit physiological responses and to comprehend what goes wrong in human disease. I hope that these findings will enable researchers to play the engineer in devising new strategies to treat disease.

To help elaborate chemical inhibitors that specifically disrupt the signalling components that we are working with, we collaborate with the pharmaceutical companies supporting the Division of Signal Transduction Therapy (DSTT) and chemical biologists such as Nathanael Gray (University of Harvard). These tool compounds greatly aid with deciphering the physiological roles that signalling pathways play and establish to what extent inhibiting specific signalling networks suppresses disease.

On all of our projects we aim to collaborate with leading clinicians to ensure that our research is addressing the most important clinical issues and where possible access patient derived cells or tissues to learn more about how disruptions of pathways are linked to disease.

Our laboratory employs state of the art biochemical and molecular technologies required to dissect signal transduction pathways. Our focus is analysing function of endogenous components and we try and avoid relying on non-robust over-expression or siRNA methodology that more often than not results in irrelevant non-physiological artefacts being observed.  To help with this we make extensive use of genetic knock-in technology and mass spectrometry analysis of endogenous proteins.

My aim is to train researchers at the PhD or Postdoc level who have the ambition to go on to become successful future research leaders.

Neurodegeneration movement disorders

One of the greatest challenges of current medical research is to understand neurodegenerative disorders such as Parkinson’s disease. There is a great need for research in this area, as effective treatments that slow the progression of these conditions do not exist. The number of patients in Europe suffering with a neurodegenerative disorder is expected to double to 14 million in the next 20 years. 

Our laboratory is currently devoting significant effort to dissecting the regulation and function of signal transduction components that have recently been found to be mutated in Parkinson’s disease (LRRK2-See Figure 1 [1-3], PINK1 [4], Parkin [4], FBXO7) and other neurodegenerative movement disorders (TTBK2 - a kinase mutated in spinocerebellar ataxia type 11 [5]).

The key of each of these projects is to understand how these mutated signalling components are controlled, what they interact with, what their physiological function is and how mutations lead to neurodegeneration.

Figure 1

Figure 1 Understanding the Parkinson's disease LRRK2 protein kinase
Protein kinases cause ~1% of all Parkinson’s disease. A goal of our research is to identify physiological substrates of LRRK2, understand how LRRK2 is controlled and help pharmaceutical companies develop inhibitors of LRRK2 as a potential future therapy for Parkinson’s disease. Our recent data strongly indicates that LRRK2 regulates a protein kinase kinase or phosphatase that targets two phosphorylated residues on LRRK2 (Ser910 and Ser935) that control 14-3-3 binding (refs 1-3).
The hunt is on to identify these and/or other LRRK2 substrates and then work out how this is linked to Parkinson’s disease. LRRK2 is also interesting as it is the only kinase that also possesses a GTPase domain of unknown function within the same polypeptide chain as a kinase.

Hypertension

Gordon’s hypertension syndrome is caused by mutations that increase expression of WNK1 protein kinase as well as specific missense mutations lying within a non-catalytic region of the WNK4 protein kinase. Patients with this condition suffer from high blood pressure and hyperkalemia (high serum potassium), and can be treated using thiazide diuretic hypertension drugs that inhibit the NCC ion co-transporter in the kidney [6].

We have discovered that WNK1 is activated in response to hyperosmotic stress and phosphorylates and activate two other closely related protein kinases termed SPAK and OSR1 [7]. We have also found that 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 [8] (Fig 2).

Our findings indicated that mutations in WNK1 and WNK4, induce hypertension by activating the SPAK/OSR1 kinases leading to the stimulation of NCC ion co-transporter activity and kidney salt retention. To validate this idea we have generated knock-in mice in which SPAK and OSR1 cannot be activated by WNK isoforms. Excitingly, these mice display low blood pressure, reduced phosphorylation of NCC and also show signs of increased salt excretion [9]. This confirms that the WNK-SPAK/OSR1 pathway plays a fundamental role in regulating blood pressure and inhibitors of SPAK/OSR1 are attractive targets for the treatment of hypertension.

 

Figure 2 Signalling networks controlled by WNK kinases

Important recent genetic studies have identified over 50 patients, in which Gordon’s syndrome was caused by mutations in Ubiquitin E3 ligase components termed Cullin-3 (CUL3) or Kelch-like 3 (KLHL3) rather than WNK isoforms. Previous work suggested that CUL3 and KLHL3 form a heterodimeric complex with CUL3 mediating the ubiquitylation of substrates and the KLHL3 subunit operating as the substrate recognition moiety.

In exciting recent work we have found that the CUL3:KLHL3 interacts strongly with and ubiquitylates WNK isoforms [10]. We have also observed that most KLHL3 disease mutations analysed that elevate blood pressure in patients strongly inhibited binding to either WNK isoforms or CUL3, indicating that interaction of WNK isoforms with KLHL3 is relevant to Gordon’s syndrome [10]. We also found that disease mutant CUL3:KLHL3 complexes failed to ubiquitylate WNK1 in vitro [10].

Interestingly, the KLHL3 binding site on WNK isoforms encompasses residues that are mutated in Gordon syndrome patients. Strikingly, the Gordon’s disease causing WNK4[E562K] and WNK4[Q565E] mutations prevent ability to interact with KLHL3 [10].

These results suggest that mutations in WNK4 causing hypertension exert their effects by hindering the interaction with KLHL3:CUL3. More work is required to establish this concept, but our prediction is that missense mutations in WNK4 exert their physiological effects by ablating KLHL3 binding thereby leading to reduced ubiquitylation and hence enhanced expression of WNK4. If this was the case it could result in inappropriate activation of the SPAK/OSR1 kinases resulting in overstimulation of the NCC/NKCC2 ion co-transporters.  A summary of how CUL3:KLHL3 regulates WNK isoforms is shown in Figure 3.

Figure 3 Signalling networks controlled by WNK kinases

A key aim for future work will be to generate and analyse CUL3:KLHL3 mutation knock-in mice to learn how mutations that prevent interaction with WNK isoforms impact on WNK signalling pathway. We also want to understand how CUL3:KLHL3 is regulated and identify novel cellular substrates for the SPAK and OSR1 protein kinases. We are also interested in elucidating the molecular mechanism by which WNK1 is activated and can sense hyperosmotic stress. Finally we would like to devote considerable effort to aiding pharmaceutical develop SPAK/OSR1 inhibitors and test whether such compounds are effective in lowering blood pressure in mice.

Cancer

Our laboratory has been heavily involved in investigating and defining the roles of the PDK1 [11, 12], mTOR [13, 14] and LKB1 signalling pathways [15,16, 17, 18]. Several PDK1-mTOR pathway inhibitors are currently being evaluated in anti-cancer clinical trials. A priority for our group will be to understand which cancers are most effectively treated with specific signalling inhibitors. We will also investigate how cancer cells acquire resistance to signalling inhibitors that are being evaluated in clinical trials and we plan to work closely with our DSTT company collaborators on these projects. A highlight of our recent work is that we have shown that overexpression of a kinase termed SGK1 and monitoring phosphorylation of its substrate NDRG1, can be used to predict whether breast cancer cells that have mutations that activate the PI 3-kinase are sensitive to Akt inhibitors [19].

Figure 4 LKB operates as a master upstream kinase controlling 12 downstream kinases (ref 15)

We are also interested in learning more about how LKB1:STRAD;MO25 complex is regulated by phosphorylation and  farnesylation. Finally we would like to define the role that the LKB1 regulated NUAK1/ARK5 kinase plays in promoting proliferation of cancer cells, based on recent work that suggests knock-down of NUAK1/ARK5 selectively induces apoptosis of myc-driven tumour cells.

Figure 5 Structure of the LKB1:STRAD:MO25 comples (ref 16)

References

  1. Dzamko, N., Deak, M., Hentati, F., Reith, A. D., Prescott, A. R., Alessi, D. R and Nichols, R. J. (2010) Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization. Biochem J. 430, 405-413 Abstract
  2. Deng, X., Dzamko, N., Prescott, A., Davies, P., Liu, Q., Yang, Q., Lee, J. D., Patricelli, M. P., Nomanbhoy, T. K., Alessi, D. R and Gray, N. S. (2011) Characterization of a selective inhibitor of the Parkinson's disease kinase LRRK2. Nat Chem Biol. 7, 203-205 Abstract
  3. Dzamko, N., Inesta-Vaquera, F., Zhang, J., Xie, C., Cai, H., Arthur, S., Tan, L., Choi, H., Gray, N., Cohen, P., Pedrioli, P., Clark, K. and Alessi, D. R (2012) The IkappaB kinase family phosphorylates the Parkinson's disease kinase LRRK2 at Ser935 and Ser910 during Toll-like receptor signaling. PloS one. 7, e39132 Abstract
  4. Kondapalli, C., Kazlauskaite, A., Zhang, N., Woodroof, H. I., Campbell, D. G., Gourlay, R., Burchell, L., Walden, H., Macartney, T. J., Deak, M., Knebel, A., Alessi, D. R and Muqit, M. M. (2012) PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65. Open biology. 2, 120080 Abstract
  5. Bouskila, M., Esoof, N., Gay, L., Fang, E. H., Deak, M., Begley, M. J., Cantley, L. C., Prescott, A., Storey, K. G. and Alessi, D. R (2011) TTBK2 kinase substrate specificity and the impact of spinocerebellar-ataxia-causing mutations on expression, activity, localization and development. Biochem J. 437, 157-167 Abstract
  6. 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
  7. Vitari, A. C., Deak, M., Morrice, N. A. and Alessi, D. R (2005) The WNK1 and WNK4 protein kinases that are mutated in Gordon's hypertension syndrome phosphorylate and activate SPAK and OSR1 protein kinases. Biochem J. 391, 17-24 Abstract
  8. 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
  9. Rafiqi, F. H., Zuber, A. M., Glover, M., Richardson, C., Fleming, S., Jovanovic, S., Jovanovic, A., O'Shaughnessy, K. M. and Alessi, D. R (2010) Role of the WNK-activated SPAK kinase in regulating blood pressure. EMBO Mol Med. 2, 63-75 Abstract
  10. Ohta, A., Schumacher, F. R., Mehellou, Y., Johnson, C., Knebel, A., Macartney, T. J., Wood, N. T., Alessi, D. R and Kurz, T. (2013) The CUL3-KLHL3 E3 ligase complex mutated in Gordon's hypertension syndrome interacts with and ubiquitylates WNK isoforms; disease-causing mutations in KLHL3 and WNK4 disrupt interaction. Biochem J Abstract
  11. Najafov, A., Shpiro, N. and Alessi, D. R (2012) Akt is efficiently activated by PIF-pocket- and PtdIns(3,4,5)P3-dependent mechanisms leading to resistance to PDK1 inhibitors. Biochem J. 448, 285-295 Abstract
  12. Najafov, A., Sommer, E. M., Axten, J. M., Deyoung, M. P. and Alessi, D. R (2011) Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1. Biochem J. 433, 357-369 Abstract
  13. 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, 375-385 Abstract
  14. Pearce, L. R., Sommer, E. M., Sakamoto, K., Wullschleger, S. and Alessi, D. R (2011) Protor-1 is required for efficient mTORC2-mediated activation of SGK1 in the kidney. Biochem J. 436, 169-179 Abstract
  15. Alessi, D. R Sakamoto, K. and Bayascas, J. R. (2006) LKB1-dependent signaling pathways. Annu Rev Biochem. 75, 137-163 Abstract
  16. Zeqiraj, E., Filippi, B. M., Deak, M., Alessi, D. R and van Aalten, D. M. (2009) Structure of the LKB1-STRAD-MO25 complex reveals an allosteric mechanism of kinase activation. Science. 326, 1707-1711 Abstract
  17. Zagórska, A., Deak, M., Campbell, D.G., Banerjee, S., Hirano, M., Aizawa, S., Prescott, A.R. and Alessi, D. R New roles for the LKB1-NUAK pathway in controlling myosin phosphatase complexes and cell adhesion. Sci Signal. 3, ra25. Abstract
  18. Filippi, B.M., de los Heros, P., Mehellou, Y., Navratilova, I., Gourlay, R., Deak, M., Plater, L., Toth, R., Zeqiraj, E., Alessi, D. R MO25 is a master regulator of SPAK/OSR1 and MST3/MST4/YSK1 protein kinases. EMBO J. 30, 1730-1741. Abstract
  19. Sommer, E.M., Dry, H., Cross, D., Guichard, S., Davies, B.R. and Alessi, D. R. (2013) Elevated SGK1 predicts resistance of breast cancer cells to Akt inhibitors. Biochemical Journal 452, 499-508. Abstract

Publications

  1. Najafov, A., Sommer, E.M., Axten, J.M. DeYoung, M.P. and Alessi, D.R. (2011) Characterisation of GSK2334470, a novel and highly specific inhibitor of PDK1.Biochemical Journal 433, 357-369 
  2. Richardson, C., Sakamoto, K., de los Heros, P., Deak, M., Campbell, D.G., Prescott, A.R. and Alessi, D.R. (2011) Regulation of NKCC2 ion cotransporter by SPAK/OSR1 dependent and independent pathways Journal of Cell Science 124, 789-800. 
  3. Wullschleger, S., Wasserman, D.H., Gray, A., Sakamoto, K. and Alessi, D.R. (2011) Role of TAPP1 and TAPP2 adaptors binding to PtdIns(3,4)P2 in regulating insulin sensitivity defined by knock-in analysis.Biochemical Journal 434, 265-274. 
  4. Filippi, B.M., de los Heros, P., Mehellou, Y., Navratilova, I., Gourlay, R., Deak, M., Plater, L., Toth, R., Zeqiraj, E. and Alessi, D.R. (2011) MO25 is a master regulator of SPAK/OSR1 and MST3/MST4/YSK1 protein kinases. EMBO J 30, 1730-1741.
  5. Pearce, L.R, Sommer, E.M., Sakamato, K. and Alessi, D.R. (2011) Protor-1 is required for efficient mTORC2-mediated activation of SGK1 in the kidney.Biochemical Journal 436, 169-179. 
  6. Bouskila, M., Esoof, N., Gay, L., Fang, E.H., Deak, M., Begley, M., Cantley, L.C., Prescott, A.R., Storey K.G. and Alessi, D.R. (2011) TTBK2 kinase substrate specificity and the impact of spinocerebellar ataxia-causing mutations on expression, activity, localisation and development.Biochemical Journal 437, 157-167 
  7. Dzamko, N., Inesta-Vaquera, F., Zhang, J., Xie, C., Cai, H., Arthur, S., Cohen, P., Pedrioli, P., Clark, K., and Alessi, D.R. (2012) The IkappaB kinase family phosphorylates the Parkinson’s disease kinase LRRK2 during Toll-like receptor signaling PloS one 7: e39132 
  8. Najafov, A., Shpiro, N. and Alessi, D. R. (2012). Akt is efficiently activated by PIF-pocket- and PtdIns(3,4,5)P3-dependent mechanisms leading to resistance to PDK1 inhibitors.Biochemical Journal 448, 285-295.
  9. Sommer, E.M., Dry, H., Cross, D., Guichard, S., Davies, B.R. and Alessi, D. R. (2013) Elevated SGK1 predicts resistance of breast cancer cells to Akt inhibitors.Biochemical Journal 452, 499-508.
  10. Houde, V,P., Ritorto, M,S., Gourlay,. R, Varghese, J., Davies, P., Shpiro, N., Sakamoto, K. and Alessi, D. R. (2014) Investigation of role that LKB1 Ser431 phosphorylation and Cys433 farnesylation play by mouse knock-in analysis reveals an unexpected role of prenylation in regulating AMPK activity.Biochemical Journal 458, 41-56.

 

Impact

Commercial Impact:

In collaboration with the pharmaceutical industry via the Division of Signal Transduction Therapy collaboration with AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Janssen Pharmaceutica, Merck Serono and Pfizer the research ouptuts from my group contribute to accelerating the development of company drug development programmes through access to research data and reagents. Reagents are also commercialised to provide access to the wider scientific community via license arrangements with companies such as Millipore, AbCam and Ubiquigent.