Professor Dario Alessi FRS FRSE
My laboratory focuses on unravelling the roles that protein phosphorylation and ubiquitylation pathway components that emerge from the genetic analysis of human disease play. We are currently focusing on dissecting signalling pathways associated with Parkinson’s Disease (LRRK2, Fbxo7) and other neurodegenerative disorders (TTBK2), cancer (SGK3 pathway) and hypertension (WNK pathway). The aim of our research is to work out how these components function and how they are regulated. We also want to understand how mutations in these components causes human disease. The ultimate goal of our research is to obtain vital new knowledge on how disruptions in signalling pathways cause human disease and to exploit these findings to develop improved strategies to better treat malady.
To ensure we maximise opportunities we frequently collaborate with pharmaceutical companies, drug discovery units and chemical biologists such as Nathanael Gray (University of Harvard) to stimulate the elaboration of chemical inhibitors that specifically target signalling components that we are working with. These tool compounds greatly aid with deciphering the physiological roles that signalling pathways play. They can also be used to determine by what extent inhibiting specific signalling networks suppresses disease. We also aim to collaborate with leading clinicians to ensure that our research is addressing the most important clinical issues of the day. Where possible we will seek access patient derived cells or tissues to learn more about how disruptions of signalling pathways are linked to disease in human patients.
Our laboratory also employs state of the art biochemical and molecular technologies. Our primary focus is to analyse the function of endogenous components. Where possible, we try to avoid having relying on non-robust over-expression or siRNA knock-down methodology that more often than not results in hard to reproduce and physiologically questionable data being obtained. To help with this we make extensive use of genetic knock-in technology including CRISPR/CAS9 gene-editing approaches. We also make extensive use of mass spectrometry analysis of endogenous protein complexes to better understand how pathways are regulated and function.
My aim is to train PhD and Postdoc researchers who have the ambition to go on to become highly successful future research leaders either in academia or pharmaceutical Industry.
Neurodegeneration movement disorders
One of the greatest challenges is to understand neurodegenerative disorders such as Parkinson’s disease. There is a great need for intensive 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 over 15 million in the next 20 years.
Our knowledge of the origins of Parkinson’s has been transformed by the identification of genes whose mutation in humans leads to Mendelian inherited disease (Figure 1). Interestingly many of these genes encode for enzymes involved in signalling pathways including Kinases (LRRK2, PINK1, GAK), components of the Ubiquitylation system (Parkin, UCHL1, Fbxo7) and GTPases (Rab7L1 and Rab39B).
Figure 1 Genes Mutated in Parkinson's disease
Our laboratory is currently devoting significant effort to dissecting the regulation and function of these components signal transduction components that have recently been found to be mutated in Parkinson’s disease. The highlight of our recent work undertaken in collaboration with Matthias Mann, identified the first physiological substrate for the LRRK2 protein kinase by showing that it directly phosphorylates a subset of the Rab GTPases on an a residue lying within the middle of the effector interacting-switch II domain (Figure 2)  . The LRRK2 phosphorylation site is conserved throughout evolution in 50 out of the 70 Rab GTPases. Thus far we have strong evidence for five Rab isoforms being phosphorylated by LRRK2 in vivo (Rab 5B, Rab7L1, Rab8A, Rab10 and Rab12)  . We found that pathogenic mutations in LRRK2 including G2019S mutation in the kinase domain as well as R1441G/C and Y1699C mutations in the GTPase regulatory domain markedly increases phosphorylation of these Rab isoforms at the switch II site  . LRRK2 phosphorylation strongly decreases the affinity of Rab isoforms we have tested to regulatory proteins that bind to the Switch II domain including Rab GDP dissociation inhibitors and the GDP/GTP exchange factor for Rab8 termed Rabin-8  . This work suggests that Parkinson’s causing mutations in LRRK2 will inhibit Rab GTPases isoforms that are phosphorylated by LRRK2.
We have also developed a robust method to rapidly assess LRRK2 phosphorylation of endogenous Rab isoforms in samples where material may be limiting without the need to use state of the art mass spectrometry to assess Rab protein phosphorylation. The new procedure termed “Phos-tag” method enables the activity of endogenous LRRK2 to be assessed by monitoring Rab10 phosphorylation for the first time in vivo  . We hope that the phos-tag assay will aide with the assessment LRRK2 signalling pathway activity in cells and to establish the impact that inhibitors, mutations and other factors have. The prediction is that elevation of LRRK2 activity leads to Parkinson’s disease and the expectation is that if a sub-group of patients can be identified with elevated LRRK2 activity, it would be important to explore whether these individuals might benefit most from LRRK2 inhibitors that are being developed.
The main focus of future work on LRRK2 will be to investigate the importance that LRRK2 phosphorylation of Rab isoforms plays in biology and to explore links to Parkinson’s disease. We will identify effector proteins specific for the most relevant Rab GTPases we select to study. We will also generate phospho-specific antibody reagents to explore whether Rab phosphorylation in blood cells or CSF can be used as a biomarker for LRRK2 activity in vivo. We would also explore how pathogenic disease-causing mutations of LRRK2 in both mice and humans impact on Rab phosphorylation and whether this can be used as a biomarker for disease progression in Parkinson’s disease.
Figure 2 LRRK2 Phosphorylates and Inhibits Rab GTPases
In collaboration with AstraZeneca we have investigated why a panel of breast cancer cells that had mutations in PTEN or Class 1 PI3Kα were resistant to Akt inhibitors. This resulted in the discovery that a majority of these Akt inhibitor resistant cells expressed high levels of SGK1, a kinase that is closely related to Akt and likely phosphorylate overlapping substrates as well as being activated by the same upstream kinases (PDK1 and mTORC2)  . We elaborated a simple assay based on monitoring the effects that Akt inhibitors have on NDRG1 phosphorylation to determine which cancer cells display high activity of SGK1 and are therefore likely to be insensitive to Akt inhibitors  .
We are also interested in SGK3 which is unique in that it is the only protein kinase known to possesses a PtdIns(3)P-binding PX domain at its N-terminus (Figure 3). We confirmed that SGK3 does indeed bind PtdIns(3)P in vitro and showed that SGK3 localises at endosomes in vivo where PtdIns(3)P is located through its PX domain  . We found that PX domain mutations that ablate binding to PtdIns(3)P, inhibit SGK3 activity by suppressing phosphorylation at the PDK1 and mTORC2 sites  . We have discovered that the lipid kinase which generates the PtdIns(3)P at the endosomal membrane required for triggering the activation of SGK3, is the class 3 PI3K family member termed hVPS34 known to localise at endosome  .
We have recently discovered that prolonged treatment of a panel of breast cancer cells lines with PI3K or Akt inhibitors, led to a marked increase in the expression of SGK3  . Our data indicated that under these conditions SGK3 is activated by PtdIns(3)P produced by hVps34 binding to the PX domain and thereby promoting PDK1 phosphorylation and hence activation of SGK3  . We characterized a reported Sanofi SGK1 inhibitor termed 14h that inhibits SGK3 with an IC50 of ~ 3nM. Our findings indicate that SGK3 and Akt are capable of phosphorylating an overlapping set of substrates. For example we showed that SGK3 was able to fully re-activate the mTORC1 signalling pathway by phosphorylating TSC2  . Working in the laboratory of José Baselga at the Memorial Sloan Kettering Cancer Center we demonstrated that a combination of Akt (MK-2206) and SGK (14h) inhibitors induced marked regression of a breast cancer (BT-474) cell derived tumours in a nude mouse xenograft model, under conditions where either inhibitor administered individually had minimal effects ( . In collaboration with José Baselga laboratory we have also contributed to the discover that certain breast cancer cell lines develop resistance to PI3K inhibitors by over-activation of SGK1 through the PDK1 pathway [5, 6] . Here again it was shown that SGK1 can mediate activation of mTOR pathway by phosphorylating TSC2 [5, 6] . These studies highlight the therapeutic potential of a strategy targeting both the Akt and SGK kinases for the treatment of cancer.
Figure 3 Can SGK3 activate mTORC1 under conditions where PI3K and Akt are inhibited?
In future work we aim to continue our characterisation of the role that SGK signalling pathways plays in cancer. This would include identifying and characterizing SGK3 substrates employing different approaches. We will explore whether SGK or more likely a combination of SGK/Akt inhibitors have therapeutic potential for treating tumours that display elevated PI3K pathway activity
We have been working a number of years in studying the regulation and function of the WNK isoforms as mutations that increased the expression of WNK1 or WNK4 isoforms caused an inherited hypertension disorder termed Gordon’s syndrome. 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  . These kinases phosphorylate and regulate the activity of ion co-transporters such as NCC, NKCC1 and NKCC2 that play critical roles in regulating blood pressure and ion homeostasis  . We have now found that SPAK/OSR1 not only activate ion co-transporters (NCC/NKCC1/NKCC2) responsible for influx of Na+ and Cl- ions into cells, but also directly phosphorylate and inhibit potassium co-transporters (KCC1,KCC2,KCC3 and KCC4) that regulate the efflux of K+ and Cl- ions from cells  . This mechanism explains how WNK signalling pathway can cordately regulate net transport of ions into cells.
Figure 4 The WNK1-regulated SPAK/OSR1 kinases stimulate net salt uptake by reciprocally activating sodium co-transporters and inhibiting potassium co-transporters
All of our data points towards SPAK/OSR1 representing good targets for drugs to treat hypertension and/or reduce chloride levels in neurons for the treatment of mood disorders and epilepsy  . There is some debate on whether conventional kinase inhibitors would be sufficiently specific to treat a largely asymptomatic condition such as hypertension, so we have explored other approached which SPAK/OSR1 function could be inhibited. We found that SPAK possesses a conserved carboxy-terminal (CCT) domain, which operates as a docking domain to recognise RFXV/I motifs present in its upstream activator WNKs as well as its substrates (NCC, NKCC1NKCC2)  . To validate the approach of generating CCT domain inhibitors, we generated knock-in mice in which the ability of the CCT domain to bind RFXI/V motif was ablated . The CCT domain defective animals displayed markedly reduced SPAK activity and phosphorylation of NCC and NKCC2 co-transporters at the residues phosphorylated by SPAK. These knock-in mice also displayed markedly reduced blood pressure, suggesting that CCT domain inhibitors would have the potential to effectively inhibit the WNK signalling pathway .
In 2012 it was reported that mutations in one of two genes (KLHL3 and Cul3) encoding a poorly studied E3 ligase complex caused Gordon’s hypertension syndrome the same conditions that was caused by mutations WNK1 and WNK4 genes. In collaboration with Thimo Kurz we discovered that KLHL3 interacted with WNK isoforms and we showed that pathogenic mutations in KLHL3 either abolished binding to CUL3 or WNK isoforms  . We identified the degron motif on WNK4 that bound to KLHL3 that interestingly encompasses the residues on WNK4 that are mutated on Gordon’s syndrome  . In collaboration with Alex Bullock we crystallised the KLHL3 Kelch domain in complex with the WNK4 degron motif which revealed the intricate web of interactions between conserved residues on the surface of the Kelch domain β-propeller and the WNK4 degron motif  . Many of the disease-causing mutations inhibit binding by disrupting critical interface contacts  . These results show that the CUL3-KLHL3 E3 ligase complex regulates blood pressure via its ability to interact with and ubiquitylate WNK isoforms.
Figure 5 Ubiquitylation of WNK isoforms is regulated by CUL3-KLHL3 E3 ligase
We plan to understand in more detail how the WNK signalling pathway senses ionic/osmotic stress. This is likely to be mediated at least in part by the uncharacterised C-terminal domains of these enzymes. We believe this project is important as the ability of mammalian cells to sense and respond to changes in ionic conditions is fundamental for survival and function of biology and this process is very poorly understood.
Steger, M., Tonelli, F., Ito, G., Davies, P., Trost, M., Vetter, M., Wachter, S., Lorentzen, E., Duddy, G., Wilson, S., Baptista, M. A., Fiske, B. K., Fell, M. J., Morrow, J. A., Reith, A. D., Alessi, D. R. and Mann, M. (2016) Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases. Elife. 5http://elifesciences.org/content/5/e12813v2
Ito, G., Katsemonova, K., Tonelli, F., Lis, P., Baptista, M. A., Shpiro, N., Duddy, G., Wilson, S., Ho, P. W., Ho, S. L., Reith, A. D. and Alessi, D. R. (2016) Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors. Biochem J. 473, 2671-2685 http://www.biochemj.org/content/473/17/2671.long
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. Biochem J. 452, 499-508 http://www.biochemj.org/content/452/3/499.long
Bago, R., Malik, N., Munson, M. J., Prescott, A. R., Davies, P., Sommer, E., Shpiro, N., Ward, R., Cross, D., Ganley, I. G. and Alessi, D. R. (2014) Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase. Biochem J. 463, 413-427 http://www.biochemj.org/content/463/3/413.long
Bago, R., Sommer, E., Castel, P., Crafter, C., Bailey, F. P., Shpiro, N., Baselga, J., Cross, D., Eyers, P. A. and Alessi, D. R. (2016) The hVps34-SGK3 pathway alleviates sustained PI3K/Akt inhibition by stimulating mTORC1 and tumour growth. EMBO J. 35, 1902-1922http://emboj.embopress.org/content/35/17/1902.long
Castel, P., Ellis, H., Bago, R., Toska, E., Razavi, P., Carmona, F. J., Kannan, S., Verma, C. S., Dickler, M., Chandarlapaty, S., Brogi, E., Alessi, D. R., Baselga, J. and Scaltriti, M. (2016) PDK1-SGK1 Signaling Sustains AKT-Independent mTORC1 Activation and Confers Resistance to PI3Kalpha Inhibition. Cancer Cell. 30, 229-242sciencedirect.com/science/article/pii/S1535610816302641
Alessi, D. R., Zhang, J., Khanna, A., Hochdorfer, T., Shang, Y. and Kahle, K. T. (2014) The WNK-SPAK/OSR1 pathway: master regulator of cation-chloride cotransporters. Science signaling. 7, re3 http://stke.sciencemag.org/content/7/334/re3.long
de Los Heros, P., Alessi, D. R., Gourlay, R., Campbell, D. G., Deak, M., Macartney, T. J., Kahle, K. T. and Zhang, J. (2014) The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K+-Cl- co-transporters. Biochem J. 458, 559-573http://www.biochemj.org/content/458/3/559.long
Zhang, J., Siew, K., Macartney, T., O'Shaughnessy, K. M. and Alessi, D. R. (2015) Critical role of the SPAK protein kinase CCT domain in controlling blood pressure. Hum Mol Genet. 24, 4545-4558 http://hmg.oxfordjournals.org/content/24/16/4545.long
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. 451, 111-122http://www.biochemj.org/content/451/1/111.long
Schumacher, F. R., Sorrell, F. J., Alessi, D. R., Bullock, A. N. and Kurz, T. (2014) Structural and biochemical characterization of the KLHL3-WNK kinase interaction important in blood pressure regulation. Biochem J. 460, 237-246http://www.biochemj.org/content/460/2/237.long
Selected Recent Publications
- Steger, M., Tonelli, F., Ito, G., Davies, P., Trost, M., Vetter, M., Wachter, S., Lorentzen, E., Duddy, G., Wilson, S., Baptista, M.A.S., Fiske, B.K., Fell, M.J., Morrow, J.A., Reith, A.D., Alessi, D. R. and Mann, M. (2016) Phosphoproteomics reveals that Parkinson’s disease kinase LRRK2 regulates a subset of Rab GTPases. eLife DOI: http://dx.doi.org/10.7554/eLife.12813 [Dario Alessi and Matthias Mann Joint corresponding authors]
- Zhang, N., Gordon, S.L., Fritsch, M.J., Esoof, N., Campbell, D.G., Gourlay, R., Velupillai, S., Macartney, T., Peggie, M., van Aalten, D.M.F., Cousin, M.A. Dario R. Alessi and Alessi, D. R. (2015) Phosphorylation of SV2A at Thr84 by CK1 family kinases controls the specific retrieval of synaptotagmin-1. Journal of Neuroscience 35, 2492-250.
- Zhang, J, Siew, K, Macartney, T, O'Shaughnessy, K,M, Alessi, D. R. (2015) Critical role of the SPAK protein kinase CCT domain in controlling blood pressure. Hum Mol Genet. 24, 4545-4558
- Perez-Oliva, A.B., Lachaud, C., Szyniarowski, P., Muñoz, I., Macartney, T., Hickson, I., Rouse, R., and Alessi, D. R. (2015) USP45 deubiquitylase controls ERCC1-XPF endonuclease mediated DNA damage responses. EMBO J. 34, 326-343.
- Bago, R., Malik, N., Munson, M.J., Prescott, A.R., Davies, P., Sommer, E., Shpiro, N., Ward, R., Cross, D., Ganley, I.G and Alessi, D. R. (2014) Characterisation of VPS34-IN1, a selective inhibitor of Vps34 reveals that the phosphatidylinositol 3-phosphate binding SGK3 protein kinase is a downstream target of Class III PI-3 kinase. Biochemical J 463, 413-427.
- Alessi, D. R., Zhang, J., Khanna, A., Hochdörfer, T., Shang, Y., and Kahle, K.T. (2014) The WNK-SPAK/OSR1 pathway: Master regulator of cation-chloride cotransporters. Science signalling 7, Re3
- Banerjee S., Zagorska A., Deak M., Campbell D.G., Prescott A., and Alessi, D. R. (2014) Interplay between Polo kinase, LKB1-activated NUAK1 kinase, PP1βMYPT1 phosphatase complex and the SCFβTrCP E3 ubiquitin ligase. Biochemical J 461, 233-245.
- van der Wijst, J., Blanchard, M.G., Woodroof, H.I., Macartney, T.J., Gourlay, R., Hoenderop, J.G., Bindels, R.J., and Alessi, D. R. (2014) Kinase and channel activity of TRPM6 are coordinated by a dimerization motif and pocket interaction. Biochemical Journal. 460,165-175.
- 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.
- 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.
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.