Understanding signalling pathways mutated in inherited disorders
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
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 , Parkin , FBXO7) and other neurodegenerative movement disorders (TTBK2 - a kinase mutated in spinocerebellar ataxia type 11 ).
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 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).
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 . 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  (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 . 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 . 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 . We also found that disease mutant CUL3:KLHL3 complexes failed to ubiquitylate WNK1 in vitro .
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 .
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.
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)
Professor Dario Alessi
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