 |
Background Research |
| Sir Philip Cohen |
| E: p.cohen@dundee.ac.uk |
| T: 44 1382 384238 |
| F: 44 1382 223778 |
| Michelle Mulligan PA to Sir Philip Cohen |
| E: m.z.mulligan@dundee.ac.uk |
| T: 44 1382 384238 |
| F: 44 1382 223778 |
Sir Philip Cohen
Research
The interplay between protein phosphorylation and protein ubiquitylation in regulating the innate immune system
The major aim of my research is to work out the signaling pathways that become activated during infection by bacteria and viruses, and to discover how they trigger the production of pro-inflammatory cytokines and interferons to combat and destroy these pathogens. Understanding this system is critical, not only because of its importance in defence against infection, but also because the uncontrolled activation of these substances is a major cause of chronic inflammatory and autoimmune diseases, such as rheumatoid arthritis, psoriasis, asthma, lupus and septic shock. Drugs that target particular components of these signaling pathways may therefore be of great benefit in the treatment of these diseases [1].
The binding of pathogen-derived molecules to many Toll-like receptors (TLRs) that are present in the plasma membranes and endosomes of immune cells, or the interaction of the pro-inflammatory cytokine interleukin-1 (IL-1) with the IL-1 receptor, recruits a signaling complex that includes the adaptor MyD88 and the protein kinases IRAK1 and IRAK4 (Fig 1). This triggers the IRAK4-catalysed activation of IRAK1, its release from the complex and interaction with the E3 ubiquitin ligases TRAF6 and Pellino with which it propagates the signal. The next step involves the activation of these E3 ligases, which can then couple with the E2 conjugating enzyme Ubc13-Uev1a to catalyse the formation of Lys63-linked polyubiquitin (K63-pUb) chains [2, 3]. The K63-pUb chains may be anchored covalently to other proteins, such as IRAK1, or remain unanchored. We have shown that IRAK1 and IRAK4 phosphorylate the three isoforms of Pellino in vitro, which activates their latent E3 ubiquitin ligase activity [3, 4]. The K63-pUb chains bind to the regulatory subunits of the protein kinases TAK1 and IKKß ( Fig 1), inducing conformational changes that lead to the activation of these “master” protein kinases. They can then switch on “downstream” signaling pathways that trigger the production of pro-inflammatory cytokines [1]. We are beginning to explore the roles of IRAK1, the related kinase IRAK2, and the three isoforms of Pellino in vivo using cells that we have generated in which functionally inactive forms of these proteins have replaced the normal, wild-type forms of these proteins.
Figure 1. Early events in signaling by IL-1. Colour key: Protein kinases are highlighted in red, E3 ubiquitin ligases in blue, polyubiquitin-binding proteins in purple, adaptors in green and receptors in orange. The polyubiquitin chains formed by TRAF6 and Pellino are linked covalently by isopeptide bonds between Lys63 of one ubiquitin and the C-terminus of the adjacent ubiquitin in the chain. Phosphorylation sites are denoted -P.
The protein NEMO, which is a regulatory subunit of IKKß (Fig 1), contains a polyubiquitin-interacting domain that binds to K63-Ub chains relatively specifically. Mutations in this domain that disrupt binding to K63-pUb chains cause a human immunodeficiency disease and greatly increase susceptibility to infection by tuberculosis-causing bacteria. Intriguingly, the polyubiquitin-binding domain found in NEMO is present in four other human proteins (Fig 2). We have established that these proteins also bind K63-pUb chains and that polyubiquitin-binding is prevented by the same mutations that prevent the interaction of K63-pUb chains with NEMO. We have recently generated cells in which polyubiquitin binding-defective mutants of three of these proteins have replaced their wild type counterparts. Studies using these cells are beginning to provide some intriguing insights into their roles in the innate immune system.
Figure 2. The Lys63-linked polyubiquitin-binding domain found in NEMO is present in four other human proteins. The mutation of the Asp or the Glu residue highlighted in red to Asn or Ala, respectively, causes an immunodeficiency disease, increases susceptibility to infection by bacteria and prevents binding to Lys63-linked polyubiquitin chains. These two residues are conserved in ABINs 1, 2 and 3 and Optineurin and the equivalent mutations in these proteins also prevents binding to polyubiquitin.
One Toll-like receptor, TLR3, is activated by double-stranded RNA (dsRNA) derived from dsRNA viruses, such as the rotaviruses that are a major cause of acute gastroenteritis in young children worldwide. TLR3 does not signal through the adaptor MyD88 but via a distinct adaptor, termed TRIF. This signaling pathway leads to the activation of two closely related protein kinases of the IKK subfamily, termed TBK1 and IKKε. TBK1 and IKKε phosphorylate and activate interferon regulatory factor 3 (IRF3), which stimulates transcription of the mRNA encoding interferon β. However, the mechanism by which TBK1 and IKKε is activated is poorly understood. We have recently identified BX795 as the first, relatively specific cell permeable inhibitor of TBK1 and IKKε and have exploited this compound to demonstrate that the activation of TBK1 and IKKε is mediated by an as yet unknown protein kinase [5] (Fig 3). Our aim is to discover the identity of this protein kinase and how it is activated by TRAF3 and other molecules. Intriguingly, we have also shown that TBK1 interacts with the K63-pUb-binding protein Optineurin [6] (Fig 2).
Figure 3. Pathway by which viral double-stranded RNA produces interferon β. Colour code is as in Fig 1.
Relevant recent references from my laboratory
[1] Cohen, P. (2009). Targeting protein kinases for the development of anti-inflammatory drugs. Current Opinion in Cell Biol. 21, 317-324.
[2] Clark, K., Peggie, M., Plater, L. and Cohen, P. (2009). Use of a pharmacological inhibitor to study the regulation and physiological roles of TBK1 and IKKε: a distinct upstream kinase mediates Ser172 phosphorylation and activation. J. Biol. Chem. 284, 14136-14146.
[3] Handoyo, H., Stafford, M., Baltzis, D., McManus, E., Peggie, M., Cohen, P. (2009). IRAK1-independent pathways required for the interleukin 1-stimulated activation of the Tpl2 catalytic subunit and its dissociation from ABIN2. Biochem. J. 424, 109-118.
[4] Ordureau, A., Smith, H., Windheim, M., Peggie, M., Carrick, E., Vandermoere, F., Morrice, N.A. and Cohen, P. (2008). The IRAK-catalysed phosphorylation of Pellino isoforms activates their E3 ligase activity. Biochem. J. 409, 43-52.
[5] Smith, H., Peggie, M., Campbell, D.G., Vandermoere, F., Carrick, E. and Cohen, P. (2009). Identification of the phosphorylaton sites on the E3 ubiquitin ligase Pellino that are critical for activation by IRAK1 and IRAK4.Proc. Natl. Acad. Sci. USA 106, 4584-4590.
[6] Morton, S., Hesson, L., Peggie, M. and Cohen, P. (2008). Enhanced binding of TBK1 by an optineurin mutant that causes a familial form of primary open angle glaucoma. FEBS Lett. 582, 997-1002.
[7] Windheim, M., Stafford, M., Peggie, M. and Cohen, P. (2008). IL-1 induces the Lys63-linked polyubiquitylation of IRAK1 to facilitate NEMO binding and the activation of IκBα kinase. Mol. Cell. Biol. 28, 1783-1791.
University of Dundee