DiMeN Doctoral Training Partnership: Evaluating ’non-canonical’ Histidine phosphorylation on human cancer-associated protein kinases

Background:
Protein phosphorylation is catalyzed by transfer of the ATP γ-phosphate to a substrate, and protein kinases have become hugely important targets for drugs desined to treat inflammation, heart disease and cancer. Protein phosphorylation controls all aspects of life, including reversible modulation of human protein kinases whose dysregulation is a cause of multiple diseases.

For several technical reasons, cellular phosphorylation analysis of proteins has traditionally focussed on ’canonical’ serine (Ser), threonine (Thr) and tyrosine (Tyr) residues. However, mounting evidence suggests that histidine (His) phosphorylation also plays a key role in regulating aspects of human cell signalling. In this studentship, we seek to examine and understand the extent and function of His-based phosphorylation in the human protein kinase superfamily.

Preliminary findings: Current Mass Spectrometry (MS)-based analytical workflows are incompatible with analysis of acid-labile phosphorylation such as pHis, and as a consequence, the extent of non-canonical phosphorylation is likely to be vastly under-estimated. Using Strong Anion Exchange Chromatography coupled to MS, we identified extensive and positional phosphorylation of multiple human kinases on a conserved His residue located in the HXN motif, which lies close to the regulatory C-helix [1, 2, 3].

Scientific challenges: His phosphorylation is established in prokaryotic and plant ‘two-component’ signalling, where pHis is employed as a signalling intermediate. Our preliminary findings in human cells, made possible by state-of-the-art Mass Spectrometry (MS) and molecular dynamics, creates an outstanding opportunity to train a next-generation cross-disciplinary scientist in state-of-the-art skills to evaluate non-canonical phosphorylation in the protein kinase superfamily. Two thirds of all human protein kinases contain a phosphorylatable His/Ser or Thr in the HXN motif in the catalytic domain, but until now, this regulatory motif was not suspected to be modified by direct phosphorylation [1].

The team: Our finding that ’non-canonical’ phosphorylation on His (alongside Asp, Glu, Arg and Lys) is widespread in human cells creates a new signaling field. This allows us to provide a truly unique interdisciplinary training environment for a highly motivated student. By marrying biochemical and biophysical data with cellular cancer models in which the function of phosphorylated His on protein kinases can be evaluated, we have created an exciting opportunity for an MRC DiMeN DTP studentship. This is a collaboration between MRC and BBSRC-funded groups at the Universities of Liverpool and Sheffield (see below), and will employ quantitative biophysical and cellular approaches to study the mechanistic aspects of protein kinase His phosphorylation. The project represents a superb training opportunity that addresses MRC strategic research areas and a variety of skills priorities. The three-way collaboration is between an experienced supervisory team of scientists who all have very successful portfolios of student training.

Scientific goals:
1) Mass spectrometry-based evaluation of pHis-regulation in the members of the human kinome.
Preliminary data and outcomes: Our initial (unfocussed) analysis demonstrated that ~10 protein kinases contain a pHis residue. By focussing on the human kinome as a whole, we will uncover the full extent of pHis modification(s).

2) Cell-based analysis of His phosphorylation in a PLK2:p53-dependent model of cell survival
Preliminary data and outcomes: Our initial analysis of PLK2 suggests strongly that His phosphorylation will be inhibitory. We will evaluate this hypothesis biochemically and in a cell survival model that relies on PLK2-dependent p53 phosphorylation.

3) Cellular analysis of CDK18 and CDK19 pHis-regulation in a panel of breast and glioma cell models.
Preliminary data and outcomes: Our published [2] and unpublished data show that CDK18/19 are phosphorylated on His. CDK18 controls genome stability, and is involved in breast cancer and glioma aetiology. We will analyse the dynamics and function of CDK18 His phosphorylation in human cancer models by MS.

Training outcomes:
1) Immersion in state-of-the art research studying the non-canonical phosphoregluation of protein kinases on His

2) Member of thriving student training networks involving Professor Patrick Eyers and Professor Claire Eyers in the Department of Biochemistry and Centre for Proteome Research at the University of Liverpool, and Dr Spencer Collis based in the Department of Oncology and Metabolism and the Sheffield Institute for Nucleic Acids.

For more information about the supervisors and our research environment see:
https://www.liverpool.ac.uk/integrative-biology/staff/patrick-eyers/
https://www.sheffield.ac.uk/sheffield-cancer-research/research
https://www.liverpool.ac.uk/integrative-biology/staff/claire-eyers//researchers/sc
https://www.liverpool.ac.uk/pfg/
http://genome.sheffield.ac.uk/

3) A portfolio of training in MRC priority skill sets, including computational/bioinformatic analysis of quantitative ‘omics data and interdisciplinary skills allied to translational medicine (we predict that His-based kinase phosphorylation will be associated with disease)