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Functional proteomics to dissect tyrosine kinase signalling pathways in cancer

Nature Reviews Cancer volume 10, pages 618–629 (2010)Cite this article

Subjects

Key Points

  • Signalling pathways are commonly deranged in cancer and quantitative proteomics offers powerful approaches to map these pathways and their aberrations in cancer.

  • Hubs in signalling pathways feature multiple protein interactions, which are involved in information processing and specification of the biological responses. These networks can be mapped by interaction proteomics to reveal molecular mechanisms of transformation and potential targets for therapeutic interventions.

  • The oncogenic actions of the epidermal growth factor receptor (EGFR) network and the breakpoint cluster region (BCR)–ABL1 oncogene rely on the dynamic assembly of multiprotein complexes, which activate multiple downstream pathways that cooperate in transformation. In EGFR networks, the oncogenic potential increases with the number of downstream pathways being activated.

  • The dynamic assembly of protein complexes is regulated by post-translational modifications (PTMs) such as phosphorylation. Advances in phosphoproteomics allow the targeted and global mapping of phosphorylation networks, confirming that kinase networks play major parts in cancer and offer numerous new targets for therapeutic intervention.

  • In addition to phosphorylation, a role for PTMs in the regulation of cancer cell biology is becoming increasingly recognized. For example, proteomic studies of ubiquitylation are beginning to unravel extensive alterations that contribute to cancer, such as growth factor receptor activation, transcription factor function, protein localization and degradation.

  • Dynamic changes in protein abundance and PTMs may also contribute to cancer cell heterogeneity, and new proteomics technologies based on optical, spectroscopic and microarray methods are being developed to analyse individual cells.

Abstract

Advances in the generation and interpretation of proteomics data have spurred a transition from focusing on protein identification to functional analysis. Here we review recent proteomics results that have elucidated new aspects of the roles and regulation of signal transduction pathways in cancer using the epidermal growth factor receptor (EGFR), ERK and breakpoint cluster region (BCR)–ABL1 networks as examples. The emerging theme is to understand cancer signalling as networks of multiprotein machines which process information in a highly dynamic environment that is shaped by changing protein interactions and post-translational modifications (PTMs). Cancerous genetic mutations derange these protein networks in complex ways that are tractable by proteomics.

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Figure 1: The epidermal growth factor receptor signalling network.
Figure 2: The BCR–ABL1 signalling network.
Figure 3: The ubiquitin system.

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Acknowledgements

We apologise for omitting many important contributions due to space constraints. We are grateful for funding from Science Foundation Ireland grant 06/CE/B1129 (W.K.) and the Biotechnology and Biological Sciences Research Council and the Engineering and Physical Sciences Research Council through the Radical Solutions for Researching the Proteome (RASOR) project BB/C511572/1 (A.P.).

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Authors and Affiliations

  1. Conway Institute, University College Dublin, Belfield, 4, Dublin, Ireland

    Walter Kolch

  2. Systems Biology Ireland, University College Dublin, Belfield, 4, Dublin, Ireland

    Walter Kolch

  3. Sir Henry Wellcome Functional Genomics Facility, University of Glasgow, Glasgow, G12 8QQ, UK

    Andrew Pitt

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Correspondence to Walter Kolch.

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DATABASES

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Glossary

Chemical biology

Use of chemicals, usually drugs or drug-like compounds, to probe biological systems to measure the response of biological systems to perturbations. In proteomics, this term also increasingly refers to the use of affinity reagents to enrich classes of proteins for further analysis.

Chemical genetics

A part of chemical biology that focuses on the use of chemicals to explore genetic systems and genetic factors that determine drug sensitivity.

Scaffold protein

A protein that can simultaneously bind two or more other proteins, and thereby facilitate physical and functional interactions between the client proteins that bind to it.

Matrix management

A flexible management approach that assigns people with the required skill sets to projects, typically drawing expertise from different departments. This comparison is used to illustrate that a protein with a defined molecular function, such as a kinase, can be used by several different pathways.

Modularization

The grouping of different functions into a single unit (module) so that the output of the module can be treated as a single functional entity, such as the ability of different combinations of components of protein complexes to achieve the same output.

Non-oncogene addiction

This occurs when the action of oncogenes needs to be supported by apparently normally functioning signalling pathways that allow the mutated oncogene to develop its transforming activity.

SH2 domain

This domain was first discovered as a conserved domain in the Src kinase family. It recognizes short peptide motifs that contain a phosphorylated tyrosine residue and thus function as phosphotyrosine-dependent protein interaction sites.

Stable isotope labelling with amino acids in cell culture (SILAC)

This method involves the in vivo metabolic labelling of samples with amino acids that carry stable (non-radioactive) heavy isotope substitutions of atoms which, when analysed by MS, produce 'conjugated' peptide peaks. These peaks originate from the same protein but show a characteristic mass shift which corresponds to the mass difference between the light and heavy label. The relative intensity of conjugated peak pairs provides the relative abundance of a protein in the two samples.

Node

This describes an object in graph form, and the connections between objects are termed edges. In signalling networks, nodes represent proteins (or genes, if they are based on genetic information) and edges represent the relationship between the nodes, such as binding, regulation or modification.

Warburg effect

This was named after a discovery made by the German biochemist Otto Warburg in the 1920s that cancer cells predominantly use anaerobic glycolysis rather than oxidative phosphorylation, even when oxygen is abundant. As a result, pyruvate is converted to lactate instead of being oxidized by the mitochondria of cancer cells.

O-linked N-acetylglucosamine acylation (O-GlcNAcylation)

A form of glycosylation found in nuclear and cytosolic proteins in which O-GlcNAc is added to the hydroxyl groups of serine and threonine residues that can also serve as phosphorylation sites.

Isobaric tag for relative and absolute quantitation (iTRAQ)

A stable isotope labelling method for the quantitation of peptides by MS, in which a molecule containing normal or heavy isotopes is used to chemically modify the proteins or peptides from each individual sample. The fragmentation of the labelled molecules then gives rise to specific reporter ions that can be used to measure the relative amounts of each protein present in each sample.

Immobilized metal ion affinity chromatography (IMAC)

A method for the enrichment of phosphopeptides that exploits the propensity of metal ions such as iron and gallium to bind phosphate groups.

Electron transfer dissociation (ETD)

A recently introduced MS method for the fragmentation of molecules by transferring electrons from anion radicals to positively charged ions. It is a non-ergodic (rapid, kinetically controlled) process, so energy is not redistributed and many bonds are broken in the molecule, not just the weakest ones as seen in collision-induced dissociation.

WD40 domain

A protein domain consisting of 4–16 repeats of an approximately 40 amino acid-long motif that ends with a W–D (tryptophan–aspartic acid) dipeptide. The WD40 domains form a circular β-sheet propeller structure that serves as a structural platform for protein interactions and the specificity of the interactions is determined by sequences outside the WD repeats.

Cyclin

A regulatory subunit that is essential for the activity of cell cycle-dependent kinases (CDKs). Its name derives from the periodic expression of cyclins during the cell cycle, which is due to the regulated degradation by the ubiquitin–proteasome system that is thought to drive the cell cycle.

Micro-engineering

The use of micro-fabricated devices that have small (micron)-scale features (such as channels, wells and vessels) to allow the processing of small volumes of fluid.

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Kolch, W., Pitt, A. Functional proteomics to dissect tyrosine kinase signalling pathways in cancer. Nat Rev Cancer 10, 618–629 (2010). https://doi.org/10.1038/nrc2900

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