Poster Presentation 23rd Annual Lorne Proteomics Symposium 2018

Charting the tumour suppressive signaling networks of CHK kinase in colorectal cancer cells with a novel tyrosine-specific phosphoproteomics approach (#88)

Andy Low 1 , Ching-Seng Ang 1 , Heung-Chin Cheng 1
  1. University of Melbourne, Melbourne, VICTORIA, Australia

Overactivation of Src is involved in tumorigenesis and metastasis of numerous cancer types, including colorectal cancer1. Src is over-activated in >80% of colorectal cancers (CRC) and is involved in the progression of CRC.

Oncogenic mutations of Src are rare, implying that overactivation of Src in cancer cells is caused by dysregulation of its upstream regulators. CSK homologous kinase (CHK) is an upstream inhibitor of Src-family kinases (SFK)2. CHK is expressed in normal mouse colon samples but significantly decreased in CRC cell lines3-4. We recently demonstrated that expression of recombinant CHK in CRC cells had significant impact on the anchorage independent growth and invasiveness of the cells, suggesting CHK as a potential CRC tumour suppressor4. As a protein tyrosine kinase, CHK exerts its tumour suppressive action by phosphorylating and inhibiting Src as well as phosphorylating other non-SFK proteins.

We aim to use quantitative phosphoproteomics approaches to define the signaling mechanisms and identify direct substrates of CHK. We have completed the analysis of the changes in phosphoproteome induced by CHK in CRC cells using conventional global phosphoproteomics by Ti4+ based-IMAX5-7. Although this approach reveals the involvement of many serine/threonine kinases in the tumour suppressive action of CHK, it failed to provide sufficient information and depth to characterise the global changes in phosphotyrosine proteome induced by CHK expression.

Therefore, we aim to utilize triple-substitution mutants of SH2 domains, termed SH2 superbinders due to their markedly increased affinity to phosphotyrosine residues, to define the changes in phosphotyrosine proteome of CRC cells induced by CHK expression8-10. Specifically, SH2 superbinders will be used to enrich phosphotyrosine-containing tryptic peptides prior to their identification and quantitation by sophisticated tandem mass spectrometry approach. These two different approaches will allow us to comprehensively identify CHK substrates and define the signaling networks governing its tumour suppressive action in CRC cells.