Characterization of the Src-regulated kinome identifies SGK1 as a key mediator of Src-induced transformation

Xiuquan Ma,Ralf B Schittenhelm,Christina A Mitchell,Fuyi Li,Cheng Huang,Jianmin Wu,Jiangning Song,Kaylene J Simpson,Samuel J Rodgers,Luxi Zhang,Kristin K Brown,Roger J Daly,Elizabeth Nguyen,Howard Chan,Rachel S Lee,Chanly Chheang

doi:10.1038/s41467-018-08154-1

Abstract

Despite significant progress, our understanding of how specific oncogenes transform cells is still limited and likely underestimates the complexity of downstream signalling events. To address this gap, we use mass spectrometry-based chemical proteomics to characterize the global impact of an oncogene on the expressed kinome, and then functionally annotate the regulated kinases. As an example, we identify 63 protein kinases exhibiting altered expression and/or phosphorylation in Src-transformed mammary epithelial cells. An integrated siRNA screen identifies nine kinases, including SGK1, as being essential for Src-induced transformation. Accordingly, we find that Src positively regulates SGK1 expression in triple negative breast cancer cells, which exhibit a prominent signalling network governed by Src family kinases. Furthermore, combined inhibition of Src and SGK1 reduces colony formation and xenograft growth more effectively than either treatment alone. Therefore, this approach not only provides mechanistic insights into oncogenic transformation but also aids the design of improved therapeutic strategies.

Highlights

Despite significant progress, our understanding of how specific oncogenes transform cells is still limited and likely underestimates the complexity of downstream signalling events
This work has been complemented and greatly extended by the application of an immunoaffinity-coupled mass spectrometry (MS)-based proteomics workflow, where tryptic tyrosine-phosphorylated peptides are enriched prior to MS analysis[18]. Application of this approach to Src-transformed fibroblasts and cancer cells exhibiting high levels of Src activity has highlighted the diversity of protein classes that are tyrosinephosphorylated upon Src-induced transformation, ranging from specific kinases and phosphatases to GEFs, GAPs, and scaffolds, and revealed novel processes regulated by Src such as RNA maturation[19,20,21,22,23]
Despite these advances in our understanding of Src-induced oncogenesis, the protein kinase pathways and networks that regulate the pleiotropic effects of active Src remain poorly characterized, since the proteomic approaches applied so far have focused on the tyrosine phosphoproteome, and do not provide insights into the expression or activation status of the large numbers of non-tyrosine phosphorylated kinases that lie downstream

Summary

Introduction

Our understanding of how specific oncogenes transform cells is still limited and likely underestimates the complexity of downstream signalling events. Application of this approach to Src-transformed fibroblasts and cancer cells exhibiting high levels of Src activity has highlighted the diversity of protein classes that are tyrosinephosphorylated upon Src-induced transformation, ranging from specific kinases and phosphatases to GEFs, GAPs, and scaffolds, and revealed novel processes regulated by Src such as RNA maturation[19,20,21,22,23] Despite these advances in our understanding of Src-induced oncogenesis, the protein kinase pathways and networks that regulate the pleiotropic effects of active Src remain poorly characterized, since the proteomic approaches applied so far have focused on the tyrosine phosphoproteome, and do not provide insights into the expression or activation status of the large numbers of non-tyrosine phosphorylated kinases that lie downstream. This is a critical issue, given the biological significance and translational relevance of this oncogene

Methods

Results

Conclusion