Quantitative Phosphoproteomics of Collagen Receptor Networks Reveals SHP-2 Phosphorylation Downstream of Wildtype DDR2 and its Lung Cancer Mutants Leo K. Iwai1, Leo S. Payne1, Maciej T. Luczynski1, Francis Chang1,Huifang Xu2, Ryan W. Clinton3, Angela Paul4, Edward A. Esposito3, Scott Gridley3, Birgit Leitinger2, Kristen M. Naegle5 and Paul H. Huang1. leo.iwai@butantan.gov.br 1. Protein Networks Team, Division of Cancer Biology, Institute of Cancer Research, London, SW3 6JB, UK. 2. National Heart and Lung Institute, Imperial College London, London SW7 2AZ, UK. 3. Blue Sky Biotech Inc., Worcester, MA 01605, USA. 4. Cancer Research UK, Tumour Cell Signalling Unit, Institute of Cancer Research, London, SW3 6JB, UK. 5. Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA. Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the β1 integrins and the Discoidin Domain Receptors (DDRs). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with in vitro kinase data. Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2, NCK1, LYN, SHIP-2, PIK3C2A and PLCL2. Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling using Selective Reaction Monitoring (SRM) of a panel of squamous cell lung cancer (SCC) DDR2 mutants demonstrated that SHP-2 is tyrosine phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, these results indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens.