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.