Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants

Leo K Iwai,Scott Gridley,Angela Paul,Kristen M Naegle,Birgit Leitinger,Maciej T Luczynski,Paul H Huang,Francis Chang,Ryan W Clinton,Huifang Xu,Leo S Payne,Edward A Esposito

doi:10.1042/bj20121750

Abstract

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 DDRs (discoidin domain receptors). 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 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 (non-catalytic region of tyrosine kinase adaptor protein 1), LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α) and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) 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 SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study 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.

Highlights

Collagen is the most abundant protein in mammals and plays a critical role in maintaining tissue structural integrity [1]
Using multiple clustering analysis methodology (MCAM), we find that a subset of phosphorylation sites on important signalling proteins such as SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1, LYN, SH2-domain-containing inositol phosphatase 2 (SHIP-2) [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], phospholipase C-like 2 (PLCL2) and phosphatidylinositol-4-phosphate 3-kinase (PIK3C2A) strongly cluster with DDR2 phosphorylation dynamics, implicating these proteins as candidate downstream effectors of DDR2 signalling
Stimulation of human embryonic kidney (HEK)-293DDR2 cells with acid-soluble collagen I resulted in the activation of DDR2 with delayed and sustained tyrosine phosphorylation over 24 h (Figure 1A) as reported previously [6]

Summary

Introduction

Collagen is the most abundant protein in mammals and plays a critical role in maintaining tissue structural integrity [1]. The resulting kinase assay reaction was run on a gel and the band corresponding to DDR2 (Supplementary Figure S1B) was excised and subjected to LC-MS/MS analysis to identify the phosphorylation sites on DDR2 that are present upon kinase activation (Table 1).

Results

Conclusion