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

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.

Genetic landscape of metastatic and recurrent head and neck squamous cell carcinoma

“…49,50). While the precise mechanism or mechanisms underlying dasatinib sensitivity are beyond the scope of this report, the general mechanisms and consequences of discoidin domain receptor signaling in cancer are areas of ongoing research (47)(48)(49)(50)56). The DDR2(R709*) mutation identified in the metachronous recurrent tumor of patient PY-3, which conferred the greatest degree of sensitization to dasatinib in the present study, has also been reported in gastric and endometrial cancers.…”

Extracellular matrix component signaling in cancer

“…However, the study did not address the contribution of wild-type DDR2 to oncogenesis, and while several more recent studies have confirmed the occurrence of DDR2 mutations in different patient populations [122][123][124], it is far from clear whether the mutations are indeed oncogenic and their roles in lung SCC cell signalling are undefined [125]. Notably, in other studies, some of the 'oncogenic' DDR2 mutants either seemed to play tumour suppressive functions [101], or did not contribute to cell proliferation in lung SCC cell lines [126].…”

“…Other molecules known to interact with phosphorylated DDR1 include the phosphatases SHP-1 [98] and SHP-2 [97,99] and members of the Stat family of transcription activators [99,100]. For DDR2, downstream effectors include SHP-2, Nck1, the Src family kinase Lyn and phospholipase C-like 2 [101], but it is not known whether any of these interact directly with the activated receptor.…”

Functional Analyses of Mutations in Receptor Tyrosine Kinase Genes in Non–Small Cell Lung Cancer: Double-Edged Sword of DDR2

“…The loss of DDR2 has also been shown to predispose hepatic tissues to colon carcinoma cell growth and metastasis (29). Moreover, wild-type DDR2 expression in the HEK293 cell line greatly reduced colony formation in the presence of collagen (30). Collagen is the major structural component of the extracellular matrix and triggers many intercellular signal pathways regulating cellular growth, migration, and differentiation (31).…”