While these previous correlative studies

While these previous correlative studies have measured tDDR1 protein by IHC, these studies have not considered that pDDR1 could also be an important marker of cancer prognosis. A phosphoproteomic survey of lung cancer tissues did identify pDDR1 as one of the most commonly phosphorylated receptor kinases invasive lung cancer, but outcome data were not available for cases in this study. Not surprisingly, we found that all cases of ESCC with high levels of pDDR1 also showed high total expression of the protein, but even greater predictive value for poor outcome was seen in those cases with high pDDR1 as well as high tDDR1. Over the past decade, activation of specific protein kinases has been found to be effective targets cancer therapy, and a wide variety of inhibitors of protein kinases have been developed. DDR1 is one such kinase activated by collagen through phosphorylation, and several multikinase inhibitors, including imatinib, nilotinib, and dasatinib, inhibit DDR1 with similar potency.30, 31 Unfortunately, however, a number of clinical trials with these kinase inhibitors in the treatment of cancers that have activated DDR1 have been disappointing, possibly because the activity of this kinase is regulated not only by kinases, but rather by an intimate balance between kinases and antagonist phosphatases. Future strategies to target kinase activation might include kinase inhibitors in combination with agents that activate tumor suppressor phosphatase.
Conclusions
Acknowledgment This study was funded by Juntendo University Shizuoka Medical Research Center for Disaster and the Commonwealth Fund of Johns Hopkins.
Introduction The Discoidin Domain Receptors (DDRs), comprising DDR1 and DDR2, are collagen-binding receptor tyrosine kinases (RTKs) that function as microenvironmental sensors at the interface of the extracellular matrix and the intracellular signal transduction machinery [1]. In response to ligand engagement, the DDRs undergo receptor tyrosine phosphorylation and recruit SH2- and PTB-containing adaptor proteins which trigger downstream signalling [2]. The DDRs are typified by a unique delayed and sustained tyrosine phosphorylation signalling profile. In mps1 kinase to growth factor activated RTKs where receptor phosphorylation is normally initiated within seconds to minutes of ligand binding and subsequently undergoes signal degradation by negative feedback regulation, the DDRs are activated by collagen with a delayed timescale of minutes to hours and tyrosine phosphorylation signalling remains sustained for several hours to days [3,4]. The mechanistic basis of this distinctive phosphorylation signalling dynamics is unknown, although it has been suggested that receptor localisation may be important [5]. Unlike other RTK family members such the ErbB, c-Met or PDGFR receptors, the spatiotemporal localisation of the DDRs remains poorly characterised. A previous study employed Förster resonance energy transfer (FRET) imaging and fluorescence microscopy to determine the cellular location of DDR1 in HEK293 cells [6]. The authors of this study found that DDR1 is rapidly internalised within 5 min of collagen presentation. A very recent study utilized a combination of fluorescence microscopy and biochemistry to demonstrate that DDR1 undergoes ligand-induced clustering at the cell surface through lateral dimer association in COS-7 cells [7]. It is unknown if DDR2 displays a similar spatiotemporal profile as DDR1.
Materials and methods
Results
Discussion In this study, we provide the first spatiotemporal characterisation of DDR2 localisation and tyrosine phosphorylation signalling. Our imaging data demonstrates that upon ligand presentation, a proportion of cellular DDR2 is localised to the cell surface. The localisation of DDR2 and cellular phosphorylated proteins is independent of the collagen-binding integrins and the presence of SHC1. Our experiments also find that tyrosine phosphorylated proteins are present at the interface where DDR2 is in contact with collagen and not in the early endosomes or lysosomes. Importantly we show that an intact collagen binding pocket is necessary for the spatiotemporal localisation of DDR2 at the cell surface, providing the first evidence that collagen binding is a key driver for receptor localisation. Since the collagen binding activity of DDR2 is independent of its kinase function [9], our data also led to the unexpected and novel finding that kinase activity is necessary for receptor localisation at the cell surface.