Integrin-mediated RON growth factor receptor phosphorylation requires tyrosine kinase activity of both the receptor and c-Src

Cooperation between integrins and growth factor receptors plays an important role in the regulation of cell growth, differentiation, and survival. The function of growth factor receptor tyrosine kinases (RTKs) can be regulated by cell adhesion to extracellular matrix (ECM) even in the absence of ligand. We investigated the pathway involved in integrin-mediated RTK activation, using RON, the receptor for macrophage-stimulating protein. Adhesion of RON-expressing epithelial cells to ECM caused phosphorylation of RON, which depended on the kinase activity of both RON itself and c-Src. This conclusion is based on these observations: 1) ECM-induced RON phosphorylation was inhibited in cells expressing kinase-inactive c-Src; 2) active c-Src could phosphorylate immunoprecipitated RON from ECM-stimulated cells but not from unstimulated cells; and 3) ECM did not cause RON phosphorylation in cells expressing kinase-dead RON, nor could active c-Src phosphorylate RON immunoprecipitated from these cells. The data fit a pathway in which ECM-induced integrin aggregation causes both c-Src activation and RON oligomerization followed by RON kinase-dependent autophosphorylation; this results in RON becoming a target for activated c-Src, which phosphorylates additional tyrosines on RON. Integrin-induced epidermal growth factor receptor (EGFR) phosphorylation also depended on both EGFR and c-Src kinase activities. This sequence appears to be a general pathway for integrin-dependent growth factor RTK activation.

Integrin-mediated growth factor receptor activation 3 Introduction.
As to the basis for collaboration between integrins and growth factor receptors, they may form macromolecular complexes on the cell membrane (7;8;13;16-18). In that case, adhesion-induced aggregation of integrins might trigger co-aggregation (5) and autophosphorylation of growth factor RTKs (13).
Integrin-induced epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) RTK phosphorylation depends on the kinase activity of the receptor (7,13). Recent data suggest that integrin association with RTKs might also protect latter against the activity of phosphatases (17;19) and/or insure the correct subcellular juxtaposition of cytoplasmic tails of dimerized growth factor receptors (17). Despite the cited progress in this area of research, the molecular mechanisms underlying growth factor receptor activation by integrins remain to be defined. Here, we investigated the pathway involved in integrin-mediated RTK activation using RON, the receptor for Macrophage Stimulating Protein (MSP) (20). RON is a RTK that mediates biological effects of MSP (20;21). MSP was discovered as a serum factor that regulates motility of macrophages (22).
Recent investigations have shown that the RON receptor is expressed in various cell types including epithelial cells (23), and MSP-mediated effects on epithelial cells are integrin-dependent (23;24). We found that plating epithelial cells on ECM induced ligand-independent RON phosphorylation and kinase activation.
Addition of ligand to ECM-adherent cells caused an additional increment of phosphorylation and kinase activity. ECM mediated RON phosphorylation required kinase activity of both RON itself and c-Src. The data fit a pathway in which ECM-induced integrin aggregation causes both c-Src activation and RON oligomerization followed by RON kinase-dependent autophosphorylation. This results in RON becoming a target for activated c-Src, which phosphorylates additional tyrosines on RON. We also observed that integrin-induced EGFR phosphorylation likewise depended on both EGFR and c-Src kinase activities.
Thus, the defined two-step sequence may represent a general pathway for integrin-dependent growth factor RTK activation.  Table 1). Effects of ECM, alone or with growth factor, on receptor phosphorylation or downstream mediators have been described for Met (25), EGF (7;8;13-16), PDGF (7,8,16), FGF (8), VEGFR-2 (17) and insulin (16) receptors in various cell types indicating that cell-ECM interactions frequently regulate growth factor-RTK responses. In thinking about a molecular mechanism mediating the ECM effect on RON, we considered two possibilities, which are not mutually exclusive: [1] inasmuch as RON is associated with β1 integrin (24) (Fig.2A).
These data were consistent with the postulated ECM-integrin-FAK-c-Src pathway, and suggested that RON was phosphorylated by activated c-Src.
We therefore determined the capacity of c-Src to phosphorylate RON in vitro by adding active purified c-Src enzyme to RON immunoprecipitated from HEK 293 cells. In addition to RON, these cells were also transfected with the kinase-inactive c-Src construct to prevent possible in vivo c-Src activity. RON was immunoprecipitated from cells that were either unstimulated or stimulated with MSP or collagen. Active c-Src in vitro phosphorylated RON from collagenstimulated cells, but failed to phosphorylate RON from unstimulated or MSP-stimulated cells (Fig. 3). These results indicate that c-Src can phosphorylate RON, but sites become available for phosphorylation by c-Src only on RON from cells stimulated with ECM. In contrast to our findings, it has been reported that c-Src can phosphorylate non-stimulated or ligand-stimulated growth factor receptors (28)(29)(30). These published results are with cultured adherent cells, the integrins of which might be engaged by fibronectin derived from cells or serum. We also found that adhesion to ECM by epithelial cells expressing endogenous EGFR caused ligand-independent EGFR tyrosine phosphorylation as well as increased EGF-dependent tyrosine phosphorylation (Fig. 4A). Like our data for RON, ECM-mediated phosphorylation of the EGFR requires kinase activity of both the EGFR itself and c-Src. Inhibition of EGFR kinase activity by tyrphostin blocked both EGF and ECM-induced EGFR phosphorylation (Fig. 4A).
In contrast, inhibition of endogeneous c-Src kinase activity by overexpression of kinase-inactive c-Src had no effect on EGF-induced phosphorylation, but prevented the collagen-mediated increment (Fig. 4B). Thus, it appears that ECM-induced EGFR phosphorylation occurs via the pathway outlined above for RON, where EGFR catalytic activity and autophosphorylation are essential for c-Src to phosphorylate additional tyrosines on EGFR. Potentiation by c-Src of the mitogenic and tumorigenic capacity of EGFR is mediated by phosphorylation of additional tyrosines in EGFR by c-Src, when c-Src interacts with phosphorylated EGFR (29). The fact that receptor phosphorylation induced by ECM-dependent adhesion occurs via similar pathways for both RON and EGFR, which belong to different growth factor receptor kinase families, suggests that by guest on July 10, 2020 http://www.jbc.org/ Downloaded from Integrin-mediated growth factor receptor activation 13 this is a common pathway that integrins may use for regulation of growth factor RTK activity.
The nature of this regulation is a subject for further investigation. The fact that c-Src can phosphorylate RON from cells stimulated by ECM, but not by MSP ( Fig.3 B)