Stimulation of tyrosine- and serine-phosphorylation of focal adhesion kinase in mouse 3T3 cells by fibronectin and fibroblast growth factor.

Phosphorylation of both tyrosine and serine residues of focal adhesion kinase (FAK) was stimulated by the adhesion of BALB/c mouse 3T3 cells to fibronectin, but phosphorylation of threonine was not detectable. Acidic and basic fibroblast growth factors also stimulated the phosphorylation of serine and tyrosine of FAK in cells adhered to poly-L-lysine, but epidermal growth factor and platelet-derived growth factor did not. A fusion protein of fibronectin and basic fibroblast growth factor effectively induced the phosphorylation of FAK. Phosphorylation of FAK in the rat myoblast L-6 cell line, which lacks fibroblast growth factor receptors, was not stimulated by fibroblast growth factors, suggesting that the interaction of fibroblast growth factors with their receptors might cause the phosphorylation of FAK.

Possible role of protein kinase C in the regulation of intracellular stability of focal adhesion kinase in mouse 3T3 cells.

Effects of various types of protein kinase inhibitor on the adhesion and spreading of BALB/c mouse 3T3 cells and on the phosphorylation and stability of focal adhesion kinase (FAK) in the cells were studied. Inhibitors of protein tyrosine kinases, methyl 2,5-dihydroxycinnamate and herbimycin A, inhibited tyrosine-phosphorylation of FAK and the adhesion of 3T3 cells to fibronectin. Among inhibitors of serine/threonine kinases tested, calphostin C, a specific inhibitor of protein kinase C, inhibited cell spreading rather than cell adhesion, and it induced the decrease of intracellular FAK within 30 min. Inhibitors of tyrosine kinase, A kinase, G kinase, and myosin light chain kinase did not induce such a rapid and specific decrease of FAK. When calphostin C (20 microM) was added to sub-confluent monolayer cultures, serine-phosphorylation of FAK was inhibited by 67% within 2 h, and decrease in the amount of FAK and rounding up of the cells began after 4 h. Label-chase experiments indicated that about 60% of 35S-labeled FAK degraded within 1-2 h after addition of calphostin C to monolayer cultures. These results indicated that serine-phosphorylation of FAK induced by protein kinase C was important in the regulation of metabolic stability of FAK.

Inhibition of cell adhesion by type V collagen.

Human umbilical vein endothelial cells grew well in dishes coated with collagen types I, II, III, or IV. However, the same cells tended to detach themselves from dishes coated with type V collagen, and cell proliferation in these dishes was inhibited. Such anti-adhesive activity was partially retained by heat-denatured type V collagen or by its alpha 1 chain, but not by its alpha 2 chain. Several other cell types did not adhere to the type V collagen substratum even in the presence of 10% serum. The cell types strongly inhibited from adhering by type V collagen included Swiss mouse 3T3 cells and their MSV-transformants, BALB/c 3T3 cells and their methylcholanthrene-transformants, NIH 3T3 cells and their ras-transformants, BHK cells, CHO-9 cells, CHO-K1 cells, and mouse melanoma B16-F10 cells. Using Swiss mouse 3T3, we studied the effects of type V collagen on cell adhesion to fibronectin in serum-free medium. When the culture dishes were coated with a mixture of fibronectin with various concentrations of type V collagen, the adhesion of the cells was inhibited depending on the concentration of type V collagen. The inhibition of cell adhesion by type V collagen was competitively overcome by increased concentrations of fibronectin. The activity that interferes with the effects of fibronectin was retained mainly by the alpha 1 chain of heat-denatured type V collagen.

Inhibition of cell adhesion by proteolytic fragments of type V collagen.

Type V collagen inhibits the cell-substratum adhesion of many types of cells. In this study, inhibitory effects of type V collagen on the adhesion of mouse melanoma B16-F10 cells to fibronectin, laminin and vitronectin were investigated. When the culture dishes were coated with a mixture of fibronectin and type V collagen, adhesion of the cells was inhibited by 50% at a fibronectin/collagen molar ratio of 10/1. At a similar molar ratio, adhesion of the cells to laminin was inhibited moderately, but that to vitronectin was not significantly affected. Type V collagen added into culture medium was less effective in inhibiting cell adhesion. The antiadhesive activity of type V collagen was partially retained in the alpha 1 (V) chain of heat-denatured collagen. The alpha 1 (V) chain was split into two large fragments, 90 kDa and 60 kDa, by limited digestion with Staphylococcus aureus V8 proteinase. The 90-kDa fragment, which was derived from the C-terminal half of the alpha 1 (V) chain, inhibited the cell adhesion more profoundly than alpha 1 (V). However, little fibronectin bound to the 90-kDa fragment, while fibronectin bound to the 60-kDa fragment, which was less antiadhesive than the 90-kDa fragment, with the same extent as alpha 1 (V). We therefore concluded that the antiadhesive effect of type V collagen was not due to its specific binding to the fibronectin molecule.

Angiogenic activity of a fusion protein of the cell-binding domain of fibronectin and basic fibroblast growth factor.

We constructed a fusion protein of the cell-binding domain of human fibronectin and human basic fibroblast growth factor, and prepared a polypeptide with both cell-adhesive activity and growth factor activity. A human gene fragment coding for basic fibroblast growth factor was amplified by the polymerase chain reaction, and introduced into the expression vector pTF7520, which encodes the cell-binding domain of human fibronectin. The resulting plasmid encoded a fusion protein in which basic fibroblast growth factor was added covalently to the C-terminal end of the fibronectin fragment. The fusion protein was expressed in Escherichia coli JM109 cells and purified from the extract by heparin affinity chromatography. The purified fusion protein had cell-adhesive activity toward BALB/c 3T3 cells, and stimulated their DNA synthesis in serum-depleted cultures. The fusion protein gave maximum mitogenic activity at the concentration of 10 nM. The fusion protein adsorbed to culture dishes, or added to collagen gels, stimulated the growth of human umbilical-vein endothelial cells. The fusion protein stimulated the angiogenesis in chorioallantoic membranes of developing chick embryos.

Effects of fibronectin-type V collagen recombinant fusion protein on cell adhesion and cell proliferation.

An expression vector pTF7520-Col-V-In, which encodes a fusion protein of the cell-binding domain of fibronectin (C277) and the insulin- and heparin-binding domain of the alpha 1 chain of human type V collagen, was constructed. E. coli transfected with this plasmid synthesized a 50-kDa fusion protein. This fusion protein, C277-V, was purified from the crude extract by a single step heparin HPLC. Similar amounts of insulin bound to purified C277-V and to the alpha 1 chain of type V collagen as judged by the binding of peroxidase-conjugated insulin. Cell-adhesive activity of C277-V was lower than that of the original fibronectin fragment C274, but similar numbers of cells adhered to both protein substrates when the culture dishes were coated with 1 mM of each protein. Insulin bound to the C277-V substratum stimulated the growth of mouse mammary tumor MTD cells in serum-free culture medium.