PHOSPHORYLATION OF VITRONECTIN BY CASEIN KINASE-II - IDENTIFICATION OF THE SITES AND THEIR PROMOTION OF CELL-ADHESION AND SPREADING

The cell adhesion protein vitronectin (Vn) was previously shown to be the major target in human blood for an extracellular protein kinase A, which is released from platelets upon their physiological stimulation with thrombin and also prevails as an ectoenzyme in several other typ es of blood cells. Because plasma Vn was shown to have only one protei n kinase A phosphorylation site (Ser(378)) but to contain similar to 3 mol of covalently bound phosphate, and because human serum and blood cells were shown to contain also a casein kinase II (CKII) on their su rface, we studied the phosphorylation of Vn by CKII attempting to find out whether such phosphorylation modulates Vn function, an acid test for its having a physiological, relevance. Here we show (i) that the C KII phosphorylation of Vn has a K-m of 0.5-2 mu M (lower than the Vn c oncentration in blood, 3-6 mu M), (ii) that it is targeted to Thr(50) and Thr(57), which are vicinal to the RGD site of Vn, and (iii) that t he phosphorylation of Thr(57) facilitates the phosphorylation of Thr(5 0). The maximal stoichiometry of the CKII phosphorylation of plasma Vn was found to be low, which, in principle, could be due to its partial prephosphorylation in vivo. However, for the detection of a functiona l modulation, we needed a comparison between a fully phosphorylated Vn (at Thr(57) and Thr(50)) and a nonphosphorylated Vn, Therefore, we ex pressed Vn in a baculovirus system and show (i) that the CKII phosphor ylation of wt-Vn enhances the adhesion of bovine aorta endothelial cel ls; (ii) that the double mutant T50E/T57E (in which the neutral Thr re sidues are replaced by the negatively charged Glu residues considered analogs of Thr-P) has a significantly enhanced capacity to promote cel l adhesion and to accelerate cell spreading when compared with either wild-type Vn or to the neutral T50A/T57A mutant; and (iii) that, at le ast in the case of bovine aorta endothelial cells, the T50E/T57E mutan t exhibits an enhanced adhesion, which seems to be due to an increased affinity toward the alpha(v)beta(3) Vn receptors.