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J Biol Chem, Vol. 273, Issue 38, 24805-24813, September 18, 1998

Phosphorylation of Vitronectin by Casein Kinase II
IDENTIFICATION OF THE SITES AND THEIR PROMOTION OF CELL ADHESION AND SPREADING

From the Department of Biological Regulation, Weizmann Institute of Science, IL-76100 Rehovot, Israel

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 types of blood cells. Because plasma Vn was shown to have only one protein kinase A phosphorylation site (Ser³⁷⁸) but to contain ~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 surface, 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 CKII phosphorylation of Vn has a K_m of 0.5-2 µM (lower than the Vn concentration in blood, 3-6 µM), (ii) that it is targeted to Thr⁵⁰ and Thr⁵⁷, which are vicinal to the RGD site of Vn, and (iii) that the phosphorylation of Thr⁵⁷ facilitates the phosphorylation of Thr⁵⁰. 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 functional modulation, we needed a comparison between a fully phosphorylated Vn (at Thr⁵⁷ and Thr⁵⁰) and a nonphosphorylated Vn. Therefore, we expressed Vn in a baculovirus system and show (i) that the CKII phosphorylation of wt-Vn enhances the adhesion of bovine aorta endothelial cells; (ii) that the double mutant T50E/T57E (in which the neutral Thr residues are replaced by the negatively charged Glu residues considered analogs of Thr-P) has a significantly enhanced capacity to promote cell adhesion and to accelerate cell spreading when compared with either wild-type Vn or to the neutral T50A/T57A mutant; and (iii) that, at least in the case of bovine aorta endothelial cells, the T50E/T57E mutant exhibits an enhanced adhesion, which seems to be due to an increased affinity toward the _v3 Vn receptors.

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