D. Seger et al., PHOSPHORYLATION OF VITRONECTIN BY CASEIN KINASE-II - IDENTIFICATION OF THE SITES AND THEIR PROMOTION OF CELL-ADHESION AND SPREADING, The Journal of biological chemistry, 273(38), 1998, pp. 24805-24813
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.