Seger D, Gechtman Z, Shaltiel S
Department of Biological Regulation, Weizmann Institute of Science, IL-76100 Rehovot, Israel.
J Biol Chem. 1998 Sep 18;273(38):24805-13. doi: 10.1074/jbc.273.38.24805.
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 approximately 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 Km of 0.5-2 microM (lower than the Vn concentration in blood, 3-6 microM), (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 alphav beta3 Vn receptors.
细胞黏附蛋白玻连蛋白（Vn）先前被证明是细胞外蛋白激酶A在人血液中的主要作用靶点，该激酶在血小板受到凝血酶的生理刺激时从血小板中释放出来，并且在其他几种血细胞类型中也作为一种胞外酶存在。由于血浆Vn被证明只有一个蛋白激酶A磷酸化位点（Ser378），但含有大约3摩尔共价结合的磷酸盐，并且由于人血清和血细胞表面也被证明含有酪蛋白激酶II（CKII），我们研究了CKII对Vn的磷酸化作用，试图弄清楚这种磷酸化是否调节Vn的功能，这是对其具有生理相关性的严峻考验。在此我们表明：（i）Vn的CKII磷酸化的Km为0.5 - 2微摩尔（低于血液中Vn的浓度，即3 - 6微摩尔）；（ii）其作用靶点是Thr50和Thr57，它们紧邻Vn的RGD位点；（iii）Thr57的磷酸化促进了Thr50的磷酸化。发现血浆Vn的CKII磷酸化的最大化学计量比很低，原则上这可能是由于其在体内的部分预磷酸化。然而，为了检测功能调节，我们需要比较完全磷酸化的Vn（在Thr57和Thr50处）和未磷酸化的Vn。因此，我们在杆状病毒系统中表达Vn，并表明：（i）野生型Vn的CKII磷酸化增强了牛主动脉内皮细胞的黏附；（ii）双突变体T50E/T57E（其中中性的Thr残基被带负电荷的Glu残基取代，后者被认为是Thr - P的类似物）与野生型Vn或中性的T50A/T57A突变体相比，具有显著增强的促进细胞黏附和加速细胞铺展的能力；（iii）至少在牛主动脉内皮细胞的情况下，T50E/T57E突变体表现出增强的黏附，这似乎是由于对αvβ3 Vn受体的亲和力增加。
