Abstract
Phosphorylation of vitronectin (Vn) by casein kinase II was previously shown to occur at Thr50 and Thr57 and to augment a major physiological function of vitronectin-cell adhesion and spreading. Here we show that this phosphorylation increases cell adhesion via the alpha(v)beta3 (not via the alpha(v)beta5 integrin), suggesting that alpha(v)beta3 differs from alpha(v)beta5 in its biorecognition profile. Although both the phospho (CK2-PVn) and non-phospho (Vn) analogs of vitronectin (simulated by mutants Vn(T50E,T57E), and Vn(T50A,T57A), respectively) trigger the alpha(v)beta3 as well as the alpha(v)beta5 integrins, and equally activate the ERK pathway, these two forms are different in their activation of the focal adhesion kinase/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) pathway. Specifically, we show (i) that, upon exposure of cells to Vn/CK2-PVn, their PKB activation depends on the availability of the alpha(v)beta3 integrin on their surface; (ii) that upon adhesion of the beta3-transfected cells onto the CK2-PVn, the extent of PKB activation coincides with the enhanced adhesion of these cells, and (iii) that both the PKB activation and the elevation in the adhesion of these cells is PI3K-dependent. The occurrence of a cell surface receptor that specifically distinguishes between a phosphorylated and a non-phosphorylated analog of Vn, together with the fact that it preferentially activates a distinct intra-cellular signaling pathway, suggest that extra-cellular CK2 phosphorylation may play an important role in the regulation of cell adhesion and migration.
Highlights
Vitronectin (Vn)1 is an adhesive glycoprotein found in the extracellular matrix (ECM) of various cells, and in circulating blood [1,2,3]
We show (i) that, upon exposure of cells to Vn/ CK2-PVn, their protein kinase B (PKB) activation depends on the availability of the ␣v3 integrin on their surface; (ii) that upon adhesion of the 3-transfected cells onto the CK2-PVn, the extent of PKB activation coincides with the enhanced adhesion of these cells, and (iii) that both the PKB activation and the elevation in the adhesion of these cells is phosphatidylinositol 3-kinase (PI3K)-dependent
The PI3K binding to Tyr397 leads to activation of PKB, whereas the Src family of kinases further phosphorylates focal adhesion kinase (FAK) on Tyr925 leading to the recruitment of additional signaling molecules that bring about an activation of the ERK pathway [31,32,33,34,35,36,37,38]
Summary
Vitronectin; ECM, extracellular matrix; FAK, focal adhesion kinase; PI3K, phosphatidylinositol 3-kinase; PKA, -B, -C, protein kinases A, B, and C; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; BAEC, bovine aorta endothelial cells; JNK, c-Jun N-terminal kinase; FITC, fluorescein isothiocyanate; PBS, phosphatebuffered saline; HA, hemagglutinin; FACS, fluorescence-activated cell sorting; RIPA, radioimmune precipitation buffer; PAGE, polyacrylamide gel electrophoresis; r-Vn, recombinant Vn; PVn, phosphorylated Vn. The cell adhesion, spreading, and migration activities of Vn are associated with its RGD sequence located near the N terminus of the protein (positions 45– 47) This sequence is recognized by the family of receptors known as the integrins: heterodimers composed of ␣ and  subunits (24 –30). ␣v1, ␣v3, ␣v5, ␣v6, and ␣v8 and the platelet-specific ␣IIb3 integrin, are known to recognize and bind Vn. It is well known that cell adhesion is a complex process that was shown to involve an activation of several Vn receptors and a variety of intra-cellular signaling pathways. One of the major obstacles in revealing the mechanism of action of CK2-phosphorylated Vn originates from the well known fact that Vn (like other adhesion proteins) can bind to several integrins, including the specific Vn-binding integrin, ␣v5, and that this family of integrins can, in turn, activate different intra-cellular pathways. We show that this enhanced adhesion coincides with a preferential activation of the FAK/PI3K/PKB cascade, rather than the ERK signaling pathway
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