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* This research was supported in part by the Israel Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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 αvβ3 (not via the αvβ5 integrin), suggesting that αvβ3 differs from αvβ5 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 αvβ3 as well as the αvβ5 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 αvβ3 integrin on their surface; (ii) that upon adhesion of the β3-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.
focal adhesion kinase
-B, -C, protein kinases A, B, and C
mitogen-activated protein kinase
extracellular signal-regulated kinase
bovine aorta endothelial cells
c-Jun N-terminal kinase
fluorescence-activated cell sorting
radioimmune precipitation buffer
polyacrylamide gel electrophoresis
Vitronectin (Vn)1 is an adhesive glycoprotein found in the extracellular matrix (ECM) of various cells, and in circulating blood (
). 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 α and β subunits (
). Several of these integrins, e.g. αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8and the platelet-specific αIIbβ3 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. For example, the focal adhesion kinase (FAK) was shown to play a central role in mediating the signal from integrins (
). It does so by its autophosphorylation on Tyr397 upon integrin stimulation. This autophosphorylation leads to the recruitment and activation of intra-cellular mediators such as PI3K, as well as the Src family kinases, by an interaction of their SH2 domain with the autophosphorylated Tyr397residue. The PI3K binding to Tyr397 leads to activation of PKB, whereas the Src family of kinases further phosphorylates FAK on Tyr925 leading to the recruitment of additional signaling molecules that bring about an activation of the ERK pathway (
). Subsequently, we showed that Vn is a substrate for CK2, which phosphorylates Vn at Thr50 and Thr57. Furthermore, we found that this phosphorylation significantly enhances the adhesion and spreading of bovine aorta endothelial cells (BAEC), presumably because the phosphorylated Vn has a higher affinity for αvβ3 (
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, αvβ5, and that this family of integrins can, in turn, activate different intra-cellular pathways. Here we extend our studies on the consequences of the CK2 phosphorylation of Vn and show that the enhanced cell adhesion involves αvβ3 (but not αvβ5). Furthermore, we show that this enhanced adhesion coincides with a preferential activation of the FAK/PI3K/PKB cascade, rather than the ERK signaling pathway.
Intra-cellular protein phosphorylation is now well established as a central regulatory mechanism. In the last few years, several reports provided evidence for the occurrence of protein kinases outside the cell, raising the possibility that protein phosphorylation may also regulate extra-cellular processes (
). This possibility was supported by the identification of specific target substrates for the kinases in the cell exterior. Some reports further indicated that the physiological function of such specific substrates is modulated upon their phosphorylation (for a review see Ref.
In addition to PKA and PKC, Vn was recently shown to be a substrate for CK2, which was found to single out and selectively phosphorylate Vn at Thr50 and Thr57 to bring about a significant enhancement of one of Vn's well known physiological functions: cell adhesion and spreading (
). In the case of Vn, the specificity in the recognition of its CK2-phosphorylated form may have a special importance in cancer, because Vn seems to be an important ligand in the αvβ3-mediated adhesion of tumor cells. In line with this fact, human melanoma cells derived from lymphatic metastases were shown to use αvβ3 to adhere to lymph node Vn (
In view of our finding that the enhanced cell adhesion onto Vn(T50E,T57E) is mediated by αvβ3 and our previous observation that this enhancement is due to an increased affinity toward this integrin, we undertook to identify the signaling pathway that is involved in this increased affinity. Several signaling cascades were previously shown to be activated by the integrin family of receptors (
). Having in our hands cells that use αvβ3 to adhere onto Vn, and essentially identical companion cells that use αvβ5 to adhere to this integrin ligand, enabled us to identify a signaling pathway, which is differentially activated upon adhesion of these cells to CK2-phosphorylated and non-phosphorylated Vn analogs. Specifically, we were able to show that the phospho and non-phospho forms of Vn trigger both αvβ3 and αvβ5, leading to a similar activation of ERK. These results suggest that the activation of ERK occurs via the α subunit (
), which has not been modified in these cells. The fact that the activation of ERK is not influenced by the introduction to the β3 subunit supports this suggestion. In contrast, the PKB activation seems to depend on the availability of the β3subunit, and therefore, is preferentially activated by the phospho form of Vn. We presume that this enhanced activation of PKB, which is αvβ3- and PI3K-dependent, results in the enhanced cell adhesion by the CK2-PVn analog (Vn(T50E,T57E)).
Based on the results presented here, we suggest that although both αvβ3 and αvβ5 share common structural elements that recognize and bind equally well the core protein shared by Vn and PVn, αvβ3 contains additional recognition elements that bind the two phosphate groups specifically introduced in Vn by its CK2 phosphorylation. Specifically, this result raises the possibility that the ligand binding site of αvβ3 possesses recognition elements to CK2-PVn that are not present in αvβ5. This suggestion, which is supported by additional experimental evidence using a set of RGD-containing peptides as inhibitors,
M. Garazi, I. Schvartz, D. Seger, and S. Shaltiel, in preparation.
can account for the distinct behavior of the αvβ3 and αvβ5 integrins and specifically for the αvβ3-mediated enhanced activation of the PI3K/PKB pathway that correlates with the increased cell adhesion.
One of the important messages reported here lies in the fact that it identifies two intracellular signaling pathways that are unequally activated upon binding of Vn and CK2-PVn, at least to the cells we tested in this study. Both pathways (one functioning via αvβ3 and αvβ5and one via αvβ3 (Scheme FS1) are activated upon adhesion of the cells onto Vns. However, although the activation of ERK (triggered by both αvβ3 and αvβ5) was not modified upon cell adhesion onto CK2-PVn, the activation of PKB (triggered by αvβ3 but not by αvβ5) is elevated upon adhesion to CK2-PVn. It is this elevation that is correlated with the enhanced cell adhesion. Therefore, we propose that the PI3K/PKB pathway (and not the ERK pathway) reflects the αvβ3-mediated enhanced cell adhesion (Scheme FS1). In line with this proposal, we found that blocking the activation of ERK by an MEK inhibitor did not have an effect on cell adhesion. In contrast, the blocking of PKB by PI3K inhibitors reduced cell adhesion.
Taken together, the results presented here together with our results reported earlier (
) indicate the occurrence of a cell surface receptor (αvβ3) and an intracellular signaling pathway that distinguish between a CK2-phosphorylated and a non-phosphorylated form of Vn. These results are based on a CK2-phospho and a non-phospho form of Vn, two mutant analogs of Vn, three different cell lines, and four independent cell clones. We believe that these findings indicate that the extra-cellular phosphorylation of Vn by CK2 may well be a physiological process with a distinct regulatory role in the control of cell adhesion and spreading.
We thank Dr. Iris Schvartz for stimulating discussions and Shoshana Lichter and Tamar Hanoch for technical assistance.