Urokinase-dependent Human Vascular Smooth Muscle Cell Adhesion Requires Selective Vitronectin Phosphorylation by Ectoprotein Kinase CK2

Abstract

Urokinase (uPA)-and urokinase receptor (uPAR)-dependent cell adhesion to the extracellular matrix protein vitronectin (Vn) is an important event in wound healing, tissue remodeling, immune response, and cancer. We recently demonstrated that in human vascular smooth muscle cells (VSMC) uPA/uPAR are functionally associated with the ectoprotein kinase casein kinase-2 (CK2). We now asked whether CK2 regulates uPA-dependent cell adhesion to Vn, since the latter is a natural CK2 substrate. We found that Vn is indeed selectively phosphorylated by CK2 and that this phosphorylation is uPA-regulated in VSMC. Vn induces release of ecto-CK2 from the cell surface via a process termed as "shedding." CK2-mediated Vn phosphorylation was decisive for the uPA-dependent VSMC adhesion. Specific inhibition of CK2 completely abolished the uPA-induced cell adhesion to Vn. This effect was specific for cell adhesion to Vn and required participation of both uPAR and ␣ v ␤ 3 integrins as adhesion receptors. CK2 localization at the cell surface was highly dynamic; Vn induced formation of clusters where CK2 colocalized with uPAR and ␣ v ␤ 3 integrins. These results indicate that the uPA-dependent VSMC adhesion is a function of selective Vn phosphorylation by the ectoprotein kinase CK2 and suggest a regulatory role for Vn phosphorylation in the uPA-directed adhesive process.The urokinase (uPA) 1 /urokinase receptor (uPAR) system has multiple functions serving as a molecular link between pericellular proteolysis and signaling cascades, regulating cell adhesion, migration, and proliferation (1). Several reports suggest a possible role for uPA/uPAR in these processes independent of the uPA enzymatic activity. For instance, uPAR has a nonproteolytic action as a cellular adhesion receptor for vitronectin (Vn). Vn is a multifunctional glycoprotein present in blood, extracellular matrix, inflammatory sites, and in atherosclerotic plaques (2). UPAR contains a high affinity Vn-binding site, which is different from the uPA-binding site. This binding site makes uPAR uniquely poised to regulate extracellular proteolysis and the adhesion process (3). Vn has been shown to contain consensus sequences for phosphorylation by various protein kinases, such as cAMP-dependent protein kinase A (PKA), protein kinase C (PKC) and, and as recently described, the protein kinase casein kinase-2 (CK2) (4). Vn can be phosphorylated at specific sites that modulate its conformation and influences its subsequent functions (5-7). However, whether or not Vn phosphorylation plays any direct role in the uPA-directed cell adhesion has not been explored.We recently demonstrated that in human vascular smooth muscle cells (VSMC), the cell surface CK2 is associated with uPAR and might be activated by uPA. Activated CK2 mediates phosphorylation of the cell-surface shuttle protein nucleolin, which translocates then in its phosphorylated form into the cell and regulates mitogenic effects of uPA (8). However, ectoprotein kinases operating on numerous cell types can provide for the pho...

Protein kinase CK2 as an ectokinase: The role of the regulatory CK2β subunit

“…Several extracellular proteins have been identified as substrates of the CK2 ectokinase activity. Vitronectin has been shown to be phosphorylated extracellularly by CK2, and evidence has been presented to demonstrate that its phosphorylation regulates the adhesion of cells to the extracellular matrix (2,3). More recently, it has been shown that CK2 phosphorylates the C9 complement and it has been suggested that this phosphorylation might control cell lysis caused by this complement protein (4).…”

Protein kinase casein kinase 2 holoenzyme produced ectopically in human cells can be exported to the external side of the cellular membrane

“…Furthermore, it has been demonstrated that this CK2-like ectokinase enzyme phosphorylates a number of external proteins or the extracellular domains of membrane proteins (5)(6)(7)(8). However, there is a dearth of evidence concerning the mechanisms through which the CK2-like enzyme or other ectokinases are exported and about the structural features that tag these proteins for export.…”

“…Specifically, there are several reports that an ectokinase has the characteristics of protein kinase casein kinase 2 (CK2) (2)(3)(4). A CK2-like ectokinase activity has been reported to be responsible for the phosphorylation of vitronectin, an extracellular matrix protein (5,6), and of the external domain of the ␤-amyloid precursor protein (7) and T lymphocyte surface proteins (8). However, there is no information about the structural features that are responsible for the export of CK2 or other ectokinases.…”

Ecto-Phosphorylation and Regeneration of the Adult Central Nervous System

“…Threonine 50 and 57 of vitronectin can be phosphorylated by CK2 [31] and the phosphorylation enhances its binding to both alpha(v)beta3 integrin [64] and urokinase receptor [65]. Furthermore, the serine 378 of vitronectin is phosphorylated by PKA, which induces a conformational change and enhances the phosphorylation by CK2 [66].…”