Calcium Oscillations Trigger Focal Adhesion Disassembly in Human U87 Astrocytoma Cells

Abstract

Integrin-associated intracellular Ca2؉ oscillations modulate cell migration, probably by controlling integrin-mediated release of the cell rear during migration. Focal adhesion kinase (FAK), via its tyrosine phosphorylation activity, plays a key role in integrin signaling. Cell migration is a cyclic process involving initial protrusion of the leading edge, formation of adhesive sites, contraction of the cell body, and release of adhesive sites at the cell rear (1). Adhesive sites are dynamic membrane structures that vary in size and composition during migration. Integrins, actin stress fibers (SFs) 1 and other structural proteins, and regulatory signaling molecules cluster at focal adhesions (2). Focal adhesions (FAs) serve as points of traction for contractile forces underlying forward cell movement and their dynamics are finely regulated. For example, FAs are highly motile in stationary fibroblasts but are largely stationary in migrating fibroblasts, thereby transducing contractile forces into movement (3). This suggests the existence of a molecular clutch that couples cytoskeleton-mediated traction and cell contraction. In human U87 astrocytoma cells, expression of the dominant negative FAK-related non-kinase domain (FRNKFocal adhesion kinase (FAK) is activated and localized at FAs upon cell adhesion to the extracellular matrix (ECM; Refs. 4 and 5). Given the abundance of FAs and the reduced migration of fibroblasts from FAK null mice (6), FAK is likely involved in FA remodeling during migration. FAK-related nonKinase (FRNK), the non-catalytic C-terminal portion of FAK containing the FA targeting sequence, is also expressed as a separate dominant negative protein (7). The differential expression of FAK and FRNK is transcriptionally regulated, each of these proteins having distinct promoters within the FAK gene (8). Although the function of endogenous FRNK is not clear, FRNK has been used to alter signaling via endogenous FAK. When overexpressed in cells, FRNK acts as a negative regulator of FAK activity, inhibiting phosphorylation of FAK and different FAK-related processes, including cell cycle progression (9, 10), cell spreading on fibronectin (7, 11), and migration (12, 13). This suggests that the inhibitory effects of FRNK in migration might arise from altered FAK localization and phosphorylation.We and others reported that migration is dependent on Ca 2ϩ signaling in astrocytoma (14)

ERK 1/2- and JNKs-dependent Synthesis of Interleukins 6 and 8 by Fibroblast-like Synoviocytes Stimulated with Protein I/II, a Modulin from Oral Streptococci, Requires Focal Adhesion Kinase

“…A universal tyrosine kinase assay kit was from Takara Biomedicals (Genevilliers, France). The FAKrelated non-kinase plasmid (FRNK-YCam) has been previously described (27), the pRk5-ShcY317F plasmid was a generous gift from Dr. Filippo G. Giancotti (Memorial Sloane-Kettering Cancer Center, New York, NY). The Nucleofector TM kit was from Amaxa (Köln, Germany).…”

“…Cells were cultured in Dulbecco's modified Eagle's medium containing penicillin (100 IU/ml), streptomycin (100 g/ml), amphotericin B (0.25 g/ml), ␤-mercaptoethanol (0.1 mM), nonessential amino acids (1%), sodium pyruvate (1%), and 10% heat-inactivated FCS. FAKϩ/ϩ cells were transfected with FRNK-YCam (27) by the calcium phosphate method. Briefly, 15 g of plasmidic DNA in 1 ml of BES-buffered saline (borate-buffered saline, pH 6.95, containing 2.5 mM of CaCl 2 ) was added to 5 ϫ 10 5 cells for 20 h at 37°C (3% CO 2 ).…”

Identification of Channels Promoting Calcium Spikes and Waves in HT1080 Tumor Cells

“…Ca 2ϩ transients have been observed in migrating neurons, neutrophils, fibroblasts, eosinophils, tumor cells, and other cell types (3)(4)(5)(6)(7)(8)(9)(10). These oscillations are thought to participate in coordinating the cyclic temporal features of cell migration, such as pseudopod extension, actin assembly, integrin regulation, the phosphorylation-mediated regulation of focal adhesion formation, and pericellular proteolysis (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). Thus, repetitive Ca 2ϩ signals likely play an important role in the repetitive structural and functional changes required for cell movement.…”

Rac1 and RhoA Promote Neurite Outgrowth through Formation and Stabilization of Growth Cone Point Contacts

“…Integrin engagement of ECM ligands results in receptor clustering and the formation of various adhesion complexes (Geiger et al, 2001) that provide a linkage to the actin cytoskeleton and support the force generation necessary for growth cone advance (Suter et al, 1998;Beningo et al, 2001). In addition to providing adhesive support for mechanical tension, studies have shown that integrin receptors activate second-messenger signaling cascades, which help coordinate assembly and disassembly of adhesion sites (Gomez et al, 2001;Giannone et al, 2002;Ivins et al, 2004).…”