CD44 Interaction with c-Src Kinase Promotes Cortactin-mediated Cytoskeleton Function and Hyaluronic Acid-dependent Ovarian Tumor Cell Migration

Abstract

In this study we have demonstrated that both CD44 (the hyaluronan (HA) receptor) and c-Src kinase are expressed in human ovarian tumor cells (SK-OV-3.ipl cell line), and that these two proteins are physically associated as a complex in vivo. Using a recombinant cytoplasmic domain of CD44 and an in vitro binding assay, we have detected a specific interaction between CD44 and c-Src kinase. Furthermore, the binding of HA to SK-OV-3.ipl cells promotes c-Src kinase recruitment to CD44 and stimulates c-Src kinase activity, which, in turn, increases tyrosine phosphorylation of the cytoskeletal protein, cortactin. Subsequently, tyrosine phosphorylation of cortactin attenuates its ability to cross-link filamentous actin in vitro. In addition, transfection of SK-OV-3.ipl cells with a dominant active form of c-Src (Y527F)cDNA promotes CD44 and c-Src association with cortactin in membrane projections, and stimulates HA-dependent/CD44-specific ovarian tumor cell migration. Finally, overexpression of a dominant-negative mutant of c-Src kinase (K295R) in SK-OV-3.ipl cells impairs the tumor cell-specific phenotype. Taken together, these findings strongly suggest that CD44 interaction with c-Src kinase plays a pivotal role in initiating cortactin-regulated cytoskeleton function and HAdependent tumor cell migration, which may be required for human ovarian cancer progression.The cell adhesion molecule, CD44, is one of the major hyaluronic acid (HA) 1 receptors (1-3). It belongs to a family of transmembrane glycoproteins which contain a variable extracellular domain, a single spanning 23-amino acid transmembrane domain, and a 70-amino acid cytoplasmic domain (4). Nucleotide sequence analyses reveal that many CD44 isoforms (derived from alternative splicing mechanisms) are variants of the standard form, CD44s (4). CD44s (molecular mass ϳ85 kDa) is the most common isoform of CD44 found in many cell types including human ovarian carcinoma cells (5-9). The presence of high levels of CD44s (often together with CD44v) is emerging as an important metastatic tumor marker in a number of carcinomas, and is also implicated in the unfavorable prognosis of a variety of cancers including human ovarian cancers (5-9).The invasive phenotype of CD44s-positive epithelial tumor cells has been linked to HA-mediated CD44 signaling and cytoskeletal activation. CD44s contains several HA-binding sites in their extracellular domain (1-3). The binding of HA to CD44s causes cells to adhere to the extracellular matrix (ECM) components (1-3), and has also been implicated in the stimulation of several different biological activities (10 -16). The intracellular domain of CD44 binds to signaling proteins such as RhoGTPases (e.g. RhoA) (17); Tiam1, a guanine nucleotide exchange factor for Rac1 (18); and cytoskeletal proteins, including ankyrin (2,3,9,(17)(18)(19)(20)(21) and the ERM proteins (ezrin, radixin, and moesin) (23). Recent studies indicate that the binding of ECM components (e.g. HA) promote CD44-mediated Tiam1-Rac1 signaling and cytoskeleton function lea...

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