Cysteine-rich protein (CRP) 2, expressed in vascular smooth muscle cells (VSMCs) of blood vessels, plays a critical role in regulating VSMC migration and vascular remodeling. To investigate the molecular mechanisms by which CRP2 regulates migration, we first determined subcellular localization of CRP2. Transfection of VSMCs with CRP2-EGFP constructs revealed that CRP2 is associated with actin cytoskeleton, suggesting a cytoskeletal function of CRP2. CRP2 null VSMCs adhered to or spread on extracellular matrix normally. In response to chemoattractant stimulation, lack of CRP2 increased VSMC lamellipodia formation. Immunofluorescence staining revealed that CRP2 colocalized with phospho-p130Cas, a scaffold protein important for lamellipodia formation, at focal adhesions (FAs) at the terminal ends of stress fibers in non-migrating cells. Mammalian 2-hybrid and coimmunoprecipitation assays further confirmed the interaction of CRP2 and p130Cas. Interestingly, in migrating cells phospho-p130Cas was present at the leading edge of lamellipodia and FAs while CRP2 was only found at FAs. Taken together, our results suggest that CRP2 may sequester p130Cas at FAs, thereby reducing lamellipodia formation and subsequent cellular migration.
