Intracellular Distribution of Gravin, a PKA and PKC Binding Protein, in Vascular Endothelial Cells

Abstract

Gravin, a high-molecular-weight protein expressed widely in tissues and cells, is upregulated in cultured endothelial cells under conditions which suggest that it may play a role in wound repair and vascular development. In the current study, we examined the intracellular distribution of gravin to determine if it is associated with the cytoskeleton or with another intracellular compartment. Immunofluorescence microscopy of human umbilical vein endothelial cells (HUVEC) revealed that gravin had a punctate staining distribution that extended to the cell margin and did not appear to colocalize with stress fibers, microtubules, and intermediate filaments. Moreover, disruption of the cytoskeletal structures with either cytochalasin D or colchicine did not alter gravin distribution. However, confocal and immunoelectron microscopy clearly revealed that gravin was concentrated at the cell margin in close association with the plasma membrane. Immunoprecipitation of gravin from endothelial cell lysates resulted in coprecipitation of protein kinase activity that could be eluted from the immunoprecipitates with cAMP and that was inhibitable with a PKA-specific inhibitor. An anti-PKA catalytic subunit antibody reacted with a 40-kD band on immunoblots of the cAMP eluate. Immunoblots of the immunoprecipitates further revealed that PKCα coprecipitated with gravin from endothelial cell lysates. This study indicates that gravin is associated with either the plasma membrane or the membrane skeleton and may play a role in endothelial wound healing by targeting PKA and PKC to specific membrane-associated sites and regulating PKA/PKC-dependent cellular activities associated with endothelial wound healing.