INVOLVEMENT OF PROTEIN PHOSPHATASE-1 IN CYTOSKELETAL ORGANIZATION OF CULTURED ENDOTHELIAL-CELLS

The phosphorylation and dephosphorylation of cytoskeletal proteins reg ulate the shape of eukaryotic cells. To elucidate the role of serine/t hreonine protein phosphatases (PP) in this process, we studied the eff ects of calyculin A (CLA), a potent and specific inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A) on the cytoskeletal structure of cultured human umbilical vein endothelial cells (HUVECs). The addition of CLA (5 min) caused marked alterations in cell morphology, such as cell constriction and bleb formation. Microtubules and F-actin were re organized, becoming markedly condensed around the nucleus. Although th e fluorescence intensity of phosphoamino acids was not significantly d ifferent according to immunocytochemistry between cells with and witho ut CLA, polypeptides of 135, 140, 158, and 175 kDa were specifically p hosphorylated on serine and/or threonine residues. There was no signif icant effect on tyrosine residues. The effects of CLA on cytoskeletal changes and protein phosphorylation were almost completely inhibited b y the non-selective kinase inhibitor, K-252a. The effect of CLA on cel l morphology was at least 100 times more potent than that of okadaic a cid, consistent with the inhibitory potency against PP-1. The catalyti c subunit of PP-1 was also identified in HUVECs by Western blotting wi th its monoclonal antibody. These results suggest that PP-1 is closely involved in sustaining the normal structure of the cytoskeleton. (C) 1995 Wiley-Liss, Inc.