Expression, subcellular localization, and cloning of the 130-kDa regulatory subunit of myosin phosphatase in porcine aortic endothelial cells

In endothelial cells in situ and in primary culture, immunoblot analysis re vealed an expression of the 130-kDa subunit of myosin phosphatase, similar to the myosin phosphatase targeting subunit (MYPT) of smooth muscle. Screen ing of an endothelial cell cDNA library yielded a clone encoding an NH2-ter minal fragment of 89.6 kDa, closely related to smooth muscle MYPT1. Two iso forms differing by a central insert of 56 residues were detected. In growin g cells, MYPT1 was localized on stress fiber, but at confluence the localiz ation pattern changed and MYPT1 was distributed close to the cell membrane and at cell-cell contacts. The membrane localization of MYPT1 suggested a t arget other than myosin and raised the possibility that MYPT1 may be involv ed in dephosphorylation of alternative substrate(s). These distinct mechani sms would also be dependent on the growth state of the endothelial cells, i .e., regulation of actin-myosin interactions in growing cells and an unknow n function in cells at confluence. (C) 1999 Academic Press.