The expression of a constitutively active form of Rho-kinase induced stress fiber and focal adhesion formation in fibroblasts and an increase in the level of myosin light chain (MLC20) phosphorylation (1). These results in smooth muscle and in non-muscle cells are attributed to an increase in MLC20 phosphorylation and this is thought to reflect the inhibition of myosin phosphatase (MP). MP is composed of three subunits: a catalytic subunit of type 1 phosphatase delta isoform, PP1c delta, and two non-catalytic subunits, 110 and 20 kDa. The 110 kDa subunit is a targeting molecule and thus has been termed myosin phosphatase target subunit 1 (MYPT1) or myosin-binding subunit (MBS). MBS (MYPT1) is the key molecule involved in regulation of MP activity (2). Phosphorylation by Rho-kinase inhibited MP activity and this reflected a decrease in Vmax. Activity of MP with different substrates also was inhibited by phosphorylation. Two major sites of phosphorylation on chicken MBS (MYPT1) were Thr695 and Thr850. Various point mutations were designed for these phosphorylation sites. Following thiophosphorylation by Rho-kinase and assays of phosphatase activity it was determined that Thr695 was responsible for inhibition.

Rho-kinase, which is activated by GTP-RhoA, phosphorylated MBS (MYPT1) and consequently inactivated myosin phosphatase (MP). Over-expression of RhoA or activated RhoA in NIH 3T3 cells increased phosphorylation of MBS (MYPT1) and MLC20. Thus, Rho appears to inhibit myosin phosphatase through the action of Rho-kinase.