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Sustained contraction of cells depends on sustained Rho-associated kinase (Rho-kinase) activation. We developed a computational model of the Rho-kinase pathway to understand the systems characteristics. Thrombin-dependent in vivo transient responses of Rho activation and Ca2+ increase could be reproduced in silico. Low and high thrombin stimulation induced transient and sustained phosphorylation, respectively, of myosin light chain (MLC) and myosin phosphatase targeting subunit 1 (MYPT1) in vivo. The transient phosphorylation of MLC and MYPT1 could be reproduced in silico, but their sustained phosphorylation could not. This discrepancy between in vivo and in silico in the sustained responses downstream of Rho-kinase indicates that a missing pathway(s) may be responsible for the sustained Rho-kinase activation. We found, experimentally, that the sustained phosphorylation of MLC and MYPT1 exhibit all-or-none responses. Bromoenol lactone, a specific inhibitor of Ca2+-independent phospholipase A2 (iPLA2), inhibited sustained phosphorylation of MLC and MYPT1, which indicates that sustained Rho-kinase activation requires iPLA2 activity. Thus, the systems analysis of the Rho-kinase pathway identified a novel iPLA2-dependent mechanism of the sustained Rho-kinase activation, which exhibits an all-or-none response. [ABSTRACT FROM AUTHOR]