Ca2+ -independent phospholipase A2-dependent sustained Rho-kinase activation exhibits all-or-none response.
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.
Maeda
Akio
A
Undergraduate Program for Bioinformatics and Systems Biology, Graduate School of Information Science and Technology, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Ozaki
Yu-ichi
Y
Sivakumaran
Sudhir
S
Akiyama
Tetsuro
T
Urakubo
Hidetoshi
H
Usami
Ayako
A
Sato
Miharu
M
Kaibuchi
Kozo
K
Kuroda
Shinya
S
D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003198:Computer Simulation; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D054513:Group IV Phospholipases A2; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008954:Models, Biological; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D054460:rho-Associated Kinases
D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D010741:Phospholipases A; D054513:Group IV Phospholipases A2; D054467:Phospholipases A2; D043263:Myosin-Light-Chain Phosphatase; C490372:PPP1R12A protein, human; D013917:Thrombin; D020741:rho GTP-Binding Proteins; D002118:Calcium