Dynamic Regulation of Myosin Light Chain Phosphorylation by Rho-kinase

Myosin light chain (MLC) phosphorylation plays important roles in various cellular functions such as cellular morphogenesis, motility, and smooth muscle contraction. MLC phosphorylation is determined by the balance between activities of Rho-associated kinase (Rho-kinase) and myosin phosphatase. An impaired balance between Rho-kinase and myosin phosphatase activities induces the abnormal sustained phosphorylation of MLC, which contributes to the pathogenesis of certain vascular diseases, such as vasospasm and hypertension. However, the dynamic principle of the system underlying the regulation of MLC phosphorylation remains to be clarified. Here, to elucidate this dynamic principle whereby Rho-kinase regulates MLC phosphorylation, we developed a mathematical model based on the behavior of thrombin-dependent MLC phosphorylation, which is regulated by the Rho-kinase signaling network. Through analyzing our mathematical model, we predict that MLC phosphorylation and myosin phosphatase activity exhibit bistability, and that a novel signaling pathway leading to the auto-activation of myosin phosphatase is required for the regulatory system of MLC phosphorylation. In addition, on the basis of experimental data, we propose that the auto-activation pathway of myosin phosphatase occurs in vivo. These results indicate that bistability of myosin phosphatase activity is responsible for the bistability of MLC phosphorylation, and the sustained phosphorylation of MLC is attributed to this feature of bistability

Disturbed intramitochondrial phosphatidic acid transport impairs cellular stress signaling

Lipid transfer proteins of the Ups1/PRELID1 family facilitate the transport of phospholipids across the intermembrane space of mitochondria in a lipid-specific manner. Heterodimeric complexes of yeast Ups1/Mdm35 or human PRELID1/TRIAP1 shuttle phosphatidic acid (PA) mainly synthesized in the endoplasmic reticulum (ER) to the inner membrane, where it is converted to cardiolipin (CL), the signature phospholipid of mitochondria. Loss of Ups1/PRELID1 proteins impairs the accumulation of CL and broadly affects mitochondrial structure and function. Unexpectedly and unlike yeast cells lacking the CL synthase Crd1, Ups1-deficient yeast cells exhibit glycolytic growth defects, pointing to functions of Ups1-mediated PA transfer beyond CL synthesis. Here, we show that the disturbed intramitochondrial transport of PA in ups1. cells leads to altered unfolded protein response (UPR) and mTORC1 signaling, independent of disturbances in CL synthesis. The impaired flux of PA into mitochondria is associated with the increased synthesis of phosphatidylcholine and a reduced phosphatidylethanolamine/phosphatidylcholine ratio in the ER of ups1. cells which suppresses the UPR. Moreover, we observed inhibition of target of rapamycin complex 1 (TORC1) signaling in these cells. Activation of either UPR by ER protein stress or of TORC1 signaling by disruption of its negative regulator, the Seh1-associated complex inhibiting TORC1 complex, increased cytosolic protein synthesis, and restored glycolytic growth of ups1. cells. These results demonstrate that PA influx into mitochondria is required to preserve ER membrane homeostasis and that its disturbance is associated with impaired glycolytic growth and cellular stress signaling

Mathematical modeling and simulation of MLC phosphorylation regulated by Rho-kinase.

<p>(A) Schematic overview of MLC phosphorylation regulated by Rho-kinase signal. Arrows, dashed line and - indicate stimulatory enzymatic reactions, myosin phosphatase auto-dephosphorylation signaling pathway and phosphorylation, respectively. (B) The graph shows that the transient Rho-kinase activations (red and blue line) are given as the input signal in our simulation. (C)(D) The simulation results of MLC and MYPT1 phosphorylation induced by the transient Rho-kinase activation in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039269#pone-0039269-g001" target="_blank">Figure 1B</a>. (E)(F) The phosphorylation of MYPT1 and MLC against time t was calculated using the various kcat₂ values. (G) The phosphorylation of MLC against time t was calculated using the different kcat₃ values.</p

Activity of myosin phosphatase is bistable.

<p>[MP] denotes the concentration of activated myosin phosphatase. The concentration of activated myosin phosphatase is assumed to represent that of non-phosphorylated MYPT1. d[MP]/dt indicates the differentiation of [MP] against time t. (A) The plots are d[MP]/dt against [MP]. In the steady states, the reaction velocity is zero, whereby the steady states are d[MP]/dt = 0. Three intersections with the horizontal axis represent three steady states (S1, S2, and S3). (B) d[MP]/dt was plotted against [MP] using the various kcat₂ values. (C) d[MP]/dt was plotted against [MP] using the various kcat₁ values.</p