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. 2004 Sep;87(3):1417-25.
doi: 10.1529/biophysj.104.042721.

A Signal Transduction Pathway Model Prototype II: Application to Ca2+-calmodulin Signaling and Myosin Light Chain Phosphorylation

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Free PMC article

A Signal Transduction Pathway Model Prototype II: Application to Ca2+-calmodulin Signaling and Myosin Light Chain Phosphorylation

Thomas J Lukas. Biophys J. .
Free PMC article

Abstract

An agonist-initiated Ca(2+) signaling model for calmodulin (CaM) coupled to the phosphorylation of myosin light chains was created using a computer-assisted simulation environment. Calmodulin buffering was introduced as a module for directing sequestered CaM to myosin light chain kinase (MLCK) through Ca(2+)-dependent release from a buffering protein. Using differing simulation conditions, it was discovered that CaM buffering allowed transient production of more Ca(2+)-CaM-MLCK complex, resulting in elevated myosin light chain phosphorylation compared to nonbuffered control. Second messenger signaling also impacts myosin light chain phosphorylation through the regulation of myosin light chain phosphatase (MLCP). A model for MLCP regulation via its regulatory MYPT1 subunit and interaction of the CPI-17 inhibitor protein was assembled that incorporated several protein kinase subsystems including Rho-kinase, protein kinase C (PKC), and constitutive MYPT1 phosphorylation activities. The effects of the different routes of MLCP regulation depend upon the relative concentrations of MLCP compared to CPI-17, and the specific activities of protein kinases such as Rho and PKC. Phosphorylated CPI-17 (CPI-17P) was found to dynamically control activity during agonist stimulation, with the assumption that inhibition by CPI-17P (resulting from PKC activation) is faster than agonist-induced phosphorylation of MYPT1. Simulation results are in accord with literature measurements of MLCP and CPI-17 phosphorylation states during agonist stimulation, validating the predictive capabilities of the system.

Figures

FIGURE 1
FIGURE 1
Downstream signaling elements in the cytoplasmic compartment. Shown are the details of the MLC phosphorylation/dephosphorylation module. The simulation system contains dynamic regulation of MLCK activity through Ca2+-CaM and MLCP activity through phosphorylation by Rho kinase and inhibition by phosphorylated CPI-17. CPI-17 is phosphorylated by PKC that is activated by DAG produced by activated phospholipase C. RhoA that is generated by G-protein exchange with receptor activated G proteins activates Rho kinase, and cG kinase is activated by cGMP.
FIGURE 2
FIGURE 2
Predicted effects of calmodulin buffering on peak MLC phosphorylation and Ca2+-CaM-MLCK complex formation. (A) Effect of changing the Ca2+ dissociation constant for the CaM-buffer protein complex on the peak of MLC phosphorylation. (B) Profile of MLC phosphorylation (MLC_P) and Ca2+-CaM-MLCK complex formation (inset) in the presence (solid line) and absence (dotted line) of CaM buffering. Simulations were run at a fixed ligand (100-nM) concentration.
FIGURE 3
FIGURE 3
Binding of myosin Ic IQ-domain peptide analogs to CaM. Dansyl-bovine brain calmodulin (200 nM) was titrated with peptide IQ1, RKHSIATFLQARWRGYHQRQKFL (squares) or IQ2, HMKHSAVEIQSWWRGTIGRRKAA (circles) in 50 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.5. Error bars are from duplicates run in the titration assay. Fluorescence emission was measured at 535 nm with excitation at 335 nm (10-nm band pass) using a Victor 2 96-well plate reading fluorometer. Data are corrected for background fluorescence in the absence of Dansyl-CaM.
FIGURE 4
FIGURE 4
Predictions of effects of myosin phosphatase regulatory elements on MLC phosphorylation (MLC_P). (A) Simulation of MLC phosphorylation as a function of the percent of MYTP prephosphorylated in a model cell stimulated with 100 nM G protein-coupled receptor ligand. MLC_P production increases with MYPT phosphorylation at 0 (thinnest line), 25%, 50%, and 75% (thickest line) prephosphorylated enzyme. (B) Simulation of MLC_P production in the presence and absence of CPI-17P inhibitor generation, and stimulated MYPT phosphorylation: control (solid line), no CPI-17 phosphorylation (dashed line), no additional MYTP1 phosphorylation (dotted line), and no additional MYPT phosphorylation or CPI-17P (dot-dash line).
FIGURE 5
FIGURE 5
Ligand dependence of MLCP regulatory activities. Illustrated are examples of simulated output of MLC_P production (solid lines), MYPT phosphorylation (dashed lines), and CPI-17P-MLCP complex (dotted lines) at (A) 1 nM, (B) 10 nM, and (C) 100 nM ligand.
FIGURE 6
FIGURE 6
Predicted effects of MLCK prephosphorylation on MLC_P production in agonist-stimulated cells. Models were modified to contain phosphorylated MLCK with enhanced specific activity (ERK phosphorylation) or decreased Ca2+-CaM sensitivity (CaMKII phosphorylation). Simulations were done at 100 nM ligand (10× Kd) with no MLCK phosphorylation (solid line), stoichiometric ERK phosphorylation (dashed line), stoichiometric CaMKII phosphorylation (dotted lines), and a combination of both (dot-dash lines).

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