Dominant affectors in the calmodulin network shape the time courses of target responses in the cell.
In endothelial cells nitric oxide synthase is a dominant affector in the calmodulin network by virtue of its ability to bind a significant fraction of limiting intracellular calmodulin. We have investigated how this affector function influences the kinetics of calmodulin-dependent signaling in cells co-expressing the synthase and a fluorescent calmodulin target analog similar in its interactions with calmodulin to myosin light chain kinase. The synthase binds (Ca(2+))(4)-calmodulin with a K(d) value of approximately 0.2 nM and an association rate constant of approximately 1.5 x 10(5) M(-1) s(-1). These values are, respectively, 10- and 100-fold smaller than the corresponding values for the analog. Thus, when Ca(2+) is added to a mixture of calmodulin, target analog and synthase in vitro a large fluorescence transient with a relaxation time of approximately 600 s is observed as (Ca(2+))(4)-calmodulin is rapidly bound to the analog and then slowly captured by the higher affinity synthase. A rapid increase in the free Ca(2+) concentration elicits similar transient analog responses in cells expressing the cytoplasmic target analog and either a wild-type membrane bound or mutant cytoplasmic synthase. Transient responses are not observed in cells co-expressing the fluorescent analog and a mutant T497D synthase unable to bind calmodulin. These results demonstrate that dominant affectors in the calmodulin network shape both the magnitudes and time courses of target responses in the cell.
Tran
Quang-Kim
QK
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri at Kansas City, 64110-2499, USA.
Black
D J
DJ
Persechini
Anthony
A
D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D005456:Fluorescent Dyes; D007700:Kinetics; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D002147:Calmodulin; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D009219:Myosin-Light-Chain Kinase